Ion Channel Factsbook, Volume 4: Voltage-Gated Channels

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THE ION CHANNEL FactsBook IV

Voltage-Gated Channels

Other books in the FactsBook Series: Edward C. Conley The Ion Channel Factsbook I: Extracellular Ligand-Gated Channels Edward C. Conley The Ion Channel Factsbook H: Intracellular Ligand-Gated Channels A. Neil Barclay, Albertus D. Beyers, Marian L. Birkeland, Marion H. Brown, Simon J. Davis, Chamorro Somoza and Alan F. Williams The Leucocyte Antigen FactsBook, 1st edn Robin Callard and Andy Gearing The Cytokine FactsBook Steve Watson and Steve Arkinstall The G-Protein Linked Receptor FactsBook Rod Pigott and Christine Power The Adhesion Molecule FactsBook Grahame Hardie and Steven Hanks

The Protein Kinase FactsBook The Protein Kinase FactsBook CD-Rom Kris Vaddi, Margaret Keller and Robert Newton The Chemokine FactsBook Marion E. Reid and Christine Lomas-Francis The Blood Group Antigen FactsBook A. Neil Barclay, Marion H. Brown, S.K. Alex Law, Andrew J. McKnight, Michael G. Tomlinson and P. Anton van der Merwe The Leucocyte Antigen FactsBook, 2nd edn Jeff Griffiths and Clare Sansom The Transporter FactsBook Robin Hesketh The Oncogene and Thmour Suppressor Gene FactsBook, 2nd edn Shirley Ayad, Ray P. Boot-Handford, Martin J. Humphries, Karl E. Kadler and C. Adrian Shuttleworth The Extracellular Matrix FactsBook, 2nd edn TakW. Mak The Gene Knockout FactsBook

THE ION CHANNEL

FactsBook IV Voltage-Gated Channels Edward C. Conley Molecular Pathology, c/o Ion Channel/Gene Expression University of Leicester/Medical Research Council Centre for Mechanisms of Human Toxicity, U.K.

William

J. Brammar

Department of Biochemistry, University of Leicester, UK

Academic Press SAN DIEGO LONDON BOSTON NEW YORK SYDNEY TOKYO TORONTO

This book is printed on acid-free paper Copyright © 1999 by ACADEMIC PRESS

All rights reserved No part of this publication may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press 24-28 Oval Road, London NWI 7DX, UK http://www.hbuk.co.uk/ap/ Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com ISBN 0-12-184453-6 A catalogue record for this book is available from the British Library

Library of Congress Catalogue Card Number: 98-86472

Typeset in Great Britain by Alden Bookset, Oxford Printed in Great Britain by WBC, Bridgend, Mid Glamorgan 99 00 01 02 03 04 WB 9 8 7 6 5 4 3 2 1

Contents Cumulative table of contents for Volumes I to IV (entry 01 resume) Acknowledgements Introduction &. layout of entries (entry 02 resume) How to use The Ion Channel FactsBook Guide to the placement criteria for each field Abbreviations (entry 03 resume)

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VIII

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xn xm

xv

XVII XXXIX

VOLUME IV VOLTAGE-GATED CHANNELS VLG Key facts (entry 41) Voltage-gated channel families - key facts References

3 21

VLG Ca (entry 42) Voltage-gated calcium channels Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References Note Added in Proof

22 22 39 55 69 84 101 131 140 153

VLG CI (entry 43) Voltage-gated chloride channels Nomenclatures Expression Sequence Analysis Structure and Functions Electrophysiology Pharmacology Information Retrieval References

154 154 157 166 173 177 186 189 193

VLG K A-T [native] (entry 44) Rapidly inactivating, transient outward i A-type' K+ currents in native cell types of vertebrates Nomenclatures Expression Electrophysiology Pharmacology Information Retrieval References

196 196 201 206 211 219 223

VLG K DR [native] (entry 45) 'Delayed rectifier'-type K+ currents in native cell types of vertebrates Nomenclatures Expression Structure and Functions Electrophysiology Pharmacology Information Retrieval References

226 226 231 246 249 252 268 269

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VLG K eag/elk/erg (entry 46) K+ channels encoded by genes related to Drosophila eag (ether-a-go-go) (gene subfamilies eag, elk, erg) Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

275 275 284 291 298 306 313 321 323

VLG K Kv-beta (entry 47) Cytoplasmic (Kv,B) subunits co-assembling with pore-forming (Kvo) voltage-gated potassium channel subunits Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

327 327 332 346 352 359 363 366 372

VLG K Kvl-Shak (entry 48) Vertebrate K+ channels related to Drosophila Shaker (Kvo subunits encoded by gene subfamily Kvl) Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

374 374 383 417 436 460

484 507 513

VLG K Kv2-Shab (entry 49) Vertebrate K+ channel subunits related to Drosophila Shab (Kvo subunits encoded by gene subfamily Kv2) Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

524 524 528 537 541 545 550 554 556

VLG K Kv3-Shaw (entry 50) Vertebrate K+ channels related to Drosophila Shaw (Kvo subunits encoded by gene subfamily Kv3) Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

559 559 565 582 591 599 607 610 614

II

"------------VLG K Kv4-Shal (entry 51) Vertebrate K+ channel subunits related to Drosophila Shal (Kvo: subunits encoded by gene subfamily Kv4) Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

617 617 621 629 632 634 638 642 644

VLG K Kvx [unassigned] (entry 52) Listing of cDNA clones encoding Kv channels with unassigned gene family relationships Nomenclatures Expression Sequence Analyses Structure and Functions Information Retrieval References

647 647 651 653 654 655 656

VLG K M-i [native] (entry 53) 'Muscarinic-inhibited' K+ channels underlying 1M (M-current in native cell types) Nomenclatures Expression Structure and Functions Electrophysiology Pharmacology Information Retrieval References

657 657 661 665 669 679 699 699

VLG (K) minK (entry 54) 'Minimal' protein subunits (minK, IsK) eliciting 'slow-activating' voltage-gated currents in oocytes Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

703 703 708 723 728 743 750 760 765

VLG Na (entry 55) Voltage-gated sodium channels Nomenclatures Expression Sequence Analyses Structure and Functions Electrophysiology Pharmacology Information Retrieval References

768 768 772 788 796 806 816 827 831

Rubrics (entry 13 resume)

839

Index

842

III

Cumulative table of contents for Volumes I to IV Contents Cumulative table of contents for Volumes I to IV (entry 01) Acknowledgements Introduction and layout of entries (entry 02) How to use The Ion Channel FactsBook Guide to the placement criteria for each field Abbreviations (entry 03)

VOLUME I

EXTRACELLULAR LIGAND-GATED CHANNELS

ELG Key facts (entry 04)

Extracellular ligand-gated receptorchannels - key facts

ELG CAT 5-HTa (entry 05) Extracellular 5-hydroxytryptaminegated integral receptor-channels ELG CAT ATP (entry 06) Extracellular ATP-gated receptorchannels (P2xR) ELGCATGLUAMPA!KAIN(entry07) AMPA / kainate-selective (nonNMDA) glutamate receptor-channels ELG CAT GLU NMDA (entry 08) N-Methyl-D-aspartate (NMDA)selective glutamate receptor-channels

VOLUME II

ELG CAT nAChR (entry 09) Nicotinic acetylcholine-gated integral receptor-channels ELG Cl GABAA (entry 10) Inhibitory receptor-channels gated by extracellular gamma-aminobutyric acid ELG Cl GLY (entry 11) Inhibitory receptor-channels gated by extracellular glycine Feedback and access to the CellSignalling Network (entry 12) Rubrics (entry 13) Entry and field number rubrics

INTRACELLULAR LIGAND-GATED CHANNELS

ILG Key facts (entry 14) The intracellular ligand-gated channel group - key facts ILG Ca AA-LTC4 [native] (entry 15) Native Ca2 + channels gated by the arachidonic acid metabolite leukotriene C4 incorporating general properties of ion channel regulation by arachidonate metabolites ILG Ca Ca InsP4 S [native] (entry 16) Native Ca2 + channels sensitive to inositol l,3,4,5-tetrakisphosphate (InsP4)

ILG Ca Ca RyR-Caf (entry 17) Caffeine-sensitive Ca2 + -release channels (ryanodine receptors, RyR) ILG Ca CSRC [native] (entry 18) Candidate native intracellularligand-gated Ca2 + -store repletion channels ILG Ca InsPa (entry 19) Inositol l,4,5-trisphosphatesensitive Ca2 + -release channels (InsPaR)

1...----

_

ILG CAT Ca [native] (entry 20) Native calcium-activated nonselective cation channels (NS ca ) ILG (CAT) cAMP (entry 21) Cation channels activated in situ by intracellular cAMP ILG CAT cGMP (entry 22) Cation channels activated in situ by intracellular cGMP

ILG Cl Ca [native] (entry 25) Native calcium-activated chloride channels (Clca) ILG K AA [native] (entry 26) Native potassium channels activated by arachidonic acid (KAA ) incorporating general properties of ion channel regulation by free fatty acids

ILG Cl ABC-CF (entry 23) ATP-binding and phosphorylationdependent CI- channels (CFTR)

ILG K Ca (entry 27) Intracellular calcium-activated K+ channels (Kca )

ILG Cl ABC-MDR/PG (entry 24) Volume-regulated CI- channels (multidmg-resistance P-glycoprotein)

ILG K Na [native] (entry 28) Native intracellular sodium-activated K+ channels (KNa )

VOLUME III

INWARD RECTIFIER AND INTERCELLULAR CHANNELS

INR K Key facts (entry 29) Inwardly-rectifying K+ channels key facts INR K ATP-i [native] (entry 30) Properties of intracellular ATPinhibited K+ channels in native cells INR K G/ACh [native] (entry 31) Properties of muscarinic-activated K+ channels underlying l KAch in native cells INR K [native] (entry 32) Properties of 'classical' inward rectifier K+ channels in native cells (excluding types covered in entries 30 & 31) INR K [subunits] (entry 33) Comparative properties of protein subunits forming inwardlyrectifying K+ channels (heterologously-expressed cDNAs of the KIR family) INR (KINa) IfhQ (entry 34) Hyperpolarization-activated cation channels underlying the inward currents if, ih, i Q

TUN [connexins] (entry 35) Intercellular gap junction channels formed by connexin proteins

MEC [mechanosensitive] (entry 36) Survey of ion channel types activated by mechanical stimuli MIT [mitochondrial] (entry 37) Survey of ion channel types expressed in mitochondrial membranes NUC [nuclear] (entry 38) Survey of ion channel types expressed in nuclear membranes OSM [aquaporins] (entry 39) The vertebrate aquaporin (water channel) family

SYN [vesicular] (entry 40) Channel-forming proteins expressed in synaptic vesicle membranes (synaptophysin)

II

_L.-...-VOLUME IV

_

VOLTAGE-GATED CHANNELS

VLG Key facts (entry 41) Voltage-gated channels - key facts VLG Ca (entry 42) Voltage-gated calcium channels VLG Cl (entry 43) Voltage-gated chloride channels VLG K A-T (entry 44) Properties of native'A-type' (transient outward) potassium channels in native cells VLG K DR (entry 45) Properties of native delayed rectifier potassium channels in native cells VLG K eag/elk/erg (entry 46) K+ channels related to Drosophila gene subfamilies eag,elk,erg VLG K Kv-beta (entry 47) Beta subunits associated with Kv (alpha subunit) channel complexes VLG K Kvl-Shak (entry 48) Vertebrate K+ channel subunits related to Drosophila Shaker (Kv subfamily I) incorporating general features of Kv channel expression in heterologous cells

VLG K Kv2-Shab (entry 49) Vertebrate K+ channel subunits related to Drosophila Shab (Kv subfamily 2) VLG K Kv3-Shaw (entry 50) Vertebrate K+ channel subunits related to Drosophila Shaw (Kv subfamily 3) VLG K Kv4-Shal (entry 51) Vertebrate K+ channel subunits related to Drosophila Shal (Kv subfamily 4) VLG K Kvx (Kv5.1/Kv6.1) (entry 52) Features of the 'non-expressible' cDNAs IK8 and KI3 VLG K M-i [native] (entry 53) Properties of native 'muscarinicinhibited' K+ channels underlying 1M

VLG (K) minK (entry 54) 'Minimal' protein subunits inducing 'slow-activating' voltagegated K+ currents VLG Na (entry 55) Voltage-gated sodium channels

ION CHANNEL RESOURCES

Resource documents and/or links supporting their scope will appear on the Ion Channel Network web site (www.le.ac.uk/csn/ ) from January 1999. Resource A Resource C G protein-linked receptors Compounds and proteins used in ion regulating ion channel activities channel research (alphabetical listing) Resource D Resource B 'Generalized' electrical effects of endogeneous receptor agonists

II

'Diagnostic'tests

Resource E Ion channel book references (sorted by year of publication)

"-----------Resource F Supplementary ion channel reviews (listed by subject)

Resource I Framework of cell-signalling molecule types (preliminary listing)

Resource G Reported 'consensus sites' and 'motifs' in primary sequences of ion channels

Resource' Search criteria &. CSN development

Resource H Listings of cell types

Resource K Framework for a multidisciplinary glossary

Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the coverage/contents can be sent to the e-mail [email protected]. (see field 57 of most entries for further details)

II

Acknowledgements Thanks are due to the following people for their time and help during compilation of the manuscripts: Professors Peter Stanfield, Nick Standen and Gordon Roberts (Leicester), and Ole Petersen (Liverpool) for advice; to Chris Hankins and Richard Mobbs of the Leicester University Computer Centre, and to Dr Tessa Picknett and Chris Gibson of Academic Press for their enthusiasm and patience. Gratitude is also expressed to all of the anonymous manuscript readers who supplied much constructive feedback, as well as the following who provided advice, information and encouragement: Ihab Awad (Center for Neuroscientific Databases, Minnesota), Jonathan Bard (Edinburgh), Mark Boyett (Leeds), David Brown (London), Cecilia Canessa (Yale), Marty Chalfie (Columbia), K. George Chandy (UC Irvine), David Clapham (Mayo Foundation), Noel Davies (Leicester), Dario DiFrancesco (Milano), Ian Forsythe (Leicester), Harry Fozzard (Chicago), Klaus Groschner (Graz), George Gutman (UC Irvine), Mike Huerta (NIMH, HBP), Rolf Joho (Texas SMC), Benjamin Kaupp (Julich), Steve Kozlow (NIMH, HBP), Jeremy Lambert (Dundee), Neil Marrion (OHSU), Shigetada Nakanishi (Kyoto), Jitendra Patel (Zeneca USA), Martin Ringwald (Jackson Labs), Gordon Shepherd (Yale), David Spray (Yeshiva), Zhong-ping Sun (Columbiail, Steve Watson (Oxford) and George Wilcox (Center for Neuroscientific Databases, Minnesota). Thanks are also due to the Department of Pathology at the University of Leicester, Harcourt Brace, the Medical Research Council, the British Heart Foundation and Zeneca Pharmaceuticals, for providing generous sponsorship, equipment and facilities. We would like to acknowledge the authors of all those papers and reviews

which in the interest of completeness we have quoted, but have not had space to cite directly. ECC would like to thank Professors Denis Noble in Oxford, Anthony Campbell in Cardiff and Richard Gregory in Bristol for help and inspiration, and would like to dedicate his contributions to Paula, Rebecca and Katherine for all their love and support.

Left: Edward Conley, Right: William Brammar

Introduction & layout of entries Edward C. Conley

Entry 02 resume

The Ion Channel FactsBook is intended to provide a 'summary of molecular properties' for all known types of ion channel protein in a cross-referenced and 'computer-updatable' format. Today, the subject of ion channel biology is an extraordinarily complex one, linking several disciplines and technologies, each adding its own contribution to the knowledge base. This diversity of approaches has left a need for accessible information sources, especially for those reading outside their own field. By presenting 'facts' within a systematic framework, the FactsBook aims to provide a 'logical place to look' for specific information when the need arises. For students and researchers entering the field, the weight of the existing literature, and the rate of new discoveries, makes it difficult to gain an overview. For these readers, The Ion Channel FactsBook is written as a directory, designed to identify similarities and differences between ion channel types, while being able to accommodate new types of data within the framework. The main advantages of a systematic format is that it can speed up identification of functional links between any 'facts' already in the database and maybe provide a raison d'etre for specific experiments where information is not known. Although such 'facts' may not go out-of-date, interpretations based on them may change considerably in the light of additional, more direct evidence. This is particularly true for the explosion of new information that is occurring as a direct consequence of the molecular cloning of ion channel genes. It can be anticipated that many more ion channel genes will be cloned in the near future, and it is also likely that their functional diversity will continue to exceed expectations based on pharmacological or physiological criteria alone.

An emphasis on properties emergent from ion channel molecular functions Understanding how the interplay of currents through many specific ion channel molecules determines complex electrophysiological behaviour of cells remains a significant scientific challenge. The approach of the FactsBook is to associate and relate this complex cell phenotypic behaviour (e.g. its physiology and pharmacology) to ion channel gene expression-control wherever possible even where the specific gene has not yet been cloned. Thus the ion channel molecule becomes the central organizer, and accordingly arbitrates whether information or topics are included, emphasized, sketched-over or excluded. In keeping with this, ion channel characteristics are described in relation to known structural or genetic features wherever possible (or where they are ultimately molecular characteristics). Invariably, this relies on the availability of sequence data for a given channel or group of channels. However, a number of channel types exist which have not yet been sequenced, or display characteristics in the native form which are not precisely matched by existing clones expressed in heterologous cells (or are otherwise ambiguously classified). To accommodate these channel types, summaries of characteristics are included in the standard entry field format, with inappropriate fieldnames omitted. Thus the present 'working arrangement' of entries and fields is broad enough to include both the 'cloned' and 'uncloned' channel types, but in due course will be gradually supplanted by a comprehensive classification based on gene locus, structure, and relatedness of primary sequences. In all cases, the scope of the FactsBook entries is limited to those proteins forming (or predicted to form) membrane-bound, integral ionic channels

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entry 02 resume

by folding and aSSocIatIon of their primary protein sequences. Activation or suppression of the channel current by a specified ligand or voltage step is generally included as part of the channel description or name (see below). Thus an emphasis is made throughout the book on intrinsic features of channel molecule itself and not on those of separately encoded, co-expressed proteins. In the present edition, there is a bias towards descriptions of vertebrate ion channels as they express the full range of channel types which resemble characteristics found in most eukaryotes.

Anticipated development of the dataset - Integration of functional information around molecular types Further understanding of complex cellular electrical and pharmacological behaviour will not come from a mere catalogue of protein properties alone. This book therefore begins a process of specific cross-referencing of molecular properties within a functional framework. This process can be extended to the interrelationships of ion channels and other classes of cell-signalling molecules and their functional properties. Retaining protein molecules (i.e. gene products) as 'fundamental units of classification' should also provide a framework for understanding complex physiological behaviour resulting from co-expressed sets of proteins. Significantly, many pathophysiological phenotypes can also be linked to selective molecular 'dysfunction' within this type of framework. Finally, the anticipated growth of raw sequence information from the human genome project may reveal hitherto unexpected classes and subtypes of cell-signalling components - in this case the task then will be to integrate these into what is already known (see also description of Field number 06: Subtype c.lassifications and Field number 05: Gene family).

The Cell-Signalling Network (CSN) From the foregoing discussion, it can be seen that establishment and consolidation of an integrated 'consensus database' for the many diverse classes of cell signalling molecules (including, for example, receptors, G proteins, ion channels, ion pumps, etc.) remains a worthwhile goal. Such a resource would provide a focus for identifying unresolved issues and may avoid unnecessary duplication of research effort. Work has begun on a prototype cell-signalling molecule database cooperatively maintained and supported by contributions from specialist groups: The Cell-Signalling Network (CSN) in mid-1996 has been designed to disseminate consensus properties of a wide range of molecules involved in cell signal transduction. While it will take some time (and much good-will) to establish a comprehensive network, the many advantages of such a co-operative structure are already apparent. Immediately, these include an 'open' mechanism for consolidation and verification of the dataset, so that it holds a 'consensus' or 'validated' set of information about what is known about each molecule and practical considerations such as nomenclature recommendations (see, for example, the IUPHAR nomenclature sections under the CSN 'home page'). The CSN also allows unlimited cross-referencing by pointing to related information sets, even where these are held in multiple centres. On-line descriptions of technical terms (glossary items, indicated by dagger symbols (t) throughout the text) and reference to explanatory references (e.g. on associated signalling components such

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as G protein t -linked receptors t ) are being written for use with this book. Eventually, applications could include (for instance) direct 'look-up' of graphical resources for protein structure, in situ and developmental gene expression atlases t, interactive molecular models for structure/function analysis, DNA/protein sequences linked to feature tables, gene mapping resources and other pictorial data. These developments (not presently supported) will use interactive electronic media for efficient browsing and maintenance. For a brief account of the Cell-Signalling Network, see Feedback eiJ CSN access, entry 12. For a full specification, see Resource T- Search criteria eiJ CSN development.

HOW TO USE THE ION CHANNEL FACTSBOOK

Common formats within the entries A proposed organizational hierarchy for information about ion channel

molecules Information on named channel types is grouped in entries under common headings which repeat in a fixed order - e.g. for ion channel molecules which have been sequenced, there are broad sections entitled NOMENCLATURES, EXPRESSION, SEQUENCE ANALYSES, STRUCTURE &. FUNCTIONS, ELECTROPHYSIOLOGY, PHARMACOLOGY, INFORMATION RETRIEVAL and REFERENCES, in that order. Within each section, related fieldnames are listed, always in alphabetical order and indexed by a field number (see below), which makes electronic cross-referencing and 'manual' comparisons easier. While the sections and fields are not rigid categories, an attempt has been made to remain consistent, so that corresponding information for two different channels can be looked up and compared directly. If a field does not appear, either the information was not known or was not found during the compilation period. Pertinent information which has been published but is absent from entries would be gratefully received and will be added to the 'entry updates' sections within the CSN (see Feedback eiJ CSN access, entry 12). Establishment of this 'field' format has been designed so that most 'facts' should have their logical 'place'. In the future, this arrangement may help to establish 'consensus' properties of any given ion channel or other cell-signalling molecule. This validation process critically depends on user feedback to contributing authors. The CSN (above) establishes an efficient electronic mechanism to do this, for continual refinement of entry contents.

Independent presentation of lacts' and conventions for cross-referencing The FactsBook departs from a traditional review format by presenting its information in related groups, each under a broader heading. Entries are not designed or intended to be read lfrom beginning to end', but each Ifact' is presented independently under the most pertinent fieldname. Independent citation of 'facts' may sometimes result in some repetition (redundancy) of general principles between fields, but if this is the case some effort has been made to 'rephrase' these for clarity (suggested improvements for presentation of any 'fact' are welcome - see Field number 57: Feedback).

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For readers unfamiliar with the more general aspects of ion channel biology, some introductory information applicable to whole groups of ion channel molecules is needed, and this is incorporated into the 'key facts' sections preceding the relevant set of entries. These sections/. coupled with the glossary items (available on-line, and indicated by the daggert symbol, see below) provide a basic overview of principles associated with detailed information in the main entries of the book. Extensive cross-referencing is a feature of the book. For example, cross-references between fields of the same entry are of the format (see Fieldname, xx-yy). Crossreferences between fields of different channel type entries are generally of the format see fieldname under SORTCODE, xx-yy; for example - see mRNA distribution, under ELG Cl GABAA , 10-13. This alphabetical 'sortcode' and numerical 'entry numbers' (printed in the header to each page) are simply devices to make crossreferencing more compact and to arrange the entries in an afproximate running order based on physiological features such as mode of gating, ionic selectivityi, and agonist t specificity. A 'sort order' based on physiological features was judged to be more intuitive for a wider readership than one based on gene structure alone, and enables 'cloned' and 'uncloned' ion channel types to be listed together. The use and criteria for sortcode designations are described under the subheading Derivation of the sortcode (see Field number 02: Category (sortcode)). Entry 'running order' is mainly of importance in book-form publications. New entries (or mergers/subdivisions between existing entries) will probably use different serial entry numbers as 'electronic pointers' to appropriate files. Cross-references are frequently made to an on-line index of glossary items by dagger symbols t wherever they might assist someone with technical terms and concepts when reading outside their own field. The glossary is designed to be used side-byside with the FactsBook entries and will be accessible in updated form over the Internet with suitable software (for details, see Feedback eiJ CSN access, entry 12).

Contextual markers and styles employed within the entries Throughout the books, a six-figure index number (xx-yy-zz, e.g. 19-44-01: ) separates groups of facts about different aspects of the channel molecule, and carries information about channel type/entry number (e.g. 19InsP3 receptorchannels), information type/field number (e.g. -44-, Channel modulation) and running paragraph number (datatype) (e.g. -01). This simple 'punctate' style has been adopted for maximum flexibility of updating (both error-correction and consolidation with new information), cross-referencing and multi-authoring. The CSN specification includes longer term plans to further structure field-based information into convenient data-types which will be indexed by a zz numerical designation. r-..I

Italicized subheadings are employed to organize the facts into related topics where a field has a lot of information associated with it:. Specific illustrated points or features within a field are referenced to adjacent figures. Usage of abbreviations and common symbols are defined in context and/or within the main abbreviations index at the front of each book. Abbreviated chemical names and those of proprietary pharmaceutical compounds are listed within Resource C - Compounds eiJ proteins. Generally, highlighting of related subtopics emergent from the molecular properties ('facts') associated with the ion channel under description are indicated within a field

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by lettering in bold. Throughout the main text, italics draw attention to special cases, caveats, hypotheses and exceptions. The 'Note: ' prefix has been used to indicate supplemental or comparative information of significance to the quoted data in context.

Special considerations for integrating properties derived from tcloned' and tnative' channels While a certain amount of introductory material is given to set the context, the emphasis on molecular properties means the treatment of many important biological processes or phenomena is reduced to a bare outline. References given in the Related sources and reviews field and Resource F - Supplementary ion channel reviews are intended to address this imbalance. For summaries of key molecular features, a central channel 'protein domain topography model' is presented. Individual features that are illustrated on the protein domain topography model are identified within the text by the symbol [PDTM]. Wherever molecular subtype-specific data are quoted (such as the particular behaviour of a ion channel gene familyt member or isoformt) a convention of using the underlined trivial or systematic name as a prefix has been adopted - e.g. mIRKl: i RCKl: i Kv3.1: etc.

GUIDE TO THE PLACEMENT CRITERIA FOR EACH FIELD

Criteria for NOMENCLATURES sections This section should bring together for comparison present and previous names of ion channels or currents, with brief distinctions between similar terms. Where systematic names have already been suggested or adopted by published convention, they should be included and used in parallel to trivial names. Field number 01: Abstract/general description: This field should provide a summary of the most important functional characteristics associated with the channel type. Field number 02: Category (sortcode): The alphabetical1sortcode' should be used for providing a logical running order for the individual entries which make up the book. It is not intended to be a rigorous channel classification, which is under discussion, but rather a practical index for finding and cross-referencing information, in conjunction with the six-figure index number (see above). The Category (sortcode) field also lists a designated electronic retrieval code (unique embedded identifier or VEl) for 'tagging' of new articles of relevance to the contents of the entry. For further details on the use and implementation of UEIs, see the description for Resource T(in this entry) and for a full description, see Resource T- Search criteria eiJ CSN development.

Derivation of the sortcode: Although we do not yet have a complete knowledge of all ion channel primaryt structures, knowledge of ion channel gene familyt and superfamilyt structure allows a working sort order to be established. To take an

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example, the extracellular ligand-gated (ELG) receptor-channels share many structural features, which reflects the likely duplication and divergent evolution of an ancestral gene. The present-day forms of such channels reflect the changes that have occurred through adaptive radiation t of the ancestral type, particularly for gatingt mechanism and ionic selectivityt determinants. Thus, the entry running order (alphabetical, via the sortcode) of the FactsBook entries should depend primarily on these two features. The sortcode therefore consists of several groups of letters, each denoting a characteristic of the channel molecule: Entries are sorted first on the principal means for channel gatingt (first three letters), whether this is by an extracellular ligandt (ELG), small intracellular ligandt (ILG) or transmembrane voltage (VLG). For convenience, the ILG entries also include certain channels which are obligately dependent on both ligand binding and hydrolysis for their activation e.g. channels of the ATP-binding cassette (ABC) superfamily. Other channel types may be subject to direct mechanical gating (MEC) or sensitive to changes in osmolarity (OSM) - see the Cumulative tables of contents and the first page of each entry for descriptions and scope. Due to their unusual gating characteristics, a separate category (INR) has been created for inward rectifier-type channels. The second sort (the next three letters of the sortcode) should be on the basis of the principal permeant ions, and may therefore indicate high selectivity for single ions (e.g. Ca, Cl, K, Na) or multiple ions of a specified charge (e.g. cations - CAT). Indefinite sortcode extensions can be assigned to the sortcode if it is necessary to distinguish similar but separately encoded groups of channels (e.g. compare ELG Cl GABAA , entry 10 and ELG Cl GLY, entry 11). Field number 03: Channel designation: This field should contain a shorthand designation for the ion channel molecule - mostly of the form X y or X(y) where X denotes the major ionic permeabilities t (e.g. K, Ca, cation) and Y denotes the principal mechanism of gatingt where this acts directly on the channel molecule itself (e.g. cGMP, voltage, calcium, etc.). Otherwise, this field contains a shorthand designation for the channel which is used in the entry itself. Field number 04: Current designation: This field should contain a shorthand designation for ionic currents conducted by the channel molecule, which is mostly of the form IXI Y ), Ix,Y or I x -y where X and Yare. defined as above. Field number 05: Gene family: This field should indicate the known molecular relationships to other ion channels or groups of ion channels at the level of amino acid primary sequence homologyt, within gene familiest or gene superfamilies t. Where multiple channel subunits are encoded by separate genes, a summary of their principal features should be tabulated for comparison. Where the gene family is particularly large, or cannot be easily described by functional variation, a gene family tree t derived by a primary sequence alignment algorithm t (see Resource D - IDiagnostic' tests) may be included as a figure in this field. Field number 06: Subtype classifications: This field should include supplementary information about any schemes of classification that have been suggested in the literature. Generally, the most robust schemes are those based on complete knowledge of gene familyt relationships (see above) and this method can identify

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similarities that are not easily discernible by pharmacological or electrophysiological criteria alone - see, for example, the entries TUN (connexins), entry 35, and JNR K (subunits), entry 33. Note, however, that some native t channel types are more conveniently 'classified' by functional or cell-type expression parameters which take into account interactions of channels with other co-expressed proteins (see, for example, discussion pertaining to the cyclic nucleotide-gated (CNG-) channel family in the entries JLG Key facts, entry 14, JLG CAT cAM~ entry 21, and JLG CAT cGM~ entry 22. Debate on the 'best' or 'most appropriate' channel classification schemes is likely to continue for some time, and it is reasonable to suppose that alternative subtype classifications may be applied and used by different workers for different purposes. Since the 'running order' of the FactsBook categories depends on inherent molecular properties of channel cDNAs t, genes t or the expressed proteins, future editions will gradually move to classification on the basis of separable gene locit. Thus multiple channel protein variants resulting from processes of alternative RNA splicingt but encoded by a single gene locust will only ever warrant one 'channel-type' entry (e.g. see BKci variants under JLG K Ca, entry 27). Distinct proteins resulting from transcription T of separable gene loci, for example in the case of different gene family members, will (ultimately) warrant separate entries. For the time being, there is insufficient knowledge about the precise phenotypic t roles of many 'separable' gene family members to justify separate entries (as in the case of the VLG K Kv series entries). Classification by gene locus designation (see Field number 18: Chromosomal location) can encompass all structural and functional variation, while being 'compatible' with efforts directed to identifying phenotypic and pathophysiologicali roles of individual gene products (e.g. by gene-knockout t, locus replacement t or disease-linked gene mapping t procedures - see Resource D - tDiagnostic' tests). Subtype classifications based on gene locus control can also incorporate the marked developmental changes which pertain to many ion channel genes (see Field number 11: Developmental regulation) and can be implemented when the 'logic' underlying gene expression-control t for each family member is fully appreciated. A 'genome-based' classification of FactsBook entries may also help comprehend and integrate equivalent information based for other ('non-channel') cell-signalling molecules (see Resources G, Hand J). Field number 07: Trivial names: This field should list commonly used names for the ion channel (or its conductance t ). Often a channel will be (unsystematically) named by its tissue location or unusual pharmacological/physiological properties, and these are also listed in this field. While unsystematic names do not indicate molecular relatedness, they are often more useful for comparative/descriptive purposes. For these and historical reasons, trivial names (e.g. clone/isolate names for K+ channel isoforms) are used side-by-side with systematic names, where these exist. A standardized nomenclature for ion channels is under discussion, e.g. see the series of articles by Pongs, Edwards, Weston, Chandy, Gutman, Spedding and Vanhoutte in Trends Pharmacol Sci (1993) 14: 433-6. Future recommendations on standardized nomenclature will appear in files accessible under the IUPHAR entry of the CellSignalling Network (see Feedback etJ CSN access, entry 12).

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Criteria for EXPRESSION sections This section should bring together information on expression patterns of the ion channel gene, indicating functional roles of specific channels in the cell type or organism. The complex and profound roles of ionic currents in vertebrate development (linking plasma membrane signalling and genome activation) are also emphasized within the fields of this section. Field number 08: Cell-type expression index: Comprehensive systems relating the expression of specified molecular components to specified anatomical and developmentalloci ('expression atlases') are being developed in a number of centres and in due course will form a superior organizational framework for this type information (see discussion below). In the meantime, the range of cell-type expression should be indicated in this field in the form of alphabetized listings. Notably, there is a substantial literature concerned with the electrophysiology of ion channels where the tissue or cell type forms the main focus of the work. In some cases, this has resulted in detailed lexpression surveys', revealing properties of interacting sets of ion channels, pumps, transporters and associated receptors. Such review-type information is of importanee when discussing the contribution of individual ion channel molecules to a complex electrophysiological phenotype t and/or overall function of the cell. For further references to 'cell-type-selective' reviews, see Resource H - Listings of cell types.

Problems and opportunities in listing ion channel molecules by cell type: Understanding the roles which individual ionic chcmnels play in the complex electrophysiological phenotypes of native t cells re:mains a significant challenge. The overwhelming range of studies covering aspects of ion channel expression in

vertebrate cells offers unique problems when eompiling a representative overview. Certainly the linking of specific ion channel gene expression to cell type is a first step towards a more comprehensive indexing, and towards this goal, cell-typeselective studies are useful for a number of reasons. First, they can help visualize the whole range of channel expression by providing an inventory of conductances t observed. Secondly, these studies generally define the experimental conditions required to observe a given conductance. Thirdly, they include much information directly relating specified ionic conductances to the functions of the cell type concerned. Collated information such as this should be of increasing utility in showing the relationship of electrophysiological phenotype to mechanistic information on their gene structure and expression-control (which largely correlates with cell-type lineage). At this time it is diffic.ult to build a definitive catalogue of ion channel gene expression patterns mapped. to cell type, not only because the determinants of gene expression are scarcely explored, but also because there remain many unavoidable ambiguities in phenotype dt~finition. Some of these problems are discussed below.

Problems of uneven coverage/omissions: Certain cell preparations have been intensely studied for ion channel expression while others have received very little attention for technical, anatomical or other reasons. Furthermore, a large number of native t ionic currents can be induced or inhibited by agonists t that bind to co-expressed G protein t -coupled receptors t. Thus a difficulty arises in deciding whether channel currents can be unambiguously defined in tenns of action at a separately encoded

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receptor protein. While it is valid to report that an agonist-sensitive current is expressed in a defined cell type, the factors of crosstalkr and receptor-transducert subtype specificities in signalling systems are complex and may produce an ambiguous classification. Receptor-coupled agonist-sensitivities are an important factor contributing to cell-pharmacological and -electrical phenotypet, but the treatment here has been limited to a number of tabular summaries of ion channel regulation through coupling to G protein-linked effectort molecules (see Resource A - G protein-linked receptors). As stated earlier, the entries are not sorted on agonist specificity except where the underlying ion channel protein sequence would be expected to form an integral ionic channel whose gating t mechanism is also part of the assembled protein complex.

Cell preparation methods are variable: A further problem inherent in classifying ion channels by their patterns of expression is that the choice of tissue or cell preparation method may influence phenotype t. The behaviour of channel-mediated ionic currents can be measured in native t cells, e.g. in the tissue slice, which has the advantages of extracellular ionic control, mechanical stability, preserved anatomical location, lack of requirement for anaesthetics and largely undisturbed intercellular communication. Cell-culture techniques show similar advantages, with the important exceptions that normal developmental context, anatomical organization and synaptic arrangements are lost and (possibly as a consequence) the 'expression profile' of receptor and channel types might change. Cultured cell preparations may also be affected by 'de-differentiationt, processes and (by definition) cell lines t are uncoupled from normal processes of cell proliferationt, differentiation t and apoptosis t. Acutely dissociated cells from native t tissue may provide cell-typespecific expression data without anomalies introduced by intercellular (gap junctional) conductances, but the enzymatic or dispersive treatments used may also affect responses in an unknown way. Verbal descriptions of cell-type expression divisions are arbitrary and are not rigorous: Definitive mapping of specific ion channel subtype expression patterns has many variables. Localization of specific gene products are most informative when in situ localizations are linked to the regulatory factors controlling their expression (see glossary entry on Gene expression-control t ). The complexity of this task can extend to processes controlling, for example, developmental regulation, co-expressed protein subunit stoichiometries and subcellular localizations. Complete integration of all structural, anatomical, co-expression and modulatory data for ion channels could eventually be accommodated within interactive graphical databases which are capable of providing 'overlays' of separately collected in situ expression data linked to functional properties of the molecules. By these methods, new data can be mathematically transformed to superimpose on fixed tissue or cell co-ordinates for comparison with existing database information. Software development efforts focused on the acquisition, analysis and exchange of complex datasets in neuroscience and mouse development have been described, and the next few years should hopefully see their implementation. For further information, see Baldock, R., Bard, J., Kaufman, M. and Davidson, D. (1992) A real mouse for your computer, Bioessays 14: 501-2

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Bloom, F. (1992) Brain Browser, v 2.0. Academic Press (Software). Kaufman, M. (1992) The Atlas of Mouse Development, Academic Press Wertheim, S. and Sidman, R. (1991) Databases for Neuroscience, Nature 354: 88-9 To help rationalize the choices available for selection of these 'prototype' classifications, see Resource H - Listings of cell types. These listings may also have some practical use for sorting the subject matter of journal articles into functionally related groups. A proposed integration of information resources relating different aspects of cell-signalling molecule gene expression is illustrated in Fig. 4 of the section headed Feedback eiJ CSN access, entry 12. Field number 09: Channel density: This field should contain information about estimated numbers of channel molecules per unit area of membrane in a specified preparation. This field lists information derived from local patch-clamp 'sampling' or autoradiographic detection in membranes using anti-channel antibodies. The field should also describe unusually high densities of ion channels ('clustering') in specified membranes where these are of functional interest. Field number 10: Cloning resource: This field should refer to cell preparations relatively 'rich' in channel-specific mRNA (although it should be noted that many ion channel mRNAs are of low abundance t ). Otherwise this field defines a 'positive control' preparation likely to contain messenger T RNA t encoding the channel. Preparations may express only specific subtypes of the channel and therefore related probes (especially peRl probes) may not work. Alternatively, a genomic t cloning resource may be cited. Field number 11: Developmental regulation: This field should contain descriptions of ion channel genes demonstrated (or expected to be) subject to developmental gene regulation - e.g. where hormonal, chemical, second messengert or other environmental stimuli appear to induce (or repress) ion channel mRNA or protein expression in native t tissues (or by other experimental interventions). Protein factors in trans t or DNA structural motifs t in cis t which influence transcriptional activation t, transcriptional enhancement t or transcriptional silencing t should also be listed under this fieldname. Information about the timing of onset for expression should also be included if available, together with evidence for ion channel activity influencing gene activation t or patterningt during vertebrate development. Field number 12: Isolation probe: This field should include information on probes used to relate distinct gene products by isolation of novel clones following lowstringency cross-hybridization screens t. The development of oligonucleotidet sets which have been used to unambiguously detect subtype-specific sequences by PCRt, RT-PCRt or in situ hybridizationt should be identified with source publication. Both types of sequence may be able to serve as unique gene isolation probes, dependent upon the library t size, target abundance t, screening stringency t and other factors. Field number 13: mRNA distribution: This field should report either quantitative/ semi-quantitative or presence/absence (±) descriptions of specific channel mRNAs in defined tissues or cell types. This type of information is generally derived from

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Northern hybridizationt, RNAase protectiont analysis, RT-peRt or in situ expression assays. See also notes on expression atlases under Field number 08: Cell-type expression index. Field number 14: Phenotypic expression: This field should include information on the proposed phenotypet or biological roles of specified ion channels where these are discernible from expression studies of native t (wild-type) genes. Phenotypic t consequences of naturally occurring (spontaneous) mutationst in ion channel genes are included where these have been defined, predicted or interpreted (see also Fields 26-32 of the STR UCTURE etJ FUNCTIONS section for interpretation of site-directed mutagenesis t procedures as well as Resource D - IDiagnostic' tests). Associations of ion channels with pathological states, or where molecular 'defects' could be 'causatory' or contribute to the progression of disease should be listed in this field (for links with established cellular and molecular pathology databases, see Fig. 4 of Feedback etJ CSN access, entry 12). The Phenotypic expression field may include references to mutations in other ('nonchannel') genes which affect channel function when the proteins are co-expressed. It is also used to link descriptions of specific (cloned) molecular components to native cell-electrophysiological phenotypes. In due course, this field will be used to hold information on phenotypict effects of transgenict manipulations of ion channel genes including those based on gene knockoutt or gene locus I" replacementt protocols. Field number 15: Protein distribution: This field should report results of expression patterns determined with probes such as antibodies raised to channel primaryt sequences or radiolabelled affinity ligandst . Field number 16: Subcellular locations: This field should describe any notable arrangements or intracellular locations related to the functional role of the channel molecule, e.g. when the channel is inserted into a specified subcellular membrane system or is expressed on one pole of the cell only (e.g. the basolateral t or apical t face). Field number 17: Transcript size: This field should list the main RNA transcriptt sizes estimated (in numbers of ribonucleotides) by Northern t hybridization analysis. Multiple transcript sizes may indicate (i) alternative processing ('splicingt ') of a primary transcriptt, (ii) the use of alternative transcriptional start sitest, or (iii) the presence of 'pre-spliced' or lincompletely spliced' transcripts identified with homologous nucleotide probest in total cell mRNAt populations. Note that probes can be chosen selectively to identify each of these categories; 'full-length' coding sequencet (exonict) probes are the most likely to identify all variants, while probes based on intronic t sequences (where appropriate) will identify 'pre-splice' variants.

Criteria for SEQUENCE ANALYSES sections This section should bring together data and interpretations derived from the nucleic acid or protein sequence of the channel molecule. The symbol {PDTM} denotes an

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illustrated feature on the channel monomer protein domain topography model, which is presented as a central figure in some entries for sequenced ion channels. These models are only intended to visualize the relative lengths and positions of features on the whole molecule (see the description for field number 30, Predicted protein topography). The PDTMs as presented are highly diagrammatic - the actual protein structure will depend on patterns of folding, compact packing and multi-subunit associations. In particular, the relative positions of motifs, domain shapes and sizes are subject to re-interpretation in the light of better structural data. Links to information resources for protein and nucleic acid sequence data are described in the Database listings field towards the end of each entry. Field number 18: Chromosomal location: This field should provide a chromosomal locust designation (chromosome number, arm, position) for channel gene(s) in specified organisms, where this is known. N'otes on interactive linking to gene mapping database resources appear under an option of the Cell-Signalling Network 'home page' (see Feedback eiJ CSN access, entry 12). Field number 19: Encoding: This field should report open reading framet lengths as numbers of nucleotides or amino acid residues encoding monomeric channel proteins (i.e. spanning the first A of the ATG translational start codont to the last base of the translational termination codont). The field should report and compare any channel protein length variants in different tissues or organisms. If considered especially relevant or informative, selected primaryt sequence alignments of different gene family members may appear under this field. Field number 2.0: Gene organization: This field should describe known intront and exon t junctions within or outside the protein coding sequence, together with positional information on gene expression-control t elements and polyadenylationt sites where known. Note: Functional changes as a result of gene expressioncontrol should be listed under the Developmental regulation field. Field number 2.1: Homologous isoforms: This field should indicate independently isolated and sequenced forms of entire channels which either show virtual identity or of such high homologyt that they can be considered equivalent should also appear in this field (but see note on percentage conservation values under Field number 28: Domain conservation). Isoformsr * of a channel protein can exist between closely related species or between different tissues of the same species (i.e. the same gene may be expressed in two or more different tissues, sequenced by two groups but named independently). Some tissue-specific variation may also result from alternative splicingt, yielding subtly distinct forms of channel protein. Since small numbers of amino acid changes may exist from individual-toindividual (as a result of normal sequence polymorphism t in populations) separate isolates may yield sequence isoforms which can be shown to be 'equivalent' by Southern hybridizationt procedures (see Field number 25: Southerns). * Note: In the entries of this book a restrictive definition of molecular identity (or

near identity) is used to define an isoformt. In this restricted sense, 'isoforms' would be expected to be the product of the same genet (or gene variant produced by, for example, alternative splicingt), and therefore have very similar or identical

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molecular constitutions and functional roles in specified cell types of closely related species. Comparative information on different gene familyt members or multiple variants affecting particular protein domainst may also be included under the Gene family and Domain conservation fields respectively. Field number 22: Protein molecular weight (purified): This field should state reported molecular weights estimated from relative protein mobilities using SDSPAGEt methods (e.g. following affinityt purification from native t or heterologous t cell membranes). Data derived from native t preparations generally includes the weight contribution from oligosaccharide t chains added during post-translational protein glycosylationt . In general, extracellular saccharider components of glycoproteinst may contribute 1-85 % by weight, ranging from a few to several hundred oligosaccharide chains per glycoprotein molecule. Field number 23: Protein molecular weight (calc.): This field should list the molecular weight of monomeric channel proteins equivalent to the summated (calculated) molecular weights of constituent amino acids in the reported sequence (e.g. derived from open reading frames t of cDNAt sequences). If 'calculated' molecular weights are less than 'purified' molecular weights (previous field) this may indicate the existence of post-translational glycosylationt on nativet expressed protein subunits in vivo. Field number 24: Sequence motifs: This field should report the position of putative regulatory sites as deduced from the protein or nucleic acid primaryt sequence (with the exception of potential phosphorylation sites for protein kinasest, which are listed under Field number 32: Protein phosphorylation). Positions of sequence motifst illustrated on the monomer protein domain topography model are denoted by the symbol [PDTM]. Typical consensust sites include those for enzymes such as glycosyl transferasest, ligand t -binding sites, transcription factort -binding sitest etc. N-glycosylationt motifs are sometimes indicated using the shorthand designation Ngly:. Signal peptide cleavage sites (sometimes designated by Sig:) can be derived by comparing sizes of the signal peptidet and the mature chaint. Field number 25: Southerns: This field should include information which reports the existence of closely related DNA sequences in the genomet or reports the copy numbert of individual genes via Southern hybridization t procedures. Note that nativet diploid somaticr cells will generally maintain two copies of a given ion channel gene locust, but stable t heterologoust expression procedures may result in mUltifle locus insertiont. Multiple locus insertion can be quantitated in Southern hybridization procedures using two probes of similar length and hybridization affinityt, one specific for a native locus (which will identify two copies) and one for the heterologous gene (which will yield a hybridization signal proportional to the copy number). Note also that the copy number parameter can not be equated to the physiological expression level of the recombinantt protein unless locus control regions are incorporated as part of the channel expression construct (for details, see the section entitled Gene copy number under Resource D - IDiagnostic' tests, and the section describing heterologous ion channel gene expression under Resource H - Listings of cell types).

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Criteria for STRUCTURE

&,

FUNCTIONS sections

This section should bring together information based on functional analysis or interpretation of ion channel structural elements. This section includes data derived from functional studies following site-directed mutagenesis t of ion channel genes and molecular modelling studies at atomic scale. Future developments linking on-line information resources for protein structure to 'functional datasets' are illustrated in Fig. 5 of Feedback etJ CSN access, entry 12, and in Resource J Search criteria etJ CSN development. Field number 26: Amino acid composition: This field should include information on channel protein hydrophilicityt or hydrophobicityt where this is of structural or functional significance. Similarities to other related proteins should be emphasized. Field number 27: Domain arrangement: This field should describe the predicted number and arrangement of protein domainst when folded in the membrane as determined by hydropathicity analysist of the primaryt sequence. Note that structural predictions of transmembrane domainst on the basis of hydrophobicityt plots may be misleading and prematurely conclusive. For example, high resolution ( rv 9 A) structural studies of the nicotinic acetylcholine receptor (nAChR, see ELG CAT nAChR, entry 09) predict that only one membrane-spanning a-helixt (likely to be M2, a pore-lining domain) is present per subunit, with the other hydrophobic regions being present as /1-sheets t (see Unwin, J Mol Biol (1993) 229: 1101-24). By contrast, extracellular ligand-gated (ELG) channels such as the nAChR display four predicted membrane-spanning regions (MI-M4) on the basis of hydrophobicity plots. From the foregoing it must be emphasized that all assignments given for

the number or arrangement of 'predicted' domains in this field are tentative. Field number 28: Domain conservation: This field should point out known structural and/or functional motift sequences which have been conserved as protein subregions of ion channel primaryt sequences during their evolution (such as those encoding a particular type of protein domain t ). Cross-references should be made to functionally related domains conserved in different proteins including 'non ion channel' proteins. Note that 'percentage conservation' values are not absolute as they depend on which particular subregions of channel sequences are aligned, the numbers and availability of samples, and/or which sequence alignment algorithms t are used. Field number 29: Domain functions (predicted): This field should indicate predicted functions of channel molecular subregions based on structural or functional data e.g. regions affecting properties such as voltage-sensitivity, ionic selectivityt, channel gating t or agonisti binding. Field number 30: Predicted protein topography: This field should include information on the stoichiometric t assemblyT patterns of protein subunits derived from the same or different genes. This field indicates whether channel monomers are likely to form homomultimerst, heteromultimerst or both, and lists estimated physical dimensions of the protein if these have been published. Note: 'topography' is a convenient term borrowed from cartography which when applied to proteins, implies a 'map' at a level of detail or scale intermediate between that

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of an amino acid sequence and a larger-scale representation such as a protein multimeric complex. Topographic maps (or 'models') are therefore particularly useful for displaying selected sets of (inter-related) datatypes within a single 'visual framework'. The protein domain topography models (symbolized by [PDTM] throughout the entries) provide prototypes for this form of data representation. The considerable scope for further development of 'shared' topographical models which interactively report and illustrate many different features in the text are described in Search Criteria etJ CSN Development (Resource J). The terms 'protein topography' and 'protein topology' are often used interchangeably (sic), but the latter should be reserved for those physical or abstract properties of a molecule which are retained when it is subjected to 'deformation'. Field number 31: Protein interactions: This field should report well-documented examples of the channel protein working directly in consort with separate proteins in its normal cellular role(s). The 'protein interactions' described need not involve physical contact between the proteins (generally referred to as 'protein-protein' interactions), but may involve a messenger t molecule. The scope of this field therefore includes notable examples of protein co-localization or functional interaction. For instance, reproduction of nativet channel properties in heterologous t cell expression systems may require accessory subunit expression (e.g. see VLG K Kv-beta, entry 47). Common channel-receptor or G protein-channel interactions are described in principle under Resource A - G protein-linked receptors, and Field number 49: Receptor/transducer interactions. Field number 32: Protein phosphorylation: This field should describe examples of experimentally determined 'phosphomodulation' of ion channel proteins, and if possible list sites and positions of phosphorylation motifst within the channel sequence. Only those consensus sitest explicitly reported in the literature are shown, and these may not be a complete description and may not be based on functional studies. Examples of primaryt sequence motifst for in vitro phosphorylation by several kinasest are listed in Resource C - Compounds etJ proteins and Resource G - Reported tConsensus sites' and tmotifs'. Abbreviations used within this field for various enzyme motifst (e.g. Phos/PKA) are listed in Abbreviations, entry 03. Electrophysiological or pharmacological effects of channel protein phosphorylation in vitro by use of purified protein kinasest should also be described or cross-referenced in this field.

Criteria for ELECTROPHYSIOLOGY sections This section should bring together information concerning the electrical characteristics of ion channel molecules - how currents are turned on and off, which ions carry them, their sensitivity to applied membrane voltage or agonists, and how individual molecules contribute to total membrane conductance in specified cell types. Field number 33: Activation: This field should contain information on experimental conditions or factors which activate (open) the channel, such as the binding of ligandst, membrane potential changes or mechanical stimulation. Descriptions of characteristic gating f behaviour such as flickering t, bursting t, activation latency t

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or thresholdt of opening are also included. Applicable models of activation and the time course of current flow are briefly described here or referred to Field number 38: Kinetic model. Field number 34: Current type: Where clarification is required, this field should contain general descriptive information on the type, shape, size and direction of ionic current. Field number 35: Current-voltage relation: This field should report the behaviour of the channel current passed in response to a series of specified membrane potential shifts from a holding potentialt under a specified recording configurationt. For ligand t -gated channels (i.e. those with sortcodes beginning ELG and ILG) entries should report the current evoked by specific concentrations of agonistt applied at various holding potentials. This field should attempt to illustrate channel behaviour by listing a range of parameters such as slope conductancet, reversal potentialst and steepnesst of rectifyingt (non-ohmict) behaviour. The conventions used for labelling the axes of I-V relations for different charge carrierstare outlined in the on-line glossary. Field number 36: Dose-response: This field should contain information relating activator 'dose' (e.g. concentration) to channel 'response' parameters (e.g. open timet , open probabilityt) and whether there are maxima or minima in the response. Agonistt dose-response experiments are used to derive parameters such as the Hill coefficient t and Equilibrium dissociation constant t . Field number 37: Inactivation: This field should describe any inactivationt behaviour of the channel in the continued presence of activating stimulus. The

field includes information on voltage- and agonistt -dependence, with indications of time course and treatments which extend or remove the inactivation response. Where known, this field will distinguish channel inactivation from receptor desensitizationt processes, which are of partic.ular significance for the extracellular ligandt -gated (ELG) channel types (see ELG Key facts, entry 04). Field number 38: Kinetic model: This field should contain references to major theoretical and functional studies on the kinetic behaviour of selected ion channels. The field contents is limited to a simple description of parameters, terms and fundamental equations. Field number 39: Rundown: This field should collate information on channel 'rundownt, ('washout') phenomena observed during whole-cellt voltage clampt / cytoplasm dialysist or patch-clampt experiments. Conditions known to accelerate or retard the development of rundown should also be listed. Field number 40: Selectivity: This field should report data on relative ionic permeabilities t under stated conditions by means of permeability ratio t and/or selectivity ratio t parameters. The field may also compare measured reversal potentials t in response to ionic equilibrium potentials t with specified charge carriers under physiological conditions. This field also lists estimated physical dimensions of ionic selectivity filterst where derived from ion permeationt or electron micrographic studies.

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Field number 41: Single-channel data: This field should report examples of singlechannel current amplitudes and single-channel conductances t measured under stated conditions. In the absence of authentic single-channel data, estimates of channel conductances t derived from whole-cell recording t and fluctuation analysis t may be listed.

Field number 42: Voltage sensitivity: This field should describe the behaviour of the channel in terms of parameters (e.g. Popen t) which are directly dependent upon applied membrane voltage. A distinction should be made between 'voltage sensitivity' resulting from intrinsic voltage-gating t phenomena (i.e. applicable to channels possessing integral voltage sensors r) and indirect effects of applied membrane voltage influencing general physical parameters such as electrochemical driving force t .

Criteria for PHARMACOLOGY sections This section should bring together information concerning pharmacological or endogenous modulators of ion channel molecule activity. Regulatory cascades in cells may simultaneously activate or inhibit many different effector proteins, including ion channels. Analysis of patterns of sensitivity to messengers t and exogenous compounds can help elucidate the molecular signalling pathway in the context of defined cell types.

Field number 43: Blockers: This field should list compounds which reduce or eliminate an ionic current by physical blockade of the conductance t pathway. The field should include notes on specificity, sidedness and/or voltage sensitivity of block, together with effective concentrations and resistance to classes of blockers where approrriate. Where sites of block have been determined by site-directed mutagenesis , these should be cross-referenced to Domain functions, field 29.

Field number 44: Channel modulation: This field should summarize information on effects of important pharmacological or endogenous modulators, including descriptions of extracellular or intracellular processes known to modify channel behaviour. Loci of modulatory sites on the channel protein primary t sequence (as determined by site-directed mutagenesis t procedures) should be cross-referenced to

Domain functions, field 29.

Field number 45: Equilibrium dissociation constant: This field should list published values of Kd for agents whose concentration affects the rate of a specified process. See also on-line glossary entry for equilibrium dissociation constant t .

Field number 46: Hill coefficient: This field records calculated Hill coefficients t of ligand t -activated processes. The Hill coefficient (n) generally estimates the minimum number of binding/activating ligands although the actual number could be larger. For example, a Hill coefficient reported as n ~ 3 suggests that complete channel activation requires co-operative binding of at least four ligand molecules (e.g. see ILG CAT cGMp, entry 22). See also Field number 36: Dose-response. Field number 47: Ligands: This field should include principal high-affinity radioligands t which have been used to investigate receptor-channel function and that

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are commercially available. Note that numbers of ligand t -binding sites cannot be equated to functional receptors because they only indicate the presence of a ligand-binding entity that may not necessarily be linked to an effectort moietyt. Field number 48: Openers: This field should list compounds (or other factors) which increase the open probabilityt (Popen ) or open timet of the channel in native t tissues. Field number 49: Receptor/transducer interactions: This field should briefly discuss known links to discrete (i.e. separately encoded) receptor and G protein molecules (see also Resource A - G protein-linked receptors, accessible via the CSN). Types of lreceptor/transducer/channel' interactions account for many of the physiological responses of ion channel molecules within complex signalling systems. Note: Many pharmacological agents acting at receptor or transducer proteins (beyond the scope of these entries, but see Watson, S. and Arkinstall, S. (1994) The GProtein Linked Receptor FactsBook. Academic Press, London) partially exert their biological effects because these receptor/transducers have ion channel molecules as an ultimate effectort protein. Field number 50: Receptor agonists (selective): For the extracellular ligandt -gated (ELG) receptor-channels, this field should list compounds which selectively bind to the ligand receptor fortion of the molecule and thereby increase the open timet, open probability or conductancet of the integral channel. Antagonistst should be categorized as competitivet, non-competitiver or uncompetitivel where this has been determined. Field number 51: Receptor antagonists (selective): This field should list agents that selectively bind to the ligand t receptor portion of integral receptor-channel molecules but do not activate a response. Field number 52: Receptor inverse agonists (selective): This field should list compounds which selectively bind (extracellular ligand-gated) receptor-channels but which initiate an opposite response to that of an agonistt, i.e. tending to reduce the open timet, open probabilityt or conductancet of the integral channel.

Criteria for INFORMATION RETRIEVAL sections This section should provide links to other sources of information about the ion channel type, particularly accession to sequence database, gene expression, structure-function and bibliographic resources operating over the Internet or available on CD-ROM. A full discussion of the potential scope for integration of these resources with molecular-based entries appears in Resource T- Search criteria etJ CSN development. Brief details are given in Feedback etJ CSN access, entry 12. Field number 53: Database listings/primary sequence discussion: This field should tabulate separately listed items of relevance to the channel type and may include 'retrieval strings' such as locus names, accession numbers, keyword-containing identifiers and other miscellaneous information. Note that terms used by databases are often abbreviated (e.g. K for potassium, Na for sodium etc., therefore only specific identifiers (such as the accession numbers, locus and author names)

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should be used for retrieval. The actual names and numbers quoted have been sourced from NCBI-GenBank® (prefixed gb:) or EMBL (prefixed em:). Since there is now a high concordance between the contents of the EMBL and NCBI-GenBank® nucleic acid databases, the NCBI-GenBank® accession numbers given should retrieve the information from either database. Note that in all of the Database listings sections, the lower case prefixes are not part of the locus name or accession number, but merely indicate the relevant database. Sources of pre-translated protein sequences are indicated by references to the following databases (given in alphabetical order following the NCBI-GenBank® nucleic acid reference): SWISSPROT (prefixed sp:), Protein Identification Resource (prefixed pir:). The journal-scanning component of GenBank uses the NCBI 'Backbone' database (prefixed bbs: for backbone sequence, composed of several individual sequence segments; bbm: for backbone molecule) - these are maintained by the NCBIT (National Center of Biotechnology Information).

General notes on sequence retrievals: Updating and error-correction procedures for public domain databases may modify a protein or nucleic acid sequence (retrievable by a given accession number) between releases of a database. Thus, two users performing an analysis on a given database record may come to different conclusions depending upon which release was used. Note also that (i) accession numbers sometimes disappear with no indication of whether a new record has replaced the old one, (ii) multiple databases sometimes each give a different accession number to a single record, and (iii) some databases do not respect the ranges of accession numbers 'reserved' by other databases. Although the 'traditional' format of accession numbers has been a letter followed by five digits (with a maximum space of 2.6 million identifiers), the rapid rate of sequence accumulation will eventually force a different format to be used. Because of these problems, the NCBI now uses unique integer identifiers (UIDs) to identify sequence records and encourages their use as the 'real' accession numbers for sequence records. Reference numbers prefixed 'gim' can be read from CD-ROM media, but only refer to a 'GenInfo Import ill' - a temporary identifier unique only to a given release of the CD-ROM compilation (such as a numbered release of Entrez - see below). Should a sequence supplied by a database change, the record will usually be allocated a new 'gim' number, but the old one will still be available under its UID from the ill database. Because of the transient nature of 'gim' identifiers, they are not recommended as search/retrieval parameters and are generally not listed in the Database listings field (except where an accession number proper has not been found). In compiling The Ion Channels FactsBook, extensive listings of aligned protein or nucleic acids to show sequence relatedness have been avoided in some entries (as these were judged to be best served by development of on-line data resources specializing in sequence alignments - for a prototype, see Hardison et al. (1994) Genomics 21: 344-53). Alternatively or in addition, alignments can be performed according to need by dedicated sequence-manipulation software. Presently available compilations of sequences (e.g. Entrez can perform powerful'neighbouring t analyses' based on pre-computed alignments of any sequence against the remainder of the existing database. Establishment of homologous alignments t can proceed by finding a match between the query sequence and any member of the 'neighbouring set'. In practice, comprehensive retrievals can be performed interactively by just

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one or two rounds of neighbouring analysis. As indicated at the beginning of each Database listings field, the range of accession numbers provided can be used to initiate relevant searches, but following on from this, neighbouring analysis is strongly recommended to identify newly reported and related sequences. Descriptions of features based on primaryt sequence data listed within fields of the SEQUENCE ANALYSES or STRUCTURE &. FUNCTIONS sections can be more readily interpreted if an interactive sequence analysis program is available. Electronic mail servers t at the NCBI can receive specially formatted e-mailt queries, process these queries, and return the search results to the address from which the message was sent out. No specific password or account is needed for these, only the ability to send e-mail to an Internet t site. For local searches, alignment programs such as BLAST can also be retrieved by anonymous file-transfer protocol l or FTP. Detailed information on interactive linking to remote nucleic acid and protein database resources will appear under an option of the Cell-Signalling Network 'home page' (see Feedback eiJ CSN access, entry 12). Accession numbers can be issued for newly submitted sequences (normally within 24 hours) by remote Internet connection or by formatting/submission software (e.g. Seqwin, obtainable from the NCBI using an anonymous l FTPt). NCBI-GenBank® can also be accessed over the World Wide Webt (http://www.ncbLnlm.nih.gov). Sample retrievals in the absence of a CD-ROM resource: For a nucleic acid sequence from the EMBL database, use the e-mailt address below exactly as shown, specifying the appropriate accession number (nnnnnn) by the GET NUC command. For example, a database entry can be automatically e-mailed to you by the EMBL servert : [email protected] GET NUC:nnnnnn An analogous procedure can be used to retrieve protein sequences from the

SWISSPROT database, substituting the GET NUC: command with GET PROT:. Nucleic acid sequences from NCBI-GenBank® can be retrieved using the servert at the NCBI. In this case, send an e-mailt message to the service (address below) specifying the name of the database, the command BEGIN and. the accession numbers or key words. A sample request is shown below for an accession number nnnnnn: [email protected] DATALIB genbank BEGINnnnnnn Protein sequences from the Protein Identification Resource (pir:) can be obtained using an e-mailt request containing the command GET followed by the database code. The database code is distinct from the accession number but can be obtained by typing the command ACCESSION and then the number. For example, to specify a request for an entry of database code XXXX containing the accession number nnnnnn, you would send an e-mail IIlessage as follows: [email protected] GET XXXX ACCESSION nnnnnn

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General information on using these file serverst can be obtained using the above e-mailt addresses followed by the single command HELP. The Database listings tables contain short-form references to original research articles which have discussed features of the channel protein and/or nucleic acid primaryt sequence(s). Sequences are retrievable with the specified accession number or the author name shown in the short-form reference.

Field number 54: Gene mapping locus designation: This field should list references to human gene mapping locit using terms defined by a human genome mapping workshop (HGMW)I convention where possible. Notes on interactive linking to gene mapping database resources appears under an option of the Cell-Signalling Network 'home page' (see Fig. 4 of Feedback etJ CSN access, entry 12). The opportunities for linking to a wide range of genetic information resources are discussed in Resource T- Search criteria etJ CSN development. Field number 55: Miscellaneous information: This is a 'catch-all' field used within the entry to reference relevant peripheral information or perspectives on the channel molecule or its function. This field also should be used to contain information about ion channels showing partial functional relatedness to those in the main entry, but which also possess some features indicating the expression of a distinct genet (for example, description of potassium-selective ligand-gated t channels within an entry describing non-selective cation channels gated by the same ligand t , or vice versa). Normally, ion channels with distinct properties are covered in 'their own' entry whenever there is sufficient information available to make a clear set of 'defining characteristics'; the Miscellaneous information field therefore encompasses those channels which either have been infrequently reported, show only minor variations with the channel type under description, or are otherwise beyond the scope of the (present) collection of (largely) vertebrate channel-type entries. Field number 56: Related sources eY reviews: For reasons of space, the FactsBook cannot provide citations for every 'fact' within individual entries. Citations within this field should provide a starting point for locating key data through major reviews and other primaryt sources where these have been quoted extensively within the entry. A full discussion of how future entries will be linked to established on-line bibliographic resources appears in entry 12 and Resource J. Field number 57: Feedback: Information supplementary to the entries will be accessible from the CMHT server following publication of the complete book series (see below). An aim in compiling this book is that the scope and arrangement of the information should, in time, be refined towards containing what is most useful, authoritative and up-to-date: Feedback from individual users is an essential part of this process. The Feedback field identifies the appropriate address for e-mail feedback of significant corrections, omissions and updates for the contents of a specified entry and fieldname. Comments regarding new or modified field categories (or supplementary reference-type material for incorporation into entries and appendices) would also be most welcome from users (for details on accessing entry updates via the Cell-Signalling Network, see Feedback etJ CSN access, entry 12).

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_ entry 02 resume ~ - - - - - - -

In-press updates: (inserted at appropriate points): This field has been used occasionally (at the most relevant points in the printed versions of the book) to index publications containing important (direct) evidence which may significantly alter several statements or conclusions in the 'finalized' entry as sent to the publishers. It is acknowledged that no 'book-form' information index can ever be completely up-to-date, and it is in the nature of scientific progress that 'interpretations' based on reported 'facts' may change considerably in the light of additional or more direct experimental approaches to a problem. Using literature 'neighbouring' techniques, on-line companion entries enable users to be directed towards citations containing the 'latest' interpretations (or important 'additional facts'). The pace of change across all of the fields touched-on by the FactsBook means that 'specialists' in a given area can help 'speed-up' this indexing process by e-mailtnotificationwhere.reinterpretation' is justified (see Feedback etJ CSN access, entry 12, and Resource J). According to the original aims and 'philosophy' of the project, the entries will probably never be 'complete' as such. More appropriately, the framework will continue to evolve towards one which is hopefully more useful, authoritative, and able to comprehensively relate tconsensus' knowledge on ion channel molecular signalling.

Criteria for REFERENCES sections This section should contain 'short-form' references for numbered citations within the entry. For textbook coverage, refer to the Book references listed under Related sources and Reviews (field 56), Resource E - Ion channel book references, Resource F Supplementary ion channel reviews and Resource H - Listings of cell types. Plans for 'hyperlinking' to full bibliographic databases within the CSN framework are

described in Resource J - Search criteria etJ CSN development.

Criteria used for compilation of supporting computer-updatable resources The following reference appendices are referred to within the text and figures of the main entries. Updated versions of these files will be accessible via the thorne page' of the Cell-Signalling Network from January 1999 - for further details, see Feedback etJ CSN access, entry 12 and Resource J - Search criteria etJ CSN development. Resource A - G protein-linked receptors: A large number of ion channels are regulated as part of signalling cascades initiated by activation of G protein-coupled receptor proteins. This appendix should describe the basic principles associated with this type of regulation, limiting descriptions to those most relevant to ion channels. Tabulations of known receptort and G proteint molecules should form a framework of possible regulatory mechanism.s based on specific protein subtypes. The entry may clarify or suggest likely interactions between receptors, transducerst (e.g. G proteins) and ion channel molecules described under the fieldnames Developmental regulation, field 11, Protein interactions, field 31, Protein phosphorylation, field 32, Channel modulation, field 44, and Receptor/transducer interactions, field 49.

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Resource B - 'Generalized' electrical effects of endogenous receptor agonists: This resource should present a 'visual summary' of general patterns of agonistt induced ionic current fluxes that have been reported across a large number of studies, predominantly in the central nervous system. By summarizing patterns of channel modulation and gating, Resource B may help to indicate whether receptort agonists tend to act in an excitatoryt or inhibitoryt fashion 'or both'. Resource B forms a prototype for 'mapping' textual and bibliographic information to hyperlinked t visual frameworks. Resource C - Compounds etJ proteins: Compounds and proteins mentioned in the entries which are commonly used to investigate ion channel function and modulation should be listed, including those used to analyse interactions with other cell-signalling molecules. In general, only frequently reported compounds which are commercially available are described in this appendix. Resource D - 'Diagnostic' tests: This appendix is intended to be a 'field-referenced' listing of common experimental manipulations used to 'implicate or exclude' the contribution of a given signalling component or phenomenon associated with ion channel signalling. For the most part, these approaches use the pharmacological tools listed under Resource C, but may also include sections describing common molecular biological and electrophysiological 'diagnostic' procedures. Resource E - Ion channel book references: This appendix should list details of published books which have addressed themes in ion channel biology or closely related topics. These references complement those of the main entries, which are almost entirely based on citations from scientific journals. Resource F - Supplementary ion channel reviews: The ion channel literature contains a large number of useful 'minireviews' which summarize the development of defined subjects and which do not necessarily fall into a single channel 'molecular type' category. This appendix should therefore list these 'supplementary' sources, indexed by topic. Updated Ire-writes' of subject reviews covering similar areas may replace earlier listings. Note: Subject reviews dedicated to aspects of an ion channel type or family can usually be found under the Related sources eiJ reviews field of appropriate entries. 'Topic-based' reviews making reference to the basic properties in the 'molecular type' entries are planned for expansion within the CSN framework. Resource G - Reported 'Consensus sites' and 'motifs': Based on extensive analysis of primaryt sequences and determination of substrate specificities for various enzymes1 a number of 'consensus' recognition sequences for post-translational modification I of proteins (including ion channels) have been determined. While these sites are not absolute, they can be highly conserved across whole families of ion channel proteins and in many cases (e.g. following phosphorylation) can lead to profound changes in ion channel function. However, the presence of 'consensus' sites or motifst (or even demonstrations of substrate specificity in vitro) does not necessarily prove that such modifications operate in vivo. This appendix should list 'consensus' motifs that are well-characterized, giving examples of 'authentic' sites for comparison. This appendix also contains references to subsets of

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consensust sites from genomic DNA sequences associated with mechanisms of ion channel gene expression-control t (e.g. in trans t protein factors which act at DNA structural motifs t in cis t, influencing transcriptional activationt, transcriptional enhancementt or transcriptional silencing t of ion channel genes t ).

Resource H - Listings of cell types: Studies of ion channels within the context of cell-type function often reflect 'recruitment' of selected genes from the genomet in a cell-developmentallineaget . Because of this, similar 'sets' of ion channel molecules can often be observed in cell types with broadly similar functions. This appendix should describe a framework for describing how integrated sets of ion channel molecules (and their associated signalling components) have co-evolved for specific functions in terminally differentiatedf cell-types. To begin with, a tentative classification of functional cell types should be employed, used to cross-reference 'surveys' of ion channel expression wherever possible. This appendix should also contain available information pertaining to efficient and appropriate heterologous expression of ion channel genes in selected cell types, as this is often a limiting factor in biophysical characterization of clonedt ion channel cDNAt or gene products. Resource I - Framework of cell-signalling molecule types: The flow of information into, within and between cells (signal transduction) generally depends on a multiplicity of co-expressed cell-signalling molecules which provide 'measured' responses to stimuli. Communication between different cellular compartments (e.g. between the cytoplasm and the nucleus) often requires 'interconversion' or 'transduction' of chemical, electrical (ionic), metabolic and enzymatic signals, with receptors and ion channels playing key roles in transducing such stimuli. For example, the 'activation' of signal transduction molecules such as kinases t or transcription factors t appear to 'sense' 'activated' conditions which resembles Ca2 +-, voltage- or ligandt -gating phenomena commonly observed for ion channels. These modes of protein activation t probably have rnany features in common, and understanding their interrelationship has important consequences for comprehending fundamental links between receptor signalling, cell activation and gene expression. To facilitate integration of information between these diverse fields of study, this appendix should provide a preliminary listing of signal transduction molecules, with some consideration of their inter-dependency in the 'activated' state. By making a rational 'connection' between activation of receptors, ion channels, enzymes and other effector t proteins, it is hoped that SOlne general principles will emerge on the electrical- and ligand t -control of complex cell phenotyrest (such as those affecting the cell cycle t, cell proliferationt, cell differentiation and apoptosist). The importance of ion channel activation (and activation of receptor/G protein transducerst which modulate ion channel activity) in other fundamental cell processes such as signal transmission/amplification, secretion (multiple forms), muscular contraction, endocytosist (and other cellular 'uptake' phenomena), sensory transduction (all types), cell volume control/osmotic responses, mechanotransduction (various forms), membrane potential control (multiple modes) and developmental compartment formation are well-documented and multiple examples appear in several fields, notably Developmental regulation, field 11, Phenotypic expression, field 14, Domain functions, field 29, Protein interactions, field 31, Protein phosphorylation, field 32 and Channel modulation, field 44.

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Resource T- Search criteria &J CSN development. The framework of database entries which form the basis of The Ion Channel FactsBook were derived by 'scanning' primary research articles and reviews appearing in a set list of 'principal' journals dealing with ion channel and receptor signalling. A disadvantage of 'journal scanning' by 'keyword' is that search terms used are often ambiguous, and contextual or unconventional grammatical usage of keyword terms within articles often results in failure of specific retrieval. To circumvent this problem, this appendix should suggest new unique embedded identifiers (UEIs) which when specified by authors in the keywords section of submitted articles should ensure appropriate electronic retrieval from the primary literature. The adoption of finalized 'UEI' codes should be open to debate, but may eventually incorporate ion channel gene locus names (see field 54) where these are established by international convention. In the mean time, prototype UEIs will be used in the CSN pages to 'tag' new/backlogged article citation lists that contain information 'affecting' an existing or new entry. This also affords a simple 'queuing' mechanism for 'future coverage' of articles that have appeared or have been 'missed' during the main entry compilations (e.g. as notified in the feedback files, field 57). In due course, individual articles will be analysed and 'field referenced' within entry/fieldname update documents for downloading/printing. Prototype UEIs for each entry (distinguishing studies on native versus heterologous cells) are indicated in Category (sortcode), field 02. Updating by 'journal scanning' will proceed retrospectively from the 'most recent' articles to eventually 'overlap' with articles cited in the printed entries. In general, UEIs will be used to attempt systematic keywording/retrieval based on molecular criteria: the central principle of unique embedded identifiers is that they can 'automatically' find articles on topics of interest (in for example literature scans). Coupling to an 'expansion' section with further search terms in a conventional order will help enormously in data compilation/consolidation processes on strictly defined subjects within 'validated' databases. These issues will be discussed in the Resource 1 documents. Finally, Resource J should act as a forum for discussing limitations of data representation when comparing ion channel properties and suggest improved methods for facilitating information exchange (including graphical resources), diagnostic conventions, resolution of lcontroversial' results, and identification of areas or highly focused topics requiring consolidation/extension of knowledge. The importance of standardized computer software compatible with Internet t -mediated communication should be emphasized (see also Feedback eiJ CSN access, entry 12). Contents organization within each 'specialist' field of the FactsBook gives further opportunities for comparative data analysis. In due course, the -zz term of the xx-yy-zz index number will be used to indicate such structured information.

Criteria used for selection of on-line glossary and index items Consolidated versions of the FactsBook support glossary (i.e. extensions, updates and corrected items) are accessible from the Cell-Signalling Network 'home page' (see Feedback eiJ CSN access, entry 12). Index of on-line glossary items [ t]: To avoid unnecessary duplication of definitions within the text and to provide assistance to readers unfamiliar with a field, the

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on-line glossary should provide short introductions to technical terms and concepts. Throughout the text, cross-references to the on-line glossary items are shown by means of a dagger symbol t .

'Self-indexing' in The Ion Channel FactsBook, volumes I to IV For the most part, The Ion Channels FactsBook should be 'self-indexing': 1. Locate the channel 'molecular type' by sortcode, table of contents or the

Rubrics at the rear of the book. 2. Go to the appropriate section (NOMENCLATURES, EXPRESSION, SEQUENCE ANALYSES, STRUCTURE & FUNCTIONS, ELECTROPHYSIOLOGY, PHARMACOLOGY, INFORMATION RETRIEVAL or REFERENCES). 3. Look under the most appropriate fieldname (as described by the criteria above). Further 'structuring' will arise in due course, when more data are entered (see previous sections). The subject index should also allow the initial location of entries through alternative names of channels, associated signalling phenomena or commonly reported properties. Electronic cross-relation of topics is intended to be a development focus of the CSN, exploiting the principle of hyperlinking between database files stored in 'addressible' loci. For further details on how this might be achieved, see Resource T- Search criteria eiJ CSN development.

Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to this introduction can be sent to the e-mail feedback file [email protected]. (see field 57 of most entries for further details)

Abbreviations For most abbreviations of compound names in use, refer to the Resource C Compounds etJ proteins, entry 58, as well as the FactsBook entries. Abbreviations for ion channel currents are listed under the Current designation field of each entry. Terms marked with a dagger symbol appear in the on-line glossary section OCa2+ 5-HT 7TD

Ca 2+-free solution 5-hydroxytryptamine; serotonin 7 transmembrane domains

A

AV AVN

ampere t amino acidt after-hyperpolarizationt 4-aminopyridine action potential duration t atrio-ventricular atrio-ventricular nodet (of heart)

BKCa BP bp

large ('big/)-conductance calcium-activated K+ channels blood pressure base pairs t

C C-terminal CjA or C-A Ca(mech) or Camech CAv cds CF CICR CI(Ca) or Clca CNG CNS COOH CRC cRNA CTK Cx or Cxn

coulombt carboxylt terminalt (of protein) cell-attachedt (recording configuration) mechanosensitivet Ca2+ channel voltage-gated t Ca2+ channels codingt sequence (used in GenBankt® entries) cystic fibrosis t calcium-induced calcium-release calcium-activated chloride channel cyclic-nucleotide-gated (channels) central nervous system t carboxyl groupt calcium release channels complementaryt RNA cytoplasmic tyrosine kinase t (cf. RTK) connexin

Da Dephosjenzyme

daltons putative (consensus t ) site for dephosphorylation t by a specified enzyme, e.g. DephosjPP-I: endogenous protein phosphatase-I; DephosjPP-2A: protein phosphatase-2A dihydropyridine receptor Duchenne muscular dystrophyt depolarizing post-synaptic potentialt

aa AHP 4-AP APD

DHPR DMD DPSP

E EAA

E-C

potential differencet, inside relative to outside excitatory amino acidt excitation-contractiont

_L..-ELG Em EMBL EMF EPP EPSP ER Erev F

F fS

G g

G/Gmax gb: gj, Gj or GO) HGMW HH h.p. HVA

e_n_try_0_3_re_s_u_m_e_'_

500/0 effective concentration equilibrium potential t for K+ ions (analogous nomenclature for other ions) extracellular ligandt -gated (as used in FactsBook sortcode) membrane potential t European Molecular Biology Laboratoryt electromotive forcet endplate t fotentialt excitatory post-synaptic potentialt endoplasmic reticulumt reversal potentialt faradt Faraday's constantt femtosiemens (10- 15 Siemenst) conductancet conductance (unit - Siemenst, formerly reciprocal ohmst or mho t ) peak conductancet designation for GenBank® accession numbert gap-junctional conductancet Human Gene Mapping Workshopt after Hodgint -Huxleyr holding potential high voltage activated

InsP3R or IP3R IPSC IPSP ISH

current t subscript abbreviation for intracellular peakt currentt inside-outt (patcht, recording configurationt) concentration which gives 500/0 of maximal inhibition effect in a dose-inhibition response curvet intracellular ligandt -gated (as used in FactsBook sortcodest) maximal currentt collective abbreviation for inositol polyphosphatest - e.g. InsP3,InsP4 inositol l,4,5-trisphosphate-sensitive receptor-channel inhibitoryt post-synaptic currentt inhibitoryt post-synaptic potentialt in situ hybridization

JCC

junctional channel complex

k KA or K(A) kb KCa, Kca or K(Ca)

Boltzmann's constantt A-typet K+ channels kilobasest (kbp - kilobase pairs or bp x 103) calcium-activated K+ channels

I i

I/Imax I/O or 1-0 ICso

ILG I max

InsP(x) or InsPx

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1__e_n_t_ry_0_3_r_e_su_m_e

K1(0) KIR or K(IR) K(mech) or Kmech

knt

Kv LTD

LTP LVA mAChR MARCKS Mb MEPC MDa MH Mr

mRNA mV

_

equilibrium dissociation constantt kilodaltonst (daltons x 103 ) equilibrium dissociation constantt for an inhibitor for KATP channels, the ATP concentration (J.LM) that produces half-maximal inhibition of channel activity inhibition constantt at zero voltaget inward rectifier t -type K+ channels shorthand designation for mechanosensitive K+ channels kilonucleotides voltage-gated K+ channels (generally delayed rectifierst) long-term depressiont long-term potentiationt low voltaget activated muscarinic acetylcholine receptor myristoylatedt , alanine-rich C-kinase substrate megabases t (Mbp - megabase pairs) miniature endplate currents megadaltonst (daltons x 106 ) malignant hyperthermia relative molecular masst messenger RNAt millivolt (10- 3 V)

NH2 NSA NSC NSC(Ca) nt N-terminal

number of functional channels also - Avogadro's number t Hill coefficientt nicotinic acetylcholine receptor-channel shorthand designation for voltage-gated Na+ channels predicted sites for N-linked glycosylationt (e.g. N-gly: aa122, specifying amino acid number 122 from known glycosylaset substrates) aminot group non-selective anion (channel) non-selective cation (channel) non-selective cation channels (calcium-activated) nucleotides amino-terminal (of protein)

o O-gly OHC 0/0 or 0-0

subscript abbreviation for extracellular O-linked glycosylationt outer hair cells outside-outt (patcht, recording configurationt)

P

prefix for post-natal day; used to describe developmental status (e.g. P5 == post-natal day 5) permeabilityt of Ca2 + ions (analogous nomenclature for other ions, e.g. PK , PNa , PRb etc.) pituitary adenylyl cyclase-activating polypeptide phosphodiesteraset

N

n nAChR Nav N-gly:

Pea PCAP PDE

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[PDTM]

pHi Phos/enzyme

pj. PIR pir: PKA PKC PNS poly(A) poly(A)+ P open or Po pS PSC PSP PSS

protein domain topography model; within the text, use of the abbreviation in square brackets denotes a positional feature illustrated on the model intracellular pHt Putative t (consensus t ) site for phosphorylation t by a specified enzyme, e.g. Phos/CaM kinase II - multi-functional (Ca2+/calmodulin)-dependent protein kinase II; Phos/CaseKII: casein kinase II; Phos/GPK: glycogen phosphorylase kinase; Phos/MLCK: myosin light-chain kinase; Phos/PKA: cAMP-dependent protein kinase (PKA); Phos/PKC: protein kinase C (PKC); Phos/PKG: cGMP-dependent protein kinase; Phos/TyrK: tyrosine kinase (TyrK) subtypes post-injection Protein Identification Resourcet (protein sequence database) designation for Protein Identification Resourcet accession numbers protein kinase A protein kinase C peripheral nervous systemt polyadenylationt (site) polyadenylatedt (mRNA) fraction of total cellular RNA channel open probabilityt picosiemens (10- 12 Siemenst) post-synaptic currentt post-synaptic potentialt porcine stress syndrome coefficientt for a ten-degree change in temperature

R R

r.p. rRNA RTK RyR S SAN SAPs s.c.a. s.c.c. s.c.p. SCR SD SDS-PAGE

SEM SFA

-

receptor resistance t (unit - ohm t ), reciprocal of conductance t resting potentialt ribosomal RNAt receptor tyrosine kinase t (at plasma membrane,cf. CTK) ryanodine receptor-channel Siemens t (unit of conductance t ; reciprocal ohm t or mho t ) sino-atrial nodet (of heart) signal-activated phospholipasest single-channel amplitudet single-channel conductancet (symbol, ,) single-channel permeabilityt single-channel recordingt standard deviation sodium dodecyl sulphate-polyacrylamide gel electrophoresist (i) standard errort of the means t or (ii) scanning electron microscopyt spike frequency adaptationt

1L-_e_n_t_ry_0_3_r_e_s_u_m_e

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indicates the range of amino acids which form the signal peptide of a precursor protein (e.g. Sig: aal-26); alternatively, the abbreviation indicates the actual cleavage sitet forming the signal peptide t and mature chaint from the precursort protein substance P designation for SWISSPROT protein sequence database accession numbert sarcoplasmic reticulumt disulphide bond t ; in sequence database entries, the S-S: symbol is sometimes used to denote positions of a known disulphide bond linkaget or motift between two residues on a protein molecule, e.g. an experimentally determined link between residues 154 and 182 on the same chain would be written as S-S: 154-bond-182.

Sig:

SP sp: SR S-S:

TPeA+ TT

v

transmembrane melting temperaturet upper limit to the amount of material that carrier-mediated transport can move across a membrane tetrapentylammonium ions transverse tubulet

VDAC VDCC

voltt voltage t voltage-activated calcium channels; analogous nomenclature for other channelsl e.g. VACIC, VAKC, VANaC, VDAC voltage-dependent t anion channel voltage-dependentt calcium channel

W/C or W-C WCR

whole-cellt (recording configuration) whole-cellt recording

YAC

yeast artificial chromosomet

V VACaC

, ,j or

,(j)

IJ-A

n

w-CgTx

unitary (single-channel) conductance single-channel junctional conductancet microamp (10- 6 Amperes) ohmt, unit of electrical resistancet; reciprocal of conductance t omega-conotoxin

Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the abbreviations section can be sent to the e-mail [email protected]. (see field 57 of most entries for further details)

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VOLTAGE-GATED CHANNELS

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VLG Key facts Edward C. Conley

Voltage-gated channel families key facts Entry 41

Note: The ~key facts' sections are intended for readers unfamiliar with the more general aspects of ion channel biology, and contain selected introductory information applicable to whole groups of ion channel molecules. These sections, coupled with the on-line glossary items (indicated by daggert symbols attached to key terms in context) provide a basic overview of principles associated with more detailed information within the main entries of the book.

Membrane potential fluctuations represent a primary mechanism for cell signal transduction 41-01-01: A large diversity of voltage-sensitive responses in many cell types

support a central role for membrane potential t control in the evolution of complex cell signalling systems. Voltage-gated (viz. voltage-dependent, voltage-sensitive, voltage-activated) ion channels thus induce transmembrane ionic flow in response to sensed changes in transmembrane potential t . These proteins have permanently charged or dipolart regions which are forced to move by fluctuations in the local electric Heldt. Physical movement of the voltage sensor thereby couples to conformational changes associated with channel gating. The initiation of voltage gating has (by definition) little or no dependence on extracellular/intracellular ligands or protein phosphorylation (compare ELG key facts, entry 04, ILG key facts, entry 14 and INR key facts, entry 29) although these factors commonly modulate responses by altering (for example) voltage dependence of gating, current duration/amplitude, activation kinetics or channel protein interactions. During evolution, some channels have 'retained' voltage sensitivity of gating while being 'obligately' modulated by ligands (e.g. Kca channels, see ILG K Ca, entry 27; see also Voltage sensitivity under ELG CAT NMDA, 08-42). By contrast, other channels appear to have 'lost' voltage sensitivity in favour of 'obligate' ligand gating functions (e.g. cGMP-gated cation channels, see ILG CAT cGMp, entry 22). Comparative structural and functional aspects of the voltage-gated cationselective channel superfamily (Table 1) have been the subject of several reviews 1 - 10. The evolutionary origin of voltage-gated and other channels has been specifically discussed11,12.

Voltage-gated channels in excitable versus non-excitable cells 41-01-02.: Voltage-gated channels are abundant in excitable cells that pass

action potentials (such as nerve and muscle) and most information on how the different classes of voltage-gated channel interact have thus been determined in excitable cells. Although present at relatively low densities, several subtypes of voltage-gated channels have also been characterized in non-excitable cells (such as exocrine/endocrine secretory and blood cells). In any cell type, a given class of voltage-gated ion channel may have

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_1_

predictable roles associated with propagation and control of that cell type's 'excitability cycle' (for details, see specific entries). For instance, some voltage-operated Ca2+ channels (VOCC, VLC Ca, entry 42) have major roles in triggering secretory vesicular release and muscle contraction, while voltage-gated Na+ channels (VLC Na, entry 55) have received most attention for their role in propagation of regenerative action potentials. In general, K+ channels stabilize cell voltages by counteracting the depolarizing effect of Ca2+ and Na+ channels - they repolarize cells following action potentials and thus modify excitability and firing patterns. Together with several classes of inwardly rectifying K+ channel (INR-series entries, Volume ill), some classes of voltage-gated K+ channel contribute to the membrane potential of resting cells and can therefore markedly influence properties such as excitability threshold t, basal secretion rate, maintenance of vascular/muscular 'tone' and cell volume. Comprehending the molecular diversity and patterns of receptor-coupled modulation of voltage-gated Ca2+ and K+ channel effectors t (in particular) has also been of significance for those interested in molecular mechanisms of signal integration and plasticityt.

Co-evolved 'integrative' properties of voltage-gated channels in shaping action potentials 41-01-03: Patterns of propagation for 'all-or-none' signals in the nervous

system are heavily influenced by the intrinsic functional properties of the channel proteins carrying the component ionic currents which summate to action potentials. For example, a voltage-gated Na+ channel subtype able to turn on and off rapidly would promote a high frequency of firing. Alternatively, a more slowly inactivating Na+ channel subtype would broaden an action potential. Co-expression with specific subtypes of K+ channel might also limit the maximum firing frequency. A striking demonstration of the roles of specific interacting sets of voltage-gated ion channel subtypes in 'shaping' action potentials is seen in the cardiac conduction system: action potentials recorded directly from each anatomical subregion of heart (broadly, sinoatrial node, atrial, atrio-ventricular node, His-Purkinje and ventricular tissue) display robust action potential shapes 'characteristic' of their constituent ionic components/channel subtypes (see also INR key facts, entry 29). Voltage-gated ion channels possess characteristic singlechannel conductances and maintain constant ionic selectivities depending on both specificity of ion binding and pore size. Thus 'selection' of channel components to 'fit' a functional role is dependent upon their integrative properties within a cellular compartment. In consequence, the interactive properties of multiple channel subtypes are of critical importance in computational approaches to understanding neuronal function 13 . Co-ordinate regulation (cell-specific expression) of voltage-gated ion channel genes and those encoding other signalling components is ultimately determined by genetic control elements t that chiefly reside 'upstream' of protein structural genes. These may include various transcriptional activator t and transcriptional silencert elements. For example, the latter have been characterized as being responsible for 'restrictive' expression of certain voltage-gated Na+ channels to neuronal but not non-neuronal cells (for an example, see Cene organization under VLC Na, 55-20).

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_

Sequence relatedness defines voltage-gated cation channel families and superfamilies 41-01-04: The functional diversity of mammalian voltage-gated cationselective channels appears to have been generated via primordial gene duplication and selection processes, which have given rise to the coexistence of multiple related-sequence genes within the genome t (Le. gene familyt members; for a detailed discussion of possible gene family evolutionary mechanisms, see Chromosomal location under VLC K Kv1Shak, 48-18). Complete sequencing of prokaryote genomes such as Escherichia coli has uncovered important candidates for ancestral K+ channel genes with homology to voltage-gated channels of higher organisms (e.g. kch, see Miscellaneous information under VLC K Kv1-Shak, 48-55). Thus homologues of voltage-gated K+ channel genes are now known to exist in an extraordinarily wide evolutionary range of organisms from Escherichia through Streptomyces, ciliate protists (e.g. Paramecium), Arabidopsis, jellyfish, worms, squid and (as most extensively characterized) flies and vertebrates. Amino acid sequence homology comparisons have revealed the protein domain arrangements of voltage-gated and cyclic nucleotide-gated (CNG) channel families to show many gross similarities14 (see also ILC CAT cAMP and ILC CAT cCMp, entries 20 and 21). These groups can be incorporated into a larger gene superfamilyt that includes the voltage-gated Na+, Ca+ and K+ channels, cyclic nucleotide-gated cation channels, hyperpolarization-activated cation channels (INR (K/Na)IfhQ' entry 34) and Ca2+-activated K+ channels (Table 1; for the distinct domain arrangements of voltage-gated Cl- channels, see next paragraph). In general, all channels in the voltage-gated superfamily listed in Table 1 show a similar protein domain arrangement (the 'SI - S6 + P arrangement') consisting of a set of six predicted membrane-spanning segments (hydrophobic regions SI to S6) plus a pore-forming region (P-region, formerly the H5 domain between 85 and 86 - see {PDTM} Fig. 6 under VLC K Kv1 Shak, entry 48). Additionally, multiple tissue-specific variants of some voltage-gated ion channels can arise through alternative splicingt of primary RNA transcripts (e.g. see VLC K Kv3-Shaw, entry 50).

tMatching' native channel currents with those conducted by heterologously expressed channel proteins 41-01-05: Several factors underlie the difficulty of 'matching' properties of native cell voltage-gated channel currents to the 'contribution' of individual channel protein subunits. With regard to Kv channels, for example, heterologous t expression of single channel subunits (Le. in the specified

II

II Table 1. Comparative protein domain arrangements within the cation-selective voltagelsecond-messenger-gated channel gene superfamily (Sortcodes beginning VLG- are covered in this volume) (From 41-01-01) Sortcodes/class subunit (example)

Descriptive notes/cross-references/typical protein domain arrangement

Gene family designation/ selectivity (see entries)

Refs

VLGCa Voltage-gated Ca2 + channel, a: subunit (see note 3)

In Cava subunits, the Sl-S6+P arrangement is

Cav (in keeping with Kv channels, see note 2)

e.g. 15?16

present in each of foul internal repeats as shown. In Cav (and Nav) channels, the a subunit is a long, single, folded polypeptide - (cf. the extracellular ligand-gated (ELG) channel superfamily (entries 04 to 11), which are composed of multiple subunits)

Generally highly Ca2+ selective

The Cava-subunit is co-expressed with a2/8, {3 and, subunits. Many structurally and functionally distinct subtypes exist. In general, Cav channels help conduct Ca2+ influx activated by depolarization, initiating a large number of cellular functions including muscular contraction and neurotransmitter release. Key: +, positively charged voltage sensor element (S4 domain); P, pore-forming domain.

g ~ ~ ~

VLG K series

(except VLG K Kv-beta and VLG K minK) Voltage-gated K+ channel, a subunit

In Kv 0: subunits, the Sl-S6+P arrangement is present only once per K+ channel a subunit. Functional channels in vivo have been shown to be formed by association of four Kvo: subunits and four Kv{3 subunits (for details, see VLC K Kv-beta, entry 47).

Kv (as first established by Chandy and colleagues, see note 2)

e.g. 17

t"1"

~ ~

Generally highly K+ selective

In general, Kv channels have cellular repolarization functions (Le. depolarized membrane potentials opening K+ channels leading to hyperpolarization and return to rest). Differential expression and modulation of Kv channel subtypes can radically alter cell excitability, Le. the firing patterns and duration of action potentials (see various VLC K series entries). Key: +, positively charged voltage sensor element (S4 domain}j P, pore-forming domain. VLGNa

Voltage-gated Na+ channel, subunit (see note 3)

a

In Nav 0: subunits, the Sl-S6+P arrangement is Nav present in each of four internal repeats as shown. Like (in keeping with Kv channels, Cay channels, the 0: subunit is a long, single, folded note 2) polypeptide. Generally highly Na+ selective

II

g ~

e.g. 16,18

II

Table 1. Continued Sortcodes/class subunit (example)

Descriptive notes/cross-references/typical protein domain arrangement

Gene family designation/ selectivity (see entries)

Refs

Mammalian Nav Q subunits are co-expressed with two smaller f3 subunits. Functionally and structurally distinct Nav channel subtypes exist (see VLC Na, entry 55), but most are steeply voltage dependent. In general, Nav channels are activated by depolarization and induce depolarization; responsible for action potential propagation. Key: +, positively charged voltage sensor element (S4 domain); P, pore-forming domain. ILG K Ca (Volume II) Ca2 +-activated K+ channel, Q

subunit

In KCa Q subunits, the SI-S6 + P arrangement is KCa present only once per K+ channel Q subunit. Species (in keeping with Kv channels, homologues of the genes encoding vertebrate and fly note 2) large-conductance Ca2+-activated K+ channels are described in ILC K KCa, entry 27.

19

Generally highly K+ selective

Key: Grey bar, Ca2+-binding site; Ca, calcium. P, poreforming domain. The Ca2+-activated K+ channel of Drosophila is encoded by slopoke; vertebrate homologues are generally named 'slo' with a species prefix; the 'SI-S6 + P' arrangement and subunit stoichiometry has some similarity to Kv channels described above.

g ~ ~ .......

ILG CAT cAMP (Volume IT) ILG CAT cGMP (Volume IT) Cyclic nucleotide-gated cation channels, a subunits

In CNG a subunits, the 81-86 + P regions are present

only once per subunit with subunit stoichiometries and assemblies analogous to Kv channels. CNG-a/ f3 subunit co-associations described in functional channels (see entries 21 and 22).

Key: Hatched bar, cyclic nucleotide t binding site; cNUC, cyclic nucleotide; P, pore-forming domain.

Ca2 + channel for inositidemediated Ca2 + entry {putative}

81-86 + P present once per subunit. An example is the putative Ca2 + channel for phosphoinositide tmediated Ca2+ entry encoded by the trp (transient receptor potential) gene, which probably mediates invertebrate photoreception. For brief discussion of trp see Table 1 under ILG Ca CSRC (entry 18, Volume II) and Miscellaneous information under ILG CAT cGMp, 22-55.

CNG (cyclic nucleotide-gated, see entries 21 and 22)

II

S M"

~ ~

.......

Mixed cation Na+/K+ /Ca+ selective dependent upon subtype (see fields 21-40 and 22-40) In press update: Hyperpolarization-activated cation channels also conform to this pattern - for refs, INR (K/ Na)IfhQ' entry 34. (-)

(non-vertebrate, but compare vertebrate channel showing M1- M6 + P arrangement in Fig. 1 of ILG Ca InsPa, entry 19)

Generally highly Ca2+ selective

Key: Black bar, calmodulint -binding site; CAM, calmodulin; P, pore-forming domain.

1,20

21,22

II

Table 1. Continued Sortcodes/class subunit (example)

Descriptive notes/cross-references/typical protein domain arrangement

Plant K+ channel/transporter SI-S6 + P present once per subunit. Sequence similarity between these molecules (expressed in plants) and the Shaker-related channels indicates a common ancestor gene prior to the separation of the plant and animal kingdoms (ca. 700 million years ago for background to K+ channel evolution see Cmomosomallocation under VLC K Kv1-Shak, 48-18).

Gene family designation/ selectivity (see entries)

Refs

(-)

23,24

(non-vertebrate; see also E. coli kch channel gene described under field 48-55).

K+ selective

Key: Light grey bar, cyclic nucleotide t -binding site; cNUC, cyclic nucleotide; P, pore-forming domain.

Notes: 1. Comparison of protein domain topography in the voltage/second messenger-gated superfamily of ion channels and molecules of the ATP·binding cassette (ABC) superfamily reveal distant structural relationships (see also Table 1 under ILC key facts, entry 14, and also PDTM of InsPg-gated Channels, entry 19 (Fig. 1).). 2. Whereas the gene family prefix 'Kv' is well-established, analogous universal designations have not appeared for other families; the designation shown is used on the ICN web site (www.le.ac.uk/csn/ ) to stay consistent with the Kv channel nomenclature. 3. Both voltage-gated sodium channels (see VLC Na, entry 55) and calcium channels (see VLC Ca, entry 42) carry inward currents which depolarize t the membrane through flow of positive charges into the cell. Conversely, outward currents which flow following activation of potassium channels (see VLC K-series, entries 44 to 54) and inward currents which flow following activation of Clchannels (see VLC Cl, entry 43) tend to hyperpolarizet the membrane by making the interior of the cell more negative.

Ig ~ ~ ..-

~e_n_t_ry_4_1

_

absence of endogenous channel subunits capable of co-assembling with the heterologous component) is generally assumed to occur via formation of homomultimeric t (i.e. homotetrameric t) channels. However, co-assembly of 'accessory' subunits with pore-forming (alpha) subunits can significantly influence the inactivation, modulation and expression properties of voltagegated channels formed from Q; subunits alone (for further examples, see VLC K Kv-beta, entry 47 and VLC K Kv1-Shak, entry 48). Heterologous expression systems may also not 'reproduce' significant modulatory conditions found in native cells. Note: These difficulties in matching 'native' with 'cloned' channel properties have necessitated separate treatment of their literatures within this volume. For example, while it is likely that many of the native cell currents extensively described as 'A-type' (entry 44) or 'delayed rectifier-type' (entry 45) arise from the proteins described in the VLG K Kv series entries, there is often relatively little direct evidence to 'implicate' specific protein subtype(s). Applications of new computer-based analytical techniques 13 and consideration of multiple functional properties may help to improve 'concordance' between 'native' and 'cloned' channels. In general, however, data derived from native versus heterologous cell preparations have been kept separate. There is still much to learn about the regulation of expression patterns for multiple voltage-gated channel subunits in the eNS (and elsewhere) and the specific subunit compositions of heteromultimeric channels in vivo.

Minor differences in voltage-gated channel sequences can underlie major functional changes 41-01-06: Introduction of relatively minor changes in channel primary DNA sequences (e.g. by site-directed mutagenesis) can radically alter functional properties of the heterologously t expressed protein. Such structurefunction t analyses have been enormously valuable in defining protein domain 'functions' acquired through processes of divergent t evolution (where the protein encoded by a single 'progenitor' gene may be 'adapted' to perform multiple functions, for example in different cell types). Alteration (mutation) of DNA sequences encoding 'key' amino acid residues participating in highly specific protein movements or interactions (including the sensitivity to 'external' modulators) may thus markedly alter phenotypic t properties in cells. Most generally, mutations affecting native channel protein conformation t, subunit assembly t properties or equilibrium (allosteric t) behaviour can be predicted to produce functional changes. Structurefunction analyses have helped to localize 'critical' residues for channel functions such as ion selectivity, permeation and block (for examples, see Selectivity, field 40 and Blockers, field 43 in various entries), inter- and intra-channel protein domain interactions (see Protein interactions, field 31), intracellular 'targeting'/localization mechanisms (see Subcellular localization, field 16) and various phosphomodulatory mechanisms (see Protein phosphorylation, field 32). A large number of studies have used mutagenesis to generate channel variants that show altered activation properties (see Activation, field 33), rectification properties (see Current-voltage relation, field 35), inactivation mechanisms (see Inactivation, field 37), voltage-gating or the voltage-dependence t of various processes (see Voltage sensitivity, field

III

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en _ t_ry_4_1_

42). Notwithstanding these striking functional effects, many mutations

introduced experimentally (or 'accumulated' during evolution) may be 'silent' or produce a 'non-functional' protein according to the criteria employed. Judgements as to the 'functional' effects of ion channel gene mutations may thus be subjective: effects may have some dependence on the heterologous t or native cell 'background' (and other, co-expressed, interacting proteins) (see also notes on selection of heterologous gene expression systems in the footnote to Table 1 under ILG key facts, entry 14).

Non-exhaustive examples of mutations in voltage-gated channel genes implicated in genetic disorders 41-01-07: As further described in Phenotypic expression (field 14) of relevant

entries, mutations in genes encoding voltage-gated Ca2+, CI-, K+ and Na+ channels have been shown to be responsible for a number of human and animal genetic disorders: Ca2+ channels: Mice with mdg mutations have muscular dysgenesis, where lack of the 0lS subunit of the skeletal muscle L-type Ca2+ channel affects excitation-contraction coupling. Mutations at the mouse tottering (tg) locus affect the 0lA subunit of a P/Q-type Ca2 + channel and induce a neurological disorder resembling petit mal epilepsy in humans. The human autosomal t dominant t disease hypokalaemic periodic paralysis (hypoPP) is caused by mutations in the CACNL1A3 gene that encodes the 0lS subunit of the skeletal muscle L-type Ca2 + channel. Mutations in the human CACNL1A4 gene encoding an 0IA subunit of a P /Q-type Ca2+ channel have been identified in patients with familial hemiplegic migraine (FHM). Genetic alterations affecting the channel Oli} subunit have also been shown to be the cause of autosomal t dominantT cerebellar ataxia. CI- channels: Mutations in the C1CN1 gene encoding the skeletal muscle CI- channel are responsible for dominant myotonia congenita (Thomsen's disease) and for recessive generalized myotonia (Becker's disease). Dent's disease, an X-linked hypercalciuric nephrolithiasis, is caused by mutations in the kidney-specific C1CN5 gene. K+ channels: Point mutations in the hKvl.l gene have been associated with one form of familial episodic ataxia, characterized by brief episodes of ataxia t with myokymiat (continuous movement or 'rippling' of muscles) evident between attacks consistent with reduced capacity for repolarization in affected nerve cells. Two forms of the familial cardiac long QT (LQT) syndrome have been associated with mutations in two distinct genes encoding potassiumselective voltage-gated K+ channels (KvLQTl underlying LQT1, see VLG K minK, entry 54 and HERG underlying LQT2 - for further details, see Phenotypic expression, 46-14 and Chromosome location, 46-18). Na+ channels: Missense t mutations in the human gene (SCN4A) specifying the adult skeletal muscle voltage-gated Na+ channel 0 subunit are associated with two hereditary disorders of sarcolemmal excitation, hyperkalaemic periodic paralysis (HYPP or hyperPP) and paramyotonia congenita (PC).

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Mutations in the seNSA gene encoding a cardiac sodium channel a subunit are known to underlie the LQT3 form of LQT syndrome (see cross-references above). Mutations of the med locus on mouse chromosome 15 (the ScnBa gene) produce a recessive t neurological disorder, motor endplate disease, with symptoms ranging from mild ataxia t to juvenile lethality. Neuronal defects, including lack of signal transmission at the neuromuscular junctiont, excess pre-terminal arborization t and degeneration of cerebellar Purkinje cells, are apparent in the disorder.

Distinct mechanisms underlying cellular versus subcellular distributions of channel subtypes

41-01-08: Virtually every excitable t cell type expresses multiple subtypes of voltage-dependent channels specific to the 'functions' of that cell type. Functionally specialized cells may exhibit a wide variety of 'current types' each displaying individual patterns of modulation. For example, olfactory receptor neurones display six classes of voltage-dependent ion currents including Na+ currents, L-type Ca2 + currents, Ca2 +-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current25 . Notably, multiple protein subtypes may support each class of current. For example, techniques such as single cell RT-PCRt have revealed co-expression of multiple subtypes of Kv channel within individual neurones (see also INR key facts, entry 29). Furthermore, as described in field 13 (mRNA distribution) and field 15 (Protein distribution) many studies have employed in situ hybridization and immunological techniques for localization of voltage-gated channel mRNAs and proteins across broad areas of tissue. In general, these studies have predicted the existence of elaborate developmental control mechanisms for appropriate 'placement' of ion channels in various developmental compartments (see Developmental regulation under TUN [connexins), 35-11). Once expressed within a developmental compartment or cell type, however, subcellular targeting mechanisms (normally involving discrete protein interactions) become important for assembling the 'multiprotein machines' that constitute functional ion channels in vivo (for examples of these principles, see Subcellular locations, field 16 and Protein interactions, field 31).

Structural elements determine compatibility of subunit associations 41-01-09: Full 'reconstitution' of the native properties of voltage-gated ion channels may require complex interactions of multiple protein subunits (for examples within the superfamily, see Protein interactions, field 31). Many examples exist where 'accessory' protein components other than those which form the main voltage-sensitive/ion-selective component interact to form the native channel (ibid.). Within some gene families, structural elements or regions have been defined that appear to determine compatibility of protein subunit associations. A well-characterized example of this is the region within the N-terminal domain of Kv (voltage-gated K+) channels that has been shown to mediate subunit-subunit interactions in homomultimeric t channels as well as determining compatibility of co-assembly between different K+ channel subunits26 . Translocation of this region to recipient channel monomers promotes co-assemblies characteristic of the

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donor channel subunit (see Protein interactions under VLC K Kv1-Shak, 4831 and VLC K Kv2-Shab, 49-31). These studies provided a mechanistic basis for understanding why different members of the same subfamily of Kv channels were able to co-assemble and form functional heteromultimeric t channels while subunits of different subfamilies do not co-assemble (for details see Protein interactions under VLC K Kv1-Shak, 48-31).

The fourth hydrophobic segment (S4) functions as a tvoltage-sensor' in voltage-gated cation channels 41-01-10: A common feature of voltage-gated potassium, sodium and calcium channels is that the 84 transmembrane segment contains basic t amino acid residues (e.g. Lys or Arg) at every third or fourth position (for examples, see the protein domain topography mode1s, PDTM, of various entries). This arrangement confers an intrinsic voltage sensitivity such that changes of membrane potential exert an electrostatic force on the charges or dipoles of the voltage sensor. This force induces protein conformational changes (including outwardly directed movement of the 54 element) that lead to channel opening (contrast the arrangement for voltage-gated chloride channels, next paragraph). Movements of voltage sensors can be detected as gating current t, and these currents show significant differences between inactivated and non-inactivated channels. The number of charges that are translocated during channel opening (the gating charge t ) can be estimated by examining the steepness of the voltage dependence of gating, as reviewed27 (see also panel D under Figure 1). In the original 84 hypothesis, four 54 sequences corresponded to voltage-sensitive gating particles as proposed by Hodgkin and Huxler8 . Classical measurements of Na+ channel gating current (i.e. movement of charges intrinsic to the channel) estimated that four to six charges move from one side of the membrane to another as the channel opens29. This was accounted for in the 54 model by depolarization causing each of the 54 sequences to move by approximately one helical turn. In general, voltage-gated channels differ in (i) the voltage activation range over which membrane potential changes can open the ion pore, and (ii) in the transition rates t between the open (conducting) and the various non-conducting states of the channel (see Activation, field 33, Kinetic model, field 38; also compare t.he various domain arrangements in the inset figures to Table 1). Note: During action potentials, membranes may typically undergo voltage changes on the order of "-JO.I V ("-JI06 VIm).

A distinct mechanism for voltage sensing in voltage-gated chloride channels 41-01-11: The 'voltage-gated' CI- channels lack an obvious structural motif analogous to the 54 domain which functions as a voltage sensor in the voltage-gated cation channels (see paragraph 41-01-10). Analysis of the voltage and pH dependence of channel gating suggests that a single acidic residue is primarily responsible for voltage sensing: an aspartate in the first

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transmembrane segment is a prime candidate as the voltage sensor, since its replacement by a glycine in a recessive form of myotonia congenita greatly affects voltage-dependent gating of the hCIC-l CI- channel. The gating behaviour of CIC-l channels is consistent with a slow blocking/unblocking event mediated by a cytoplasmic 'gate' that is postulated to behave as an open-channel blocker, analogous to the 'ball and chain' model for the inactivation of voltage-gated Na+ and K+ channels. The voltage-induced conformational change is postulated to alter the 'on-rate' of the blocking particle (see note) (for further details, see the VLG Cl fields Phenotypic expression, 43-14, Domain functions, 43-29 and Kinetic model, 43-38); also compare the description of the voltage-gated anion channel (VDAC) characterized in mitochondrial membranes under the MIT [mitochondrial, native}, entry 37. Note: Not all members of the CIC family are voltage gated; the CIC-3 channel, for example, is voltage independent and remains open at all membrane potentials.

Distinctions between voltage-gated and inwardly rectifying K+ channels 41-01-12: By definition, voltage-gated K+ channels have their ionic conductance (g) regulated as a function (f) of membrane potential alone (V) - i.e. g == f(V). This is to be contrasted with inward rectifier K+ channels (see the INR K series entries, Volume III) whose conductances depend on the electrochemical gradient or driving force t on the ions (Le. gK versus voltage (V) varies according to a function (f) of gK == f(V - EK), where EK is the equilibrium potential l for potassium ions. In general, therefore, the current-voltage relation t for an inward rectifier will also depend on factors like [K+]o that can influence EK and in consequence, the prevailing driving force on potassium ions (compare also the effects of [K+}o on HERG channels under VLC K eag/elk/erg, entry 46). Figure 1 illustrates the origin of certain conductance parameters for voltage-gated channels that are cited in the main entries.

Many voltage-gated channels automatically inactivate with time

41-01-13: In comparison to some ligand t -gated channels (e.g. see the ELG and ILG series entries in Volumes I and II) most voltage-gatedt channels conduct relatively short-duration currents, before intrinsic channel inactivationt or intrinsic gating t processes sensitive to changes in membrane potential 'cut off' the signal under physiological conditions (see below). Whereas the opening and closing of some voltage-gated channels appear to depend entirely on the prevailing membrane potential, many show lprogressive' inactivation (even under maintained depolarizing conditions that would initially cause the channels to open). Inactivated channels are thus refractory to further depolarization-induced openings. Agents that inhibit Na+ channel inactivation in nerve axons (e.g. the pyrethrins, natural insecticides isolated from the flowers of the genus Chrysanthemum, and their synthetic analogues, the pyrethroids) cause repetitive firing and depolarizing after-potentials t. The synthetic insecticide DDT (dichlorodiphenyltrichloroethane) acts in a similar fashion. A large body of work has analysed different inactivation mechanisms, particularly in cloned voltage-gated potassium and sodium channels

II

II . . - - - - - - - - - Panel A: Generation of current families [2] Larger depolarizations generate larger currents which activate more rapidly

[1 ] Recording of the potassium current that flows by changing the membrane potential in a stepwise manner

c

Faster

I I I

Exponential tail current } -

[3]

lO

~

vm:J--'--------

-ve

Tail currents start with an abrupt drop as the channels close In response to the change in Vm. The degree of change in current depends on where the membrane voltage is relative to the equilibrium potential for potassium ions (E K>

Figure 1. Origin of conductance parameters for voltage-gated channels as mentioned in text, exemplified for non-inactivating voltage-gated K+ channels. In these examples K+ current would be isolated (i.e. measured exclusively) from other membrane currents present (either using pharmacological blockers, appropriate voltage protocols and/or by heterologous expression a cloned K+ channel gene of interest).

g ~ .,J:::l.

......

(1)

=

~

~ I-'

Panel B: Generation of current-voltage (I-V) relations

[2] [3]

The I-V relation in this example shows step depolarizations to have little effect near EK, but having increasing effect with increasing depolarization - see panel C.

IK

[1 ] From the types of record shown in panel A, it is possible to plot the membrane current (at its peak) against the voltage at which it was recorded.

-ve

I

EK Figure 1. Continued

II

~

o

Vm

+ve

At the membrane potential defined by EK, potassium ions will move neither inwards nor outwards across the membrane - as defined by the Nernst equation. EK is the membrane voltage at which the concentration gradient is exactly balanced by the electrical gradient working on potassium ions.

II ,.....-.----- Panel C: Chord conductance and slope conductance [2] The chord conductance for potassium (gK) is higher at more positive potentials - i.e. it increases with increasing depolarization

[1 ] Conversion of potassium current into a chord conductance is obtained* as the slope of a line (dotted) joining a point on the I-V relation to the equilibrium potential for potassium ions.

*assuming Ohms law, I = VIR I K = gK (Vm-E K )

[3]

IK Chord conductance gK =

I K

, ,,""

(Vm-EK )

,,"

-ve

t~--'"

EK

,",,"

o

,,;.

More rarely, the slope __ . __ . __ . __ . __ . __ . __ .•_. __ conductance parameter is used as the simple slope of the I-V relation (~II~V). The slope conductance, unlike the chord conductance, is dependent on ionic conditions.

I

Vm

+ve

Figure 1. Continued ~

= ~ ~ ......

Panel D: Normalized conductance-voltage relations

, [1 ] The conductancevoltage curve shows how the probabi Iity of a channel being open varies with voltage.

Conversion of currents to conductances (as described in panels A-C) and plotting against membrane potential produces a sigmoid relation with a near-zero conductance at -ve membrane potentials and a maximum at +ve potentials (see note 1).

For voltage-activated channels, g-V relations can be fitted with Boltzmann functions of the form:

Relative 9

K

Relative gK

1

=

1 1 + exp [(Vh-Vm)/k]

[3] from above Vh voltage at which gK is half-maximal; k a constant related to the number of equivalent charges involved in channel gating (and that describes the steepness of the voltage-dependence). Together, Vh and k specify the voltage-dependence of the channel (see note 2).

= =

o

-ve

Vm

EK Notes: 1: The maximum level of the plateau will depend on the concentration of permeant ions, so the curves are normalized to reach the same maximum. 2: For a purely voltage-gated channel, alteration of EK to zero (Le. by raising the extracellular [K+]) should not alter the position of the g-V curve along the voltage axis - compare with inward rectifiers described under INR {key facts}, entry 29. If g-V curves do not shift with EK set to zero, the potassium conductance varies as a function of voltage alone.

Figure 1. Continued

II

!

~ ..-

[2]

_'--

e_n_t_ry_4_1_

(for details, see Inactivation, field 37, under the VLC Na and VLC K series entries). The 'hinged-lid' and 'ball-and chain' mechanisms (ibid.) have become dominant models for fast inactivation of voltage-gated Na+ and K+ channels respectively. Note: Prior to the molecular cloning of large numbers of K+ channel genes, K+ current inactivation behaviour was a principal means of channel classification (see discussion under VLC K A-T [native], entry 44 and VLC K DR [native], entry 45).

Some blockers of voltage-dependent channels act from the cytoplasmic side on open channels 41-01-14: Several chemical blockers of voltage-gated ion channels appear to bind at a site close to the vestibulet near the cytoplasmic opening of the poret - e.g. local anaesthetics for Na.+ channels (see below), quaternary D600 for some Ca2+ channels and tetraethylammonium ion (TEA) and its analogues for some K+ channels. Historically, blocking behaviour like this was used to gain a picture of channel structure in the absence of any direct molecular information. Blocking agents which became 'lodged' within the channel as it closed could be rendered less effective (i.e. 'dislodged') by increasing concentrations of the permeant ions on the extracellular side. These and other observations indicate:d that the 'gate' for closure of the channel was on the cytoplasmic side and that the pore itself did not change its shape significantly during gating. Local anaesthetics, such as lidocaine and procaine, are generally lipid-soluble tertiaryt amine compounds that inhibit propagated action potentials by blocking Na+ channels. Note: Blockade of voltage-gated Na+ channel function is of potential clinical use to augment neuroprotection during ischaemic or traumatic injury.

Voltage-gated channels are generally modulated by activation of separate receptor proteins 41-01-15: The majority of channels coupled to discrete receptor and transducert proteins can be gated by membrane potential changes even in the absence of receptor agonist. These ehannels can therefore be considered receptor modulated, where neurotransmitters enhance or suppress the primary, voltage-dependent responses. Although some neurotransmitters may have effects on voltage-dependent Na+ and CI- channels, the majority of neurotransmitter actions appear to modulate the function of Ca2+ and K+ channels via phosphorylation/dephosphorylation (see Protein phosphorylation, field 32 and Receptor/transducer interactions, field 49, under various entries). Ca2+ channel modulation reflects the role of calcium ion not only as a principal depolarizing charge carrier, but also a ubiquitous second messengert (i.e. modulating neurotransmitter release, enzyme activation and other channels). Expression of a great diversity of voltagegated channel subtypes (most capable of independent modulation) permits 'fine tuning' of functions such as firing pattern, Ca2 + influx and resting potential. The precise effect of modulation is primarily determined by characteristics of voltage gating for each channel type. In general, activation of any K+ current will bring the cell closer to the K+ reversal potential t , which in most cell types is more negative than the resting potential. Thus activation of a K+ current (or its enhancement through neurotransmitter action) will

1'--_e_n_t_ry_4_1

I

----J_

depress excitabilityt, while interactions which decrease K+ currents will enhance cell excitability (see also INR key facts, entry 29).

Feedback Error-corrections, enhancements and extensions 41-57-01: Please notify specific errors, omissions, updates and comments on this 'key facts' entry by contributing to its e-mail feedback file. For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 41-01-03). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected"replacement according to the guidelines in Feedback etJ CSN Access. Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

REFERENCES 1 2 3 4

5 6 7

8 9 10

11 12 13 14

15 16 17

18 19 20 21

22 23

24

25 26 27

28 29

Jan, Cell (1989) 56: 13-25. Catterall, Trends Neurosci (1993) 16: 500-6. Honore, Fund Clin Pharmacol (1994) 8: 108-16. Isom, Neuron (1994) 12: 1183-94. Pusch, Physiol Rev (1994) 74: 813-27. Ertel, Drug Develop Res (1994) 33: 203-13. Jentsch, Chloride Channels (1994) 42: 35-57. Keynes, Q Rev Biophys (1994) 27: 339-434. Perez-Reyes, Drug Develop Res (1994) 33: 295-318. Catterall, Annu Rev Biochem (1995) 64: 493-531. Hille, Q TExp Physiol (1989) 74: 785-804. Salkoff, Trends Neurosci (1992) 15: 161-6. Conley, Meth Enzymol (1998) In press. Jan, Cell (1992) 69: 715-18. Miller, TBioi Chem (1992) 267: 1403-6. Catterall, Science (1988) 242: 50-61. Jan, Annu Rev Physiol (1992) 54: 535-55. Trimmer, Annu Rev Physiol (1989) 51: 401-18. Atkinson, Science (1991) 253: 551-5. Jan, Nature (1990) 345: 672. Hardie, Neuron (1992) 8: 643-51. Phillips, Neuron (1992) 8: 631-42. Sentenac, Science (1992) 256: 663-5. Anderson, Proc Natl Acad Sci USA (1992) 89: 3736-40. Miyamoto, T Gen Physiol (1992) 99: 505-30. Li, Science (1992) 257: 1225-30. Hille, Ionic Channels of Excitable Membranes, 2nd edn. (1992) Sinauer Associates, Sunderland, MA. Hodgkin, TPhysiol (1952) 117: 500-44. Almers, Rev Physiol Biochem Pharmacol (1978) 82: 96-190.

II

. . . Voltage-gated calcium channels

William

J. Brammar

Entry 42

NOMENCLATURES

Abstract/general description 42-01-01: Calcium-selective voltage-gated channel types are ubiquitous in excitable membranes. In general, unlike Na+ channels, voltage-gated Ca2+ channels are not rapidly inactivating, so that they are able to maintain inward currents for longer depolarizing responses. This electrical role of Ca2+ channels is important in secretory glands and endocrine organs, where Ca2 + channels dominate the electrical response and produce a prolonged depolarization to drive the maintained secretion. It is also important in cardiac and smooth muscle, where the longer depolarization is required to maintain the contraction. 42-01-02: Calcium channels regulate a wide range of cellular events as they can convert electrical into chemical signals (e.g. intracellular calcium flux and neurotransmitter release). Since resting intracellular Ca2+ concentration is low ("-110- 7 M, see ILG key facts, entry 14), only a small amount of Ca2 + influx is required to initiate signalling. In conjunction with cell-surface receptors and the intracellular Ca2 +-release channels (e.g. see ILG Ca InsPa, entry 19 and ILG Ca Ca RyR-Cal, entry 17), voltage-gated calcium channels initiate and control diverse cellular responses. These responses are often cell-type-specific and include secretion, metabolic adjustments, cell proliferation, contraction and control of gene expression. 42-01-03: Voltage-gated Ca2+ channels are of many types, including L-, T-, N-, P-, Q- and R-types, distinguished by biophysical and pharmacological criteria (see Gene family, 42-05-03). Although sequences encoding many different Ca2 + channel proteins have been cloned and characterized, the structural basis of Ca2+ channel diversity is still not fully understood, and the relationship between native currents and expressed genes is still being established. 42-01-04: Purification and immunoprecipitationt have shown that voltagegated Ca2 + channels are hetero-oligomerict protein complexes. The L-type Ca2 + channel of skeletal muscle, for example, consists of five subunits, 01, 02, {3, 8 and '"Y. The 01 subunit contains the pore t of the channel and is the main site of action of the L-type-specific Ca2 + channel agonistst and antagonistst. There are also various isoformst of the {3 subunit which differentially affect the electrophysiological behaviour of the channels produced by heterologoust expression of 0 subunit eDNAs. 42-01-05: L-type calcium channels, which are found in virtually all excitable and many non-excitable tissues, are re~ldily blocked by 1,4-dihydropyridines (e.g. nifedipine), phenylalkylamines (e.g. verapamil) and benzothiazepines (e.g. diltiazem). Three known 01 subunits, 0lS, 0lC and OlD, encoded by separate genes, are involved in different L-type Ca2 + channels: 0lS in skeletal muscle and 0lC and OlD in brain and heart.

II

I

entry42

1..-.---

_ _

42-01-06: N-type channels, expressed in neuronal tissue and associated with neurotransmitter release, are characterized by their high voltage activated (HVA) currents and their sensitivity to block by the cone snail neuropeptide omega-conotoxin GVIA (w-conotoxin GVIA or w-CTx). The activity of N-type channels is inhibited by the action of noradrenaline at pre-synaptic 02 receptors, via a G protein/second messenger system. The 01 subunit of Ntype Ca2+ channels has been identified as 0lB. 42-01-07: P-type channels, particularly prevalent in cerebellar Purkinje cells, activate over a range of potentials positive to -50mV and inactivate very slowly (tlj2 rv I s). They are sensitive to block by nanomolar concentrations of the peptide toxin w-agatoxin IVA from the funnel web spider, Agelenopsis aperta, and to micromolar concentrations of the polyamine FTx isolated from the same source. 42-01-08: T-type (low voltage activated, LVA) channels constitute a broad class of Ca2+ channels that transiently activate at negative potentials, typically in the range -70 to -50 mV, and are relatively sensitive to changes in resting membrane potential. T-type channels have been called 'fast' because of their rapid, voltage-dependent inactivation, but they close (deactivate) 10-100-fold more slowly than other voltage-gated Ca2+ channels. Their contribution to total Ca2+ current can be assessed from the amplitude of the slow component of deactivation ('tail') currents. It is a characteristic of T-type channels that they are less sensitive to classical Ca2+ channel agonists and antagonists than the high-threshold channel types. 42-01-09: The skeletal muscle and cardiac L-type Ca2+ channels are both essential components of excitation-contraction (E-C) coupling. In skeletal muscle, the L-type channel acts as the voltage-sensor that controls release of Ca2+ from the sarcoplasmic reticulumt in response to depolarization of the membrane of the transverse tubules, without needing Ca2+ entry across the sarcolemma. In contrast, the cardiac L-type channel functions only to permit entry of Ca2+ in response to depolarization, and the elevated Ca2+ concentration subsequently triggers the release of Ca2+ from the sarcoplasmic reticulum. Dihydropyridine-sensitive L-type channels are also present in brain, where the Ole subunit is the pore-forming component. 42-01-10: Mutations affecting Ca2+ channel subunits have been implicated in a number of genetic disorders. Mice with mdg mutations have muscular dysgenesis, in which lack of the 0lS subunit of the skeletal muscle L-type Ca2+ channel affects excitation-contraction coupling. Mutations at the mouse tottering (tg) locus affect the alA subunit of a P/Q-type Ca2+ channel and cause a neurological disorder resembling petit mal epilepsy in humans. The human autosomalt dominant t disease hypokalaemic periodic paralysis (hypoPP) is caused by mutations in the CACNL1A3 gene that encodes the alS subunit of the skeletal muscle L-type Ca2 + channel. Mutations in the human CACNL1A4 gene encoding an alA subunit of a P/Q-type Ca2+ channel have been identified in patients with familial hemiplegic migraine (FHM). Genetic alterations affecting the channel OIl} subunit have also been shown to be the cause of autosomal t dominantT cerebellar ataxia (see Phenotypic expression, 42-14-01 to 42-14-09).

II

_'--

e_n_try_4_2_1

42-01-11: Ca2+ channels are modulated by a variety of neurotransmitters and hormones, acting via G protein-linked receptors. In some cases the G protein interacts directly with the Ca2+ channel, without involvement of a diffusible second messenger. The inhibitory action of G proteins is selective for some channel types, the N-type and P/Q-type Ca2+ channels being the principal targets. This modulation of Ca2+ channels is important in pre-synaptic inhibition, because the inhibition of Ca2+ entry to pre-synaptic neurones blocks transmitter release. The channels are also modulated by phosphorylation by protein kinases A, C and G (PKA, PKC and PKG) and by phosphatase action. 42-01-12: The different voltage-activated Ca2+ channels are all activated by

depolarization, but they show marked differences in their sensitivity. Currents are potentiated by depolarizing pre-pulses, and this 'facilitation' involves voltage-dependent phosphorylation by PKA. 42-01-12: The binding sites for several different classes of Ca2+ channel

agonists and antagonists have been defined, and naturally occurring peptide toxins have been used to distinguish channel subtypes. Calcium channel antagonists cause vascular relaxation and are used in the treatment of hypertension, angina and cardiac and eerebral ischaemia.

Category (sortcode) 42-02-01: VLG Ca, Le. voltage-gated calcium channels.

Channel designation A unified nomenclature for voltage-gated Ca 2+ channels 42-03-01: A unified nomenclature for voltage-gated Ca2+ channels, based on rules that allow the description of the component subunits, has been proposed1 . The heteromerict channel is described as an a1x!3n'n.a28n complex, where X is a capital letter (5, A, B, C, D, E, etc.) that identifies the gene encoding the 0.1 subunit and n is a number (I, 2, 3, etc.) that identifies the gene encoding the other subunits. Note that the 0.2 and 8 polypeptides are encoded as a single polypeptide chain that is subsequently cleaved to give the two polypeptides that are disulphide-linked. Splice t variants are denoted by y, a lower case letter (Le. alA-a, a1A-h, !31a, !31h etc.). Where no second gene is known, such as for the 0.28 subunit, the capital letter or numerical subsc.ript is omitted. In this system, the skeletal muscle L-type Ca2+ channel, for example, has the composition a1s!31a,a2 8a1.

Current designation 42-04-01: ICa; I(Ca). Currents through the different types of Ca2+ channels are

distinguished by use of subscripts designating the channel-type: for example, current carried by Ca2+ through L-type channels is designated lCa,L.

II

II...-_e_ _ _ry_42 nt

_

Gene family The voltage-gated channel superfamily 42-05-01: Calcium channels form part of the voltage-gated channel superfamilyt, which also includes most sodium channels and some potassium channels (see VLG key facts, entry 41 and compare with VLG Na and VLG K series, entries 44 to 55).

The skeletal muscle Ca 2+ channel contains four protein subunits 42-05-02: Much information about Ca2+ channel structure has come from studies on the skeletal muscle isoform, which consists of a complex of four distinct protein subunits - 01, 02/8, (3, and, (reduction of disulphide bonds can release the 8 'subunit' from the 0.26 protein (see {PDTM}, Fig. 4).

Molecular characteristics of Ca 2+ channel subunits 42-05-03: A wide range of tissue-specific cDNAs encoding calcium channel subunits have now been cloned and sequenced (see Database listings, 4253-01). Basic characteristics of the distinct gene products are given in Table 1. More detailed information specific to each subunit under each field in this entry is indicated by the prefixes 0.1 subunit:, 0.2/6 subunit:, {3 subunit:, and 1 subunit: Because of the large number of cloned variants, these prefixes are sometimes extended with a gene isolate reference name and/or a bibliographic reference where appropriate, e.g. 0.1 subunit, gene rbC2 : (see also Homologous is oform s, 42-21). Further structure - function information for cloned subunits can also be found by reference to the 'primary sequence discussion' papers cited in the Database listings, 42-53.

Subtype classifications 42-06-01: See also Gene family, 42-05 and Homologous isoforms, 42-21. By electrophysiological and pharmacological criteria, there are several subtypes of voltage-dependent Ca2+ channels, distinguishable by their voltage dependence, time course of inactivation, and susceptibility to antagonists and toxins. Briefly, these have been described as L-type, T-type, N-type, Ptype60, Q-type and R_type61 , which for comparative purposes only are listed together within the fields of this entry. Where L, T, N, P, Q or R-channel type-specific data are cited, this is made clear by using an underlined subheading (e.g. 'L-type:') and subunit-specific data are indicated in context. The defining features of these channel types, based on electrophysiological/ pharmacological criteria are given in Table 2. Independent structural and genetic data will provide a more complete classification of these broad functional categories. Table 2 is annotated with cross-references to individual fieldnames which give more detailed information.

Trivial names 42-07-01: T-type Ca2+ channels can be activated by small depolarizations t from the resting potential t, so they are known as 'low voltage activated' (LVA) or 'low-threshold' Ca2+ channels. They are also called 'fast' channels

II

_L...-

e_n_try_4_2_

Table 1. General molecular characteristics of protein subunits encoded by distinct genes forming voltage-gated calcium channels - relationship of different nomenclatures (from 42-05-03) 01 subunit, class A, e.g. encoded by rat gene rbA 2 and human gene CACNL1A4

42-05-04: Brain class A and B al subunits resemble each other, but are less closely related to the al subunits forming putative L-channels. Fulllength class A al subunits do not show homology with class C: (/L-type') al subunits in the putative DHP-binding domain, indicating that alA subunits contribute to a DHP-resistant type of Ca2+ channeI3 - s . 42-05-05: Variants of class A sequences arising from alternative splicing t have been reporteds,6. Two alA cDNA sequences cloned from rabbit brain represent splice variants t that encode channel subunits differing in the Cterminalt sequence, beginning at amino acid residue 2230. The BI-l variant has 2273 amino acids, while the BI-2 isoform has 2424 residues. These two isoforms of the alA subunit, expressed alone or in conjunction with a2, f3 and, subunits, produce functional channels with no significant differences in electrophysiological properties5 . 42-05-06: The rat brain QlA subunit cDNA expressed in Xenopus oocytes results in rapidly activating Ba2+ currents (t==3.3ms) that peak after about 20ms and inactivate slowly (43.6% current remained after 400ms). The half-point for current activation (Vl/2act) is -l.OmV. The current is insensitive to Bay K8644 (10 J.lM) and wconotoxin GVIA (1 J.lM), but is partially (70%) blocked by w-conotoxin MVIIC (5 J.lM) and wagatoxin IVA (23% at 200nM). The alA current is also sensitive to blockade by Cd2+ (760/0 blocked at 10 J.lM). Co-expression t of the alA cDNA with cDNAs encoding f3 subunits increased the average size of the whole-cell Ba2+ current, without changing the rate of activation. While the presence of the f3lb and f33 subunits increased the inactivation rate of the Ba2+ currents, the f32a subunit decreased the inactivation, so that> 70% remained after a 400 ms pulse. In addition, all f3 subunits shifted the I-V relations of the QlA subunit to more hyperpolarized potentials 7 . The electrophysiological characteristics of the channels containing the alA subunit vary with the

II

1i--_e_n_t _ry_4_2

---'_

Table 1. Continued subunit, class A, e.g. encoded by rat gene rbA 2 and human gene CACNL1A4 a1

type of the associated (3- subunit. The channels formed by co-expression of cDNAs encoding Q1A and (32a have the characteristics of P-type currents, while the co-expression of Q1A and (31b or (33 cDNAs produces currents similar to Q-type currents of cerebellar granule cells 7. 42-05-07: 0.2/8 subunits can enhance class A 0.1 subunit activity following co-expression in Xenopus oocytes, which indicates the 0.2/8 subunit may be a structural component of DHPinsensitive Ca2 + channel class, and that the subunit structure of these may resemble that of L-type channels5 . at subunit: rv190kDa (skeletal muscle, low MW form, see Protein M~ below): derived from the 210 kDa precursor by proteolytic removal of a C-terminal peptide8 .

42-05-08: at subunit: rv2l0 kDa (skeletal muscle, high MW form, see Protein M~ below) at subunit: Shows striking homology to other cloned voltage-gated channels, including Na+ (300/0 homology) and K+ - see VLG key facts. at subunit: The skeletal muscle 0.1 subunit comprises four homologous internal repeats, each containing six predicted membrane spanning sequences (see PDTM). The fourth of each of these helices contains positively charged amino acids every 3 or 4 residues and is likely to represent the voltage sensor (see VLC key facts). 42-05-09: at subunit: The Q1 subunit contains binding sites for drugs such as dihydropyridines and phenylalkylamines that have been used to define L-channels pharmacologically (see Receptor antagonists, below). Electrophysiological studies have suggested that the DHP-binding site is localized at the outer surface of the 0.1 -subunit because DHP derivatives which carry net charge (i.e. are membraneimpermeable) only have access to the 'receptor' when applied extracellularly - e.g. 9 • Affinitylabeling experiments have indicated that the DHP-binding site is formed by a combination of the extracellular portions of the S6 helices of the third and fourth repeating domains, as well as the loop linking S5 and S6 of the third domain10,11.

II

_

entry 42

L...---

_

Table 1. Continued subunit, class A, e.g. encoded by rat gene rbA2 and human gene CACNL1A4 (}:1

subunit: stable heterologous expression in mouse L-cells results in formation of DHPsensitive Ca2+ channels12 in the absence of 0.2/8, {3 or , subunits. (}:1

42-05-10: (}:1 subunit: Electrically-stimulated contraction of skeletal muscle can occur in the absence of extracellular Ca2+ (due to the voltage sensor role in this tissue - see ILG Ca Ca RyrCaf). By contrast, cardiac 0.1 subunits exhibit functional L-type Ca2+ channel properties and are dependent upon extracellular Ca2+ for initating contractile events. 42-05-11: (}:1 subunit: Electrophysiological and pharmacological studies of the Ca2+ channels produced by co-expression of rabbit cDNAs encoding o.1A, {31 and skeletal muscle 0.2/8 subunits in Xenopus oocytes 13 show characteristics consistent with those of the Q-type channels of cerebellar granule neurones 14. (see Subtype classifications, below).

subunits, class B, e.g. encoded by rat gene rbB 2 (human gene CACNL1A5)

42-05-12: See also notes on class A 0.1 subunits. The o.IB subunit is a component of N-type Ca2+

subunits, class C, e.g. encoded by gene rbC (rat)2 (human gene CACNL1Al)

42-05-13: A full-length cDNA sequence cloned from rat brain cDNA2 encodes an 0.1 subunit (rbC-I) of 2140 amino acids that is highly homologous (95% identical) to the 0.1 subunit from rabbit cardiac muscle19 and also to other sequences from rabbit lunio. cDNAs encoding a variant 0. subunit, rbC.II, with three extra amino acids in the cytoplasmic loop between domains II and ID and 13 amino acid substitutions in a stretch of 28 amino acids in the S3 region of domain IV, were shown to arise by alternative splicing of transcripts from the same gene2. The same 13 amino acid changes in the rbC-II domain IV S3 segment are also found in the rabbit lun~o and rat aorta21 L-type channel 0.1 subunit.

(}:1

(}:1

II

channels that are sensitive to the cone snail toxin

w-conotoxin GVIA15,16. Transient co-expression of cDNA sequences encoding o.1B (BID), {31a and 0.2 subunits in a human embryonic kidney (HEK-293) cell line produced characteristic N-type Ca2+ channel activity17. N-type channels were also produced by expression of o.1B cDNA in myotubes from mdg mutant mice18.

entry42

I-

_

- - - - - - -

Table 1. Continued subunits, class C, e.g. encoded by gene rbC (rat)2 (human gene CACNL1Al) al

42-05-14: The basic electrophysiological and pharmacological properties of the cardiac and smooth muscle isoforms are similar2, except that Ba2+ currents through the smooth muscle al subunit are IO-times more sensitive to the DHP nisoldipine than those through the cardiac muscle 23 al subunit . Heterologous t expression of 'CaCh2B' cDNA encoding the alC 'smooth muscle' subunit in stably transformed Chinese hamster ovary cells yielded Ca2+ channels with kinetic and pharmacological properties very close to those of native channels in smooth muscle24. 42-05-15: Bmax values are enhanced for the skeletal muscle al subunits when co-expressed with a2/8 subunits following liposome reconstitution25. All expressed alC coding sequences result in highvoltage-activated Ca2+ currents showing minimal inactivation during a depolarizing pulse and sensitive to dihydropyridines.

al

subunits, class D, e.g. encoded by gene rbD (rat)2 (human gene CACNL1A2)

42-05-16: Full-length cDNA clones corresponding to class D have been isolated from rat brain26, human neuroblastoma15, human pancreatic (3 cells27 and a hamster insulin-secreting cellline28 . Expression of the human aID coding sequence, together with cDNAs encoding (32 and a2b subunits, results in dihydropyridine-sensitive L-type Ca2 + channels15, and it is presumed that class D al subunits may be archetypes of a 'neuroendocrine' form of L-type channel. (Expression of the sequence encoding aID alone, or co-expression with cRNA encoding a2b, did not generate functional Ca2+ channels in Xenopus oocytes15.)

subunits, class E, e.g. encoded by rat gene rbE29 (human gene CACNL1A6).

42-05-17: Full-length cDNA sequences encoding a class E al subunit have been isolated from rat (rbE-IT)29, rabbit (BIT-I, BII_2)3o, mouse and human brain31 cDNA libraries. The rbE-IT sequence encodes an al subunit of 2222 amino acids with a predicted molecular weight of 252 kDa with 53 % homology to class A and class B al subunits and 230/0 identity to class C and class D proteins. I Ba carried by the channels produced by transient expression of rbE-II eDNA in Xenopus oocytes first activated at -SOmV and peaked at around -IOmV. The currents were insensitive to Bay K

al

II

_L..-

,

e_n_try_4_2_1

Table 1. Continued 01 subunits, class E, e.g. encoded by rat gene rbE29 (human gene

CACNL1A6).

8644 (10 JlM), nifedipine (10 JlM) and w-conotoxin GVIA (1 JlM), but were partially blocked by wagatoxin IVll (33% block at 200 JlM) and sensitive to block by Ni2+ (IC so = 28 JlM) and Cd2+ (>80% block at 10 JlM Cd2+)29. The presence of the rat brain (31 subunit did not affect the whole-cell current or the rate of activation, but shifted the I-V relation and the voltage dependence of inactivation to more negative potentials29. Expression of the human Q1E coding sequence in HEK-293 cells and Xenopus oocytes produced rapidly inactivating (7 ~20ms at OmV) whole-cell currents that were enhanced about 40-fold by co-expression with sequences encoding human neuroneal Q2 and (3 subunits31 .

II

Ql subunits, class S, e.g. encoded by gene CaChl or CACNL1A3 (human)

42-05-18: The cDNA encoding the Q1 subunit ('Q1S') from rabbit skeletal muscle was the first sequence corresponding to a Ca2+ channel subunit to be cloned32 • The deduced protein of 1873 amino acids has a calculated Mr of 212018 and is encoded by an mRNA of 6.5 kb in rabbit skeletal muscle32 • The mRNA encoding the Q1S subunit has been detected in kidney and brain by sensitive peR-based techniques33 • The Q1 subunit is present in skeletal muscle as two size-variants, a full-length, minor (~S%) form of ~212kDa and a major (~9S%) species of ~ 190 kDa, derived from the longer protein by post-translationalt cleavage close to amino acid residue 16908 .

llccessory/regulatory subunits (intro)

42-05-19: Although a1 subunits are capable of forming Ca2+ channels by themselves, consistent co-purification of other, distinct proteins with a1 has determined they form part of a larger multisubunit native protein complex in cells (see [PDTM), Fig. 4). The origin and arrangement of these aI-associated subunits (a2/ 8, (3, ')') exemplified by the skeletal muscle Ca2+ channel is shown in Fig. 1.

02/6 subunit

42-05-20: Co-purifies with a1 subunit. From skeletal muscle, the ~17S kDa a2/8 subunit shifts to ~ 150 kDa (= a2) upon reduction t of disulphide bonds together with the appearance of the 8 proteins of ~2S kDa, 22 kDa and 17kDa35,36.

entry42

I-

_

- - - - - - -

Table 1. Continued

Figure 1. Subunits of the skeletal muscle L-type calcium channel protein complex. All subunits except f3 have hydrophobic domains and are predicted to be transmembranal or membrane associated. The Q;2 and 8 peptides are linked by disulphide bonds (S-S) and are cleaved from a common precursor. The Q;l, Q;2, 8 and ~ subunits are glycosylated. The molecular weights of the subunits of the L-type channel in skeletal muscle are Q;l = 175kDa; Q;2 = 143kDa; f3 = 54kDa; ~ = 30kDa and 8 = 27kDa34• (Redrawn from Dunlap (1995) Trends Neurosci 18: 89-98.) (From 42-05-13) 0.2/6 subunit

42-05-21: The Q;2/8 protein is the product of a single gene, with the Q;2 portion forming the Nterminal sequence (amino acids 1-934) and the 8 portion forming the C-terminal sequence (amino acids 935-1080)35-37 with a disulphide bridget linking them. 42-05-22: Both the Q;2 and the 8 portion of the Q;2/8 subunit are heavily glycosylated 35-37, e.g. the rabbit Q;2/8 ( rv 125 kDa) contains 18 consensus glycosylation sites and two cAMP-dependent phosphorylation sites38 (see positions under the Database listings, 42-53). Although the Q;2 subunit contains consensus phosphorylation sites, this subunit has not been shown to be phosphorylated in vivo. 42-05-23: mRNA sequences related to the skeletal muscle Q;2/8 subunit are expressed in cardiac and smooth muscle and the eNS, and antiskeletal

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_L-

e_n_try_4_2_

Table 1. Continued a2/6 subunit

muscle a2/8 antibodies precipitate the DHPbinding complex from both skeletal muscle and brain39. (Note: Channel proteins purified on the basis of high affinity w-CgTx (Le. those of the N-type) are inefficiently precipitated ("-1<10%) by these anti-a2/8 antibodies). The human15 and rat40 brain isoforms, encoded by splice variants t of the skeletal muscle coding sequence, contain an insertion of seven amino acids and a deletion of a 19 amino acid segment between transmembrane domains 1 and 2. 42-05-24: Both the a2 and 8 portions of the protein contain hydrophobic sequences, implying some association with the membrane. A model has been proposed which suggests that the 8 portion acts as a membrane anchor for the Q2 portion35,36. The Q2 subunit, but not the 8 peptide, can be extracted from the membrane at high pH under reducing conditions36, and anti-a2 antibodies label cultured sensory neurones without permeabilization of the cell membrane41 , consistent with the suggestion36 that the a2 subunit may be completely extracellular. 42-05-25: There is evidence that the a1 and a'1./6 subunits interact functionally as well as structurally. Ca2+ current amplitudes are significantly enhanced, and the kinetics are altered when Q1 is co-expressed with skeletal muscle a2/ 8 subunit in oocytes5,15,17,19,34,42,43. Note: Because of the diversity of the Q1 (and potentially other subunit structures), not all co-expression combinations may be expected to produce enhancements in channel currents. 42-05-26: Although the a2/8 subunit is normally produced coordinately with the aI, {3 and 'Y subunits, this is not necessarily always the case. Differentiating BC3Hl cells, which produce skeletal muscle L-type Ca2+ channels44, contain the mRNA for skeletal muscle aI, {3 and 'Y subunits but do not produce mRNA specific for the a2/8 subunit45 .

{3 subunits

42-05-27: "-158 kDa. Four main classes of {3

subunits from mammalian tissues have been characterized by cDNA cloning. According to the nomenclature of reference46, the {3 subunit genes

II

1L--_e_n_t_ry_4_2

-----"_

Table 1. Continued {3 subunits

are named numerically, with lower case subscripts being given to subunits produced by differential splicing. (See details of f3 subunits encoded under Homologous is%rms, 42-21).

42-05-28: Co-expressiont of sequences encoding f3 subunits with those encoding al subunits in Xenopus oocytes or mammalian cell lines results in increased currents, usually associated with a shift in the voltage dependence of activation to more hyperpolarizedt potentials. There are discernable differences in the magnitude of these effects with different combinations of al and f3 subunit isoforms. The microinjection of an antisense t oligonucleotide complementary to the mRNAs of all cloned f3 subunits into cultured rat sensory neurones produced a reduction in wholecell I Ba and a shift in the voltage dependence of activation to more depolarized potentials47. {31a subunits

42-05-29: The primary structure of the rabbit skeletal muscle {3 subunit, CaBI, has been determined from cDNA clones 48 . The open reading frame encodes a protein with 524 amino acids and a predicted molecular mass of 57,868 daltons. The CaBI subunit lacks a signal sequence, membrane-spanning domains or N-glycosylation sites, but has several potential sites for phosphorylation by protein kinase A, protein kinase C and cGMP-dependent protein kinase48 . The f3la subunit is predicted to contain four ahelical domains, three of which contain a heptad repeat t motif, similar to that found in cytoskeletal proteins. It has been suggested that the f3-subunit might link the Ca2+ channel to the cytoskeleton48. 42-05-30: RNA species hybridising to CaBI subunit cDNA sequences are detectable by Northern analysis in rabbit skeletal muscle (1.6 and 1.9kb) and in brain (3.0kb) RNAs48 . No such species were detected in RNA preparations from smooth muscle or heart48 .

{3lb subunit

42-05-31: An alternatively spliced isoform of the f3I subunit, alb, has been identified by cloning from rat brain49, human heartSO and human hippocampal51 cDNA libraries. These isoforms differ from f3la in having a deletion of 45-50

II

_L-.

,

e_n_try_4_2_

Table 1. Continued f3lh subunit

amino acids at residue 209 and in having a C-terminal elongation of rv120 amino acids.

f3lc subunit

42-05-32: A third splice-variantt expressed from the {31 gene, al cl has been isolated by cDNA cloning from human heart50 and hippocampal neurones 15,51. The {3lc variant has the same deletion as I~lh at residue 209, but has the same C-terminus as {3la. 42-05-33: The CaB2a Ca2+ channel {3 subunit is expressed in heart, aorta and brain52. The cDNA

f32a subunit

sequence cloned from rabbit heart predicts a protein of 606 amino acids and a molecular weight of 68,177. The homologous sequence has also been cloned from a rat brain cDNA library46. /32b subunit

42-05-34: An alternatively spliced coding sequence, CaB2b, cloned from rabbit heart eDNA, has an extra 26 amino acids within an altered N-terminal sequence52. The CaB2b isoform, expressed in heart, aorta and brain, is predicted to contain 632 amino acids and have a molecular weight of 70,943 52. Co-expression of CaB2a and CaBB2h with QIC 46,52 or QIA 7 in Xenopus oocytes altered the functional proprties of the QI subunits.

f32c subunit

42-05-35: A third splice-variant, f32c' has been cloned as an. incomplete cDNA sequence from rabbit brain52.

/32d subunit

42-05-36: The sequence encoding a fourth variant of the {32 gene-product, {32d, has been cloned from a rat brain cDNA library46. The {32d isoform contains 604 amino acids.

133 subunit

42-05-37: cL)NA sequences encoding a3 subunits have been cloned from rabbit heart52 and rat brain53 RNA. The predicted proteins contain 477 and 484 amino acids, respectively. Transcripts hybridising to CQB3 (rabbit brain) cDNA probes are most abundant in brain, but are also detectable in aorta, trachea, lung, heart and skeletal muscle52.

134 subunit

42-05-38: Sequences encoding a 134 subunit of 519 amino acids have been cloned from rat brain cDNAs54. The {34 mRNA is found almost exclusively in neuronal tissues, with the highest levels in the cerebellum. The {34 subunit can interact with QIC and QIS subunits to increase

III

Il...--e_n_t_ry_42

---'_

Table 1. Continued

/34 subunits

Ca2+ channel activity following production in Xenopus oocytes54 . 42-05-39: Co-expression t of al and f3 subunits in Xenopus oocytes43 or mouse L cells 12 can generate Ca2+ currents with normal characteristics, including peak current amplitude, voltage-dependence and kinetics of activation and inactivation. In some combinations, however, co-expression of cDNAs encoding f3-subunits with those specifying al subunits can markedly affect the properties of the al subunit. In Xenopus oocytes, I Ba due to expression of cRNA encoding Ca2+ channel alC subunits is increased 20-fold by co-expression of f32a (CaB2a), 6-fold by that of f32b (CaB2b) and 30-fold by co-expression of f33 (CaB3)52. In all cases involving the al subunit, IBa is stimulated by Bay K 8644 (0.5 mM), but the combinations involving f3 subunits are less sensitive to stimulation than the al channel52. The presence of f3la or f3lb subunits stimulates whole cell currents produced by rabbit brain alA subunits expressed in Xenopus oocytes by about 20-fold. The combined presence of f3l and a2 subunits enhances these currents 200fold 5. 42-05-40: The purified skeletal muscle L-type Ca2+ channel contains a , subunit of 30 kDa55 that is glycosylated56 . Sequences encoding the voltage-gated Ca2+ channel, subunit have been cloned from rabbit57 and human58 skeletal muscle eDNA libraries. The proteins contain 222 amino acids (rv 25 kDa) and have four putative transmembrane regions. The corresponding mRNAs were detected in skeletal muscle and lung57, but not in brain, cardiac muscle, spleen, kidney, liver, or stomach58 . 42-05-41: The skeletal muscle, subunit does not affect the properties of the alC subunit59, but does increase whole-cell currents and the rate of activation of channels constituted from alC and f3la subunits59.

II

_ entry 42

--------Table 2. Basic classification of voltage-gated calcium channels from electrophysiological and pharmacological criteria (from 42-06-01)

II

Channel type

Characteristics of channel

L-type:

L for long-lasting. Voltage-dependent Ca2+ -selective L-channels (isoforms LI, L2, L3, L4 - Q1, Q2, {3, 1, and 8 subunits identified). See 'L-type:' prefix in other fields for main characteristics. -Wide distribution, well characterized in heart, smooth and skeletal muscle and some neurones. Highly sensitive to the dihydropyridines, phenylalkylamines and the benzothiazepines. Consists of five subunits, known as Q1, Q2, {3, 1 and 8. The 8 subunit is linked by disulphidet bonds to the (}2 subunit. The Q2 subunit is a la.rge glycoprotein, which does not contain any known drug-binding sites. The L-type Q1 subunit has an apparent size of rvl65 kDa from SDS-PAGEt. The Q1 subunit spans the membrane and consists of four large repeated regions, each containing six putative membrane-spanning domains (see {PDTM}, Fig. 4). There are at least four isoforms of the L-channel, with different susceptibilities to calcium antagonists. L-type channels have important functions in both excitation-contraction and excitation-secretion coupling. The L-channel expressed in skeletal muscle functions as a voltage sensor for caleium release from the sarcoplasmic reticulum (see ILG Ca Ca RyR-Caf, entry 17) - cf. to the cardiac L-type channel, which conducts Ca2+ capable of directly activating contractile proteins or indirectly as a trigger for Ca2+ release from the sarcoplasmic reticulum. Cardiac and skeletal rnuscle isoforms of the L-type channel are encoded by separate genes. Second messenger systems play important modulatory roles for the L-type channel, particularly in the heart. Typical conductance ,,,25 pS with 100 JlM Ba2+ as charge carrier. High-voltage activation (Vpeak rv +20mV), long-lasting current, relatively slo'w inactivation.

N-type:

N for neuronal, neither L nor T. Voltage-dependent Ca2+selective N channels. Found specifically in neurones, and correlated with control of neurotransmitter release, N-type channels are blocked by omega-conotoxin GVIA (w-CTx), a neuropeptide toxin isolated from the marine snail, Conus geographus. See 'N-type:' prefix in other fields for main characteristics. - - -

l_e_n_t_ry_42

_

Table 2. Continued Channel type

Characteristics of channel

N-type:

Typical conductance rv12-20pS with 100 JlM Ba2+ as charge carrier. Sensitive to w-conotoxins. High-voltage-activated current with moderate rate of inactivation. Although predominantly voltage activated (responding with neurotransmitter release following depolarization of nerve terminals), Ca2+ flux through the N-type channel is modulated (inhibited) by binding of noradrenaline to pre-synaptic a2 receptor, linked through a G protein/second messenger system. Expressed in neuronal tissue, associated with synaptic core complex t and involved in neurotransmitter release. Also found in endocrine cells from the anterior pituitary, pancreas and adrenal gland. The purified N-type channel consists of aI, a2- 8 and {3 subunits analogous to those of L-type channels (see above). A 95-100kDa subunit of unknown identity and function copurifies with the N-type channeI62- 64.

P-type:

P for high density in cerebellar Purkinje cells65 • Voltagedependent Ca2+ -selective P channels. High-threshold voltage-gated Ca channel. Activated by moderately high voltage and insensitive to DHP, blocked by w-Aga-IVA, a peptide toxin from the funnel web spider66, and by FTx, a low molecular weight polyamine from the same source67. See IP-type:' prefix in other fields for main characteristics. Typical conductance rv 10-12 pS with 100 JlM Ba2+ as charge carrier. Sensitive to the funnel web spider toxins w-Aga-IVA and FTx. Moderately high-voltage activation, non-inactivating. Expressed in many central and some peripheral neurones. As detected by sensitivity to block by w-Aga-IVA, present in neurones from (rat) hippocampal CAl and CA3 regions, visual cortex, spinal cord and dorsal root ganglia: absent from rat sympathetic neurones 68 • Involved in excitation-secretion coupling at glutamatergic t pre-synaptic nerve terminals, as judged by sensitivity to w-Aga-IVA69. (see Blockers, 42-43).

Q-type:

Voltage-dependent Ca2+ -selective Q-channels. Insensitive to w-conotoxin GVIA (1 JlM), w-agatoxin IVA (30nM) and nimodipine (5 JlM), but completely blocked by w-conotoxin MVIIC (1.5 JlM) and by 1 JlM w-Aga-IVA13. (Note that w-conotoxin MVIIC also blocks N-type and P-type channels, see above).

II

_'--

,

e_n_try_4_2_

Table 2. Continued Channel type

Characteristics of channel Expressed in cerebellar granule neurones 70, hippocampus 61 . Sensitive to activation by treatments that activate protein kinase C61 (see Protein phosphorylation, 42-32), and to inhibition by agonists of metabotropic t glutamate, ,-aminobutyric acid type B, adenosine and acetylcholine receptors 61 .

R for resistant. Voltage-dependent Ca2+ -selective R-channels 70. No selective blockers are known; the R-type channels are resistant to w-Aga-IVA, w-CTx-GVIA and w-CTx-MVllC. Show rapid (7 = 20-30 ms) voltage-dependent inactivation. T for transient. Voltage-dependent Ca2+-selective T-channels. See IT-type:' prefix in other fields for main characteristics. - - Typical conductance rv8 pS with 100 JlM Ba2+ as chargecarrier. Low voltage activated (Vpeak rv -30mV) transient current. Rapid (7 = 20-S0ms) voltage-dependent inactivation. Concentrated in conducting system of heart (sinoatrial node, atrioventricular node), smooth muscle, endocrine cells and some neurones. In the mature mammalian brain, they are prominent in thalamic 71,72 and hypothalamic 73 neurones. T-type channels influence spontaneous firing activity. Inhibition of T-type currents in rabbit sino-atrial node cells with 40 JlM Ni2+ induces bradycardia t74 . T-type channels are expressed in mammalian spermatogenic cells and retained in mature sperm, where their function is required for the acrosome reaction prior to sperm-egg fusion 75 . Other Ca2+ channel types

II

Compare to: intracellular Ca2+ -release channels (ILG Ca InsPg, entry 19); receptor-operated calcium channels (e.g. see ILG Ca AA-LTC4 , entry 15); excitatory amino acid receptor-linked channels (e.g. see ELG CAT series, entries 20 to 22) and mixed cation conductance channels which may pass a Ca2+ component (e.g. see ILG CAT cGMp, entry 22).

l_e_n_try_4_2

_

because of their rapid inactivation. In various preparations they are also called type I or slow-deactivating (SD) currents. L-type, N-type, P-type and Q-type channels are all 'high-threshold' or thigh voltage activated' (HVA) channels, requiring larger depolarizations for activation. Such currents are sometimes designated 'type II', 'slow', 'persistent' or 'fast-deactivating' (FD). L-type channels have been subdivided by some authors into Lm (expressed in skeletal muscle), Lh (heart and smooth muscle) and Ln (neuronal) types (see ref. 76, for example). In the light of more recent information about the sites of expression of the genes encoding Ca2+ channel subunits, this is not a helpful nomenclature and could be misleading.

EXPRESSION

Cell-type expression index Voltage-sensing function of Ca 2+ channels in skeletal muscle transverse tubules 42-08-01: The L-type Ca2+ channels of skeletal muscle transverse tubules have a voltage-sensing function (see ILG Ca Ca RyR-Caf, entry 17). In cardiac muscle and most smooth muscle, calcium influx through L-type Ca2+ channels triggers a larger Ca2+ -induced Ca2+ release from the sarcoplasmic reticulum and initiates contraction. The L-type Ca2+ channels of skeletal muscle show activation kinetics >100-fold slower than those of other L-type Ca2+ channels and would not open during the 2 ms action potential of a skeletal muscle. Extracellular Ca2+ ions are not needed for excitation-contraction coupling in skeletal muscle, because the function of the L-type channel is to act as the voltage sensor for the ryanodine receptor in the SR membrane 77. Skeletal muscle from mouse embryos homozygous for mdg (muscular dysgenesis) mutations lack the al subunit of the skeletal muscle L-type Ca2 + channel 78 and are defective in excitation-contraction coupling: they make action potentials but cannot twitch. Expression of cDNA encoding wild-type t a1 subunits restores dysgenic myotubes t to the normal phenotype 79,8o (See Phenotypic expression, 42-14-01).

Several types of voltage-gated Ca 2+ channel govern exocytosis from presynaptic terminals 42-08-02: Neurotransmitter release involves exocytosis t of vesicles in presynaptic nerve terminals, a process that requires Ca2+ influx through voltage-dependent Ca2+ channels. Exocytosis can be studied directly by fluorescence-imaging techniques following the introduction of the fluorescent styryl dye FMI-43 into pre-synaptic vesicles 81,82. Unloading of the dye from single synapses during electrical stimulation depends on the propagation of action potentials and is completely blocked by tetrodotoxin (1 JlM). The effects of various Ca2+ channel blockers on release of FMl-43 have been studied with rat hippocampal neurones in culture. Individual synapses respond differently to the blockers, implying considerable heterogeneity of Ca2+ channel distribution. Although blocking N-type channels inhibited about 60% of overall FMI-43 release, the data indicate

II

_"--

e_n_try_4_2------1

that L-, N-, P- and other channel types participate in exocytosis in synaptic boutons83 . The effects of w-conotoxin GVIA and w-agatoxin VIA on postsynaptic currents also demonstrate the presence of at least N-type and Ptype Ca2+ channels in pre-synaptic nerve terminals in the rat central nervous system84 .

Synaptic transmission at hippocampal CA3-CAl synapses involves Q-type channels 42-08-03: Selective blockade of voltage-gated Ca2+ channels has been used to determine the channel types involved in synaptic transmission between rat hippocampal CA3 and CAl neurones in brain slices. The use of wconotoxin GVIA (w-CTx-GVIA) (1 f.lM) to block N-type channels caused a rapid but incomplete depression of synaptic transmission (average inhibition of 46%). Specific blockers of L-type (nimodipine, 5 f.lM) and Ptype (w-agatoxin IVA, 30nM) channels had no effect, but transmission was reversibly eliminated by removal of external Ca2+ ions. Application of wconotoxin MVIIC (5 f.lM), a blocker of N-type and Q-type channels, eliminated synaptic transmission, both in the presence and the absence of w-CTx-GVIA, supporting the contention that N-type and Q-type channels entirely account for the Ca2+ currents required for synaptic transmission in these neurones 61 . Note that this conclusion has been both criticized as premature85 and further supported86 • (See Channel modulation, 42-44, for effects of neuromodulators on Q-type channel function).

L-type channels in cochlear hair cells 42-08-04: Voltage-gated calcium channels from chick cochlear hair cells have

the characteristics of L-type Ca2+ channels, including sensitivity to dihydropyridines and insensitivity to the peptide toxins w-Aga-IVA, w-CTx-GVIA and w-CTx-MVTIC. No differences in kinetics or voltage dependence of activation of I Ba were found between tall and short hair cells. These L-type channels are responsible for processes requiring voltage-dependent calcium entry through the basolateralt cell membrane, such as transmitter release and activation of Ca2+ -dependent K+ channels87•

P-type channels are expressed in small cell lung carcinoma cells

42-08-05: A partial cDNA clone t sharing extensive nucleotide identity with sequences encoding a P-type calcium channel 01 subunit has been isolated from a small cell lung carcinoma (SCLC) cell line. Anti-peptide antibodies generated to a unique acidic region in the IVS5-S6 linker of the putative SCLC P-type channel reacted specifically with a cell surface molecule in SCLC cells and inhibited calcium currents in SCLC cells, measured by whole-cell patch clamp t. Similar calcium currents were also inhibited by the P-type channel-specific toxin, w-Aga-IVA. The inhibitory effects of the antibody and the toxin were not additive, consistent with their acting on the same type of channel88 . (The expression of neurone-related P-type channels by SCLC cells is of interest because small-cell lung carcinoma is frequently associated with paraneoplastic t disorders affecting the nervous system.)

1L.-_e_n_t _ry_4_2

----'_

Channel density L-type channels are more abundant than T-type channels in ventricular myocytes 42-09-01: The functional density of L-type channels in guinea-pig ventricular myocytes89, canine cardiac Purkinje cells 90 and smooth muscle myocytes 91 is in the range 1-5/J.1m2. This density is approximately ten times higher than that of T-type channels in guinea-pig ventricular myocytes (0.1-0.3/J.1m2)92. In a rat adrenal medullary tumour cell line, PC12, estimates of the number of L-type channels from agonist- or antagonist-binding assays, 1200-6000 binding sites per ce11 93,94, are in good agreement with those from electrophysiological measurements of peak Ca2+ currents (2500 channels/ceI195 ).

Relative densities of L-type and T-type channels vary with cell type 42-09-02: The relative densities of L-type and T-type channels can be assessed from the maximum current amplitudes. In guinea-pig ventricular myocytes the T-type current amplitude is generally <10% of L-type current at their optimal voltage ('Vpeak'). This ratio can vary very widely in myocytes from other tissues and species. Whole-cell T-type currents can be nearly as large or larger than L-type currents in cultured rat aorta 96 or portal vein 97, for example, while T-type current has been unsuccessfully sought in myocytes from many different tissues (reviewed in reference 98 ). The density of RyR 'foot' structures, which form a complementary 'checkerboard array' with voltage-sensing L-type Ca2+ channels, has been estimated at 700 per J.1m2 (see ILG Ca Ca RyR-Caf, entry 17).

Density of N-type channels in bovine chromaffin cells 42-09-03: Bovine chromaffin cells contain DHP-sensitive L-type channels, wconotoxin (w-CTx)-sensitive N-type channels and a presumed P-type Ca2+ channel that is insensitive to both DHP and w-CTX 99 . Knowledge of the unitary channel current ("-10.3 pA) and open probability ("-10.15) of the wCTx-sensitive channel, together with measurements of whole-cell currents sensitive to w-CTx ("-I800pA), allow the estimation of "-118000 w-CTxsensitive channels per chromaffin ce11 99.

Evidence for the presence of a mobilizable intracellular pool of N-type channel proteins 42-09-04: The binding of 125I-w-conotoxin to human neuroblastoma cells, a measure of the density of N-type Ca2+ channels, continued to increase for 6-8h at 37°C, reaching seven-fold higher than that observed at 4°C. There was no change in the Ko for w-conotoxin (10-20pM) during this increase. The increase in w-CTx-binding sites was energy dependent (sensitive to 100 J.1M NaNa ), but resistant to inhibitors of protein synthesis. Blockers of exit from the Golgi apparatus t (10 J.1M brefeldin A) and microtubule t based vesicular transport (20 J.1M nocodazole) reversibly inhibited the increase in toxin-binding sites. The number of w-CTx-binding sites in cells incubated with 10 J.1M brefeldin A decreased with a half-life of 14.5 hr10o. The permeabilization of paraformaldehyde-fixed cells with 0.01 % Triton X-100 produced a three-fold increase in the number of CTxbinding sites, due to the presence of an intracellular pool of binding sites.

III

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2

----J

The measurable intracellular pool could be decreased three-fold by incubation with unlabelled w-CTx (lOOpM) for about 9h. Blockage of N-type Ca2+ channels with Cd2+ also produced a time- and dose-dependent increase in the number of surface w-CTx-binding sites, but the blockage of L-type channels had no such effect. Patch-clamp measurements showed that the induced increase in w-CTx-binding sites correlated with an increase in the number of functional Ca2+ channels per cell10o. The precise intracellular location of the internal pool of channel proteins has not been determined.

f3 Subunits do not affect channel density 42-09-05: It has been suggested that the presence of {3 subunits increases the number of functional Ca2+ channels in the plasma membrane, based on increases in the number of binding sites for w-conotoxin17 and for dihydropyridines101 . Co-production of QIC and {3la in stably transfected Chinese hamster ovary cells does produce an increase in dihydropyridine-binding and whole-cell current, but there is no corresponding elevation of the amount of QI subunit mRNA or immunologically detectable QI protein at the cell surface101 . In sympathy with these findings, there was no increase in the total gating charget in Xenopus oocytes producing QIC and {31 subunits compared with that obtained from oocytes expressing only QIC cRNA102. These observations support the conclusion that the {3 subunit does not affect the number of functional channels in the membrane, but alters the conformationt of the QI subunit, affecting both the opening probability and ligand-binding properties of the channell 01, 102. The presence of {31 subunits has been shown to increase the single-channel open probability of channels containing human QIC subunits103.

Cloning resource 42-10-01:

" 2 104; aiD, QIS, skeIetaI museIe32; ale, card"lac museIe19, Iunb,.20, brain' brain4,16,26, neuroblastoma cell line15, insulin-secreting cell line28, pancreatic islets27. N-type: cDNAs encoding the alB subunit of the N-type channel have been cloned from rat16 and human 16,17 brain libraries. P-type: alA: Cerebellar Purkinje cells4,5; human cerebellum eDNA libraryl05; rat phaeochromocytoma PC12 cells 106. Q-type: Cerebellar granule cells. T-type: The subunits constituting T-type Ca2+ channels have not yet been identified, and there is no description of cloning the sequences that encode them (see note added in proof). L -type..

Genomic clones covering the human CACNLIA4 are available on request 42-10-02: The human gene (CACNL1A4) encoding the alA subunit of a P/Qtype Ca2+ channel has been isolated on a set of ten overlapping cosmidt clones t ('contigt'). The set of ordered and restriction-mapped clones is available from the Lawrence Livermore National Laboratory (LLNL), BBRP, Livermore, California 94550, USA105.

II

l_e_n_t_ry_42

_

Developmental regulation Changing patterns of expression in developing neurones 42-11-01: Calcium currents are present in most developing embryonic preparations; after they appear, net calcium currents are generally stable or increase. In developing neurones, decreases in LVA T-type currents that are present at very early stages appear to be compensated by increases in HVA N-type and L-type currents (reviewed in ref.l07; see also this field under VLC K DR, 45-11).

The level of skeletal muscle

0',1

subunit increases post-natally

42-11-02: The levels of al subunit polypeptide detected with monoclonal antibodies are quite low in rat skeletal muscle during the first 10 days after birth, but then increase dramatically to approach those found in adult muscle by day 20. In contrast, the substantial amount of a2 subunit in rat skeletal muscle at birth increases only slightly during this same period of development108.

A two-motif isoform of the skeletal muscle developmentally regulated

0',1

subunit is

42-11-03: Two species of mRNA encoding al subunits of the voltage-regulated Ca2+ channel are detectable in skeletal muscle of newborn and adult rabbit: a 6.5 kb species that predominates in adult muscle and encodes the full-length al subunit, and a species that is 2 kb shorter due to an internal deletion. The deletion does not disturb the reading-framet, and the shorter mRNA encodes an al subunit with the IIS2 domain fused to the IVS2 domain, so that it has only two of the homologoust repeat regions. Westernt blotting data show that the shorter isoform predominates in newborn skeletal muscle, while the longer isoform is prevalent in adult tissue 109.

Alternative splicing of 0',1 subunit mRNA is developmentally regulated in heart 42-11-04: Two distinct cDNAt species corresponding to mRNAs encoding Ltype Ca2+ channel al subunits have been cloned from both rat21 and rabbit 19 cardiac tissue. In both cases, the predicted protein sequences differ only in the third membrane-spanning region of the fourth motif (IVS3). Cloning and sequencing of the rat gene encoding the al subunit has shown that the alternative IVS3 sequences arise from separate, adjacent exonst, by mutually exclusive alternative splicing t 110. The splicing pattern is developmentally regulated: cardiac musculature from neonatal rats expresses both species, whereas al subunit mRNA from adult cardiac tissue contains only the second of the two exons110.

Ca 2+ influx activates transcription of immediate early genes 42-11-05: Influx of Ca2+ through the L-type Ca2+ channels and NMDA receptors of hippocampal neurones has been shown to regulate gene transcriptionalt events in the nucleus 111 . Activation of multifunctional Ca2 +calmodulin-dependent protein kinase (CaM kinase) is evoked by stimulation of either NMDA receptors or L-type Ca2+ channels but is

II

_ _ _ _ _ _ _ _ _ _ _ _ _,

e_n_try_4_2

-----J

critical only for propagating the L-type Ca2+ channel signal to the nucleus. The NMDAR and L-type Ca2+ channel pathways activate transcription by means of different cis-actingt regulatory elements for the promotert of the immediate-earlyt gene c-fOSt 111 . Note: Among known calcium-responsive genest are those involved in the growth and proliferative responses of vascular smooth muscle cells. Cytoplasmic calcium elevations (via cellular influx and intracellular release) have roles in activating a range of cellular functions including smooth muscle contraction and growth factor release (reviewed in ref.112).

T-type currents are associated with growth 42-11-06: The flexor digitomm brevis muscle fibres in newborn mice show both T-type and L-type Ca2+ channel currents. The T-type current, 2.7 A/F on day I, decreases progressively after birth, becoming undetectable by day 17. In contrast, the specific amplitude of the sustained (L-type) current increases four-fold from day 1 (6.9A/F) to day 30 (27.7 A/F). Denervation of muscle fibres after birth does not influence the disappearance of the transient current, but suppresses the increase of the sustained current. The disappearance of the T-type current is delayed by isolation and in vitro culture of muscle fibres. In fibres isolated after the transient current had become undetectable, subsequent in vitro culture caused the transient current to reappear, while the level of the sustained current was maintainedl13 .

Endothelin enhances T-type Ca 2+ currents 42-11-07: Endothelin-l (ET-l), a 21 aa vasoconstrictive t peptide, increases intracellular Ca2 + levels and has hypertrophic action on ventricular myocytes. The effects of ET-l on L-type and T-type Ca2+ currents in cultured neonatal rat ventricular myocytes have been determined by patchclamp techniques. ET-l (10nM) increased the maximum current density of ICa,T from -3.0 JlA/cm2 to -4.4 llA/cm2 • This enhancement by ET-l was dose dependent, with the maximal response at approximately 10nM and a half-maximal dose of 1.3 nM. The stimulation of ICa,T was antagonized by protein kinase C inhibitors staurosporine (0.2 JlM) and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7, 20 JlM) in the pipette solution. Extracellular application of phorbol esters, activators of protein kinase C, also increased the maximal current density of ICa,T' an effect that was blocked by H-7 (20 JlM) in the pipette solution. The application of ET-l had no significant effect on Ica,L in this system l14 .

NGF induces expression of genes encoding Ql and {3 subunits in PC12 cells 42-11-08: The treatment of the rat phaeochromocytoma cell line PC12 with nerve growth factor (NGF; 50 ng/ml) induces morphological changes, including the development of neurites. Such treatment increased the level of mRNA transcribed from the genes encoding the alA, alB, alC and aID subunits within a dayl15. In experiments in which the level of alB subunits permg of membrane protein was increased by rv59%, the relative association of alB with specific /3 subunits (74% with /33, 21 % with /32) was not changed by NGF treatment106.

III

1L...-_e_n_t _ry_42

--"_

mRNA distribution Distribution of rbC-I and rbC-II mRNAs 42-13-01: The distributions of mRNAs encoding specific subunits of Ca2+ channels are summarized in Table 3.

The rat phaeochromocytoma cell line (PC12) expresses several voltage-gated Ca 2+ channels 42-13-02: Undifferentiated rat phaeochromocytoma (PCI2) cells display Ltype, N-type and P/Q-type Ca2+ channels, as shown by patch-clamp studies in the presence of specific channel blockers 106,119,120. The mRNAs encoding alC, alB, alA, /31, /32 and /33 subunits are detectable by RT-PCRt analysis and sequencing106.

Phenotypic expression Pathology: mutations affecting

alS

cause muscular dysgenesis

42-14-01: mdg mutations for muscular dysgenesis affect excitationcontraction (E-C) couplingt in mice such that action potentials are produced but the muscle lacks the DHP-sensitive slow lea and does not twitch. Dysgenic myotubes lack the alS subunit of the skeletal muscle Ltype Ca2+ channel78. Homozygous lethal mutant primaryt myotubes t can be restored to normal phenotype by nuclear injection of mammalian expression vectorst expressing wild-typet alS subunit mRNA 79,80. Expression of a cardiac calcium channel in skeletal muscle cells results in E-C couplingt resembling that of cardiac muscle121, but expression of BI (alA) Ca2+ channel cDNAs, cloned from rabbit brain, does not122. Dysgenic myotubes injected with BI cDNA constructs expressed high densities of Ca2+ channel currents (average 31 pA/pF) but did not spontaneously contract and only very rarely displayed evoked contractions122.

Lambert-Eaton syndrome involves autoantibodies to pre-synaptic Ca 2+ channels 42-14-02: The pathology of the autoimmunet disorder Lambert-Eaton myasthenic syndrome (LEMS) has been linked to the presence of antibodies binding to pre-synaptic Ca2+ channels. This causes the channels to cluster, leading to their down-regulation, with reduced release of acetylcholine and consequent muscle weakness123. Plasma from LEMS patients contains antibodies that immunoprecipitate N-type Ca2+ channels from solubilized rat brain synaptic membranes124. The specific antigen with which these antibodies react is a 58 kDa protein, identified as the synaptic vesicle membrane protein, synaptotagmin, by molecular cloning of cDNAs from an expressiont library124. A tight association between synaptotagmin and Ntype Ca2+ channels may play a role in docking synaptic vesicles at the plasma membrane, bringing the vesicles into ready accessibility with incoming Ca2+ ions. It is suggested that circulating antibodies in the serum of LEMS patients bind to the synaptotagmin-Ca2+ channel complex and interfere with neurotransmitter release 124.

II

_L...-

en_try_4_2_

Table 3. Distributions of MRNAs encoding specific subunits of Ca 2+ channels (From 42-13-01) mRNA

Tissue type and detailed localization

rbA

42-13-02: Cerebellar Purkinje cells: the alA mRNA is most concentrated in the Purkinje cell bodies, with less in the granule celllayer 7 . 42-13-02: The alA mRNA is also prominent in many regions of the rat CNS, including the dentate gyms and CA fields of the hippocampus, the cerebellar cortex, the pontine nucleus, the mitral cell layer of the olfactory bulb and tubercle and the cerebral cortex layers 11-VI 7. More moderate levels of alA mRNA are detected in the striatum, tuberal hypothalamus, substantia nigra, red nucleus, lateral reticular nucleus and inferior olivary nucleus. Low levels of alA mRNA could also be detected in the suprachiasmatic nucleus, supraoptic nuclei, medial habenula, pre-mammillary nuclei, interpeduncular nucleus, oculomotor nucleus, dorsal raphe nucleus, pineal gland, hypoglossal nucleus, dorsal motor nucleus of the vagus, facial nucleus and the nucleus of the solitary tract 7 • The pattern of alA mRNA detection in mouse brain by in situ hybridization has been describedl16 .

II

rbB

42-13-04: The expression of (:tIB is limited to the nervous system and neuronally derived cell lines. Northern blottingt detects a single major mRNA species of "-JIO kb in all regions of the rat CNS, including the spinal cord16. More sensitive PCR-based analysis failed to detect alB mRNA in any rat tissue other than the CNS, but did detect the mRNA in calcitonin-secreting C cell lines that originate from the neural crest, and in rat phaeochromocytoma (PCI2) cells before and after differentiation induced by nerve growth factor 16 . (There is an exact correspondence between alB (rbB-I) mRNA and N-type Ca2+ currents in the cell lines analysed, and no correlation with L-type or T-type currents.)

rbC

42-13-05: Sequences encoding alC subunits have been cloned from rabbit cardiac muscle19, rabbit lunio, rat brain2 , rat aorta21 and mouse brain104. Northern blott analyses detected two alC mRNA species in rabbit heart, brain and stomach19, and in rat heart, brain and smooth muscle of the uterus, lung, stomach, small and large intestine21 . The rat brain rbC: (alc) probe detected 8 and lOkb mRNA species representing two splicet variants that encode the rbC-I and rbC-II isoforms. Although the two variants of the mRNA are expressed in all regions of the rat CNS, with the rbC-II transcript being the more prevalent, the relative amounts of the two mature mRNA species vary between regions of the brain. There is significantly more rbC-II mRNA in the olfactory bulb, cortex, hippocampus, hypothalamus and striatum, approximately equal amounts in the cerebellum and pons/medulla, and a

---J_

IL-_e_n_t _ry_4_2 Table 3. Continued mRNA

Tissue type and detailed localization

rbC

predominance of rbC-I mRNA in the spinal cord and the trigeminal nerve. The rbC gene is also transcribed in non-neuronal tissues, including heart, pituitary, adrenal gland, liver and kidney, and at low levels in the testes and spleen. In all cases, both splice variantst of the rbC mRNA are detectable, with rbC-II mRNA usually being predominant, except in the pituitary, where rbC-I mRNA is the more prevalent2 . Note that the rbC-II variant represents the majority of the rbC mRNA in the heart, despite the fact that the first published cardiac Ca2 + channel sequence19 was derived from the rbC-I mRNA.

rbD

42-13-06: The human aID (CACN4) eDNA probe detected a single rv 11 kb mRNA species in rat brain and pancreatic islets, but failed to detect mRNA in human skeletal muscle, heart, kidney, spleen, liver, jejunum and colon27. High levels of aID mRNA were detected in the insulin-producing cell line RINm5F and a lower level in the ,BTC-3 cell line. ISH: detected aID mRNA in ,B cells of rat pancreatic islets27, most regions of rat brain, including olfactory bulb, cerebral cortex, hippocampal CA fields and dentate gyrus, cerebellar Purkinje cells, suprachiasmatic nucleus, pituitary gland and pineal gland l17. Human hippocampus and basal ganglia, habenula and thalamus, plus a human neuroblastoma cell line, IMR32, have been shown to contain aID mRNA by RT-PCRt 15 . 42-13-07: The sequence encoding the hamster aID subunit was isolated from an insulin-secreting cell line (HIT-TI5) eDNA librarrs . The corresponding mRNA was detectable in hamster pancreas, brain, heart and skeletal muscle. PCR-based screening detected the aID mRNA in rat CNS, including the cortex, cerebellum, hypothalamus and brainstem2s .

rbE

42-13-08: Northern: the 12 kb rbE-II (alE) mRNA is present throughout the rat CNS. ISH: shows large amounts of the rbE-II mRNA in olfactory bulb (periglomerular, mitral and granule cells), neocortical layers II-VI, entorhinal and piriform cortex, hippocampal pyramidal cells, dentate gyrus (granule cells), the hypothalamus (supraoptic nucleus and the tuberal region), the thalamus (intralaminar, parafascular and reticular nuclei), the medial habenula, the caudal brainstem (pontine nuclei, inferior olive and nucleus of the solitary tract), cerebellar cortex (granule and Purkinje cell layers), pineal gland, retina (ganglion cell layer and inner nuclear layer) and the pituitary (anterior and intermediate lobes). The mRNA was also detectable in striatum, lateral septum, amygdala, substantia nigra pars compacta and the dorsal raphe29. The patterns observed with mouse brain31 were similar to those obtained with rat brain.

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_1

Table 3. Continued mRNA

Tissue type and detailed localization

rbE

42-13-09: In the rabbit brain, BIT (alE) probes detect mRNA species of rv 11 and 10.5 kb. High levels of both species are found in the hippocampus and corpus striatum, and of only the 11 kb species in the cerebral cortex. The cerebellum contains only the 10.5 kb transcript30. 42-13-10: The a2/8 subunit is encoded by a single gene, but different splice variants t are expressed in skeletal muscle (a2a), brain (a2b), heart (a2e and a2d), and smooth muscle (a2d and a2e)40,118. Northern blotting demonstrates similar, but not necessarily identical, a2/8 mRNAs in skeletal muscle, heart, brain, vascular and intestinal smooth muscle38,45.

/31

42-13-11: The skeletal muscle /3 subunit, /31, is encoded by a gene, CaBl, that is also expressed via alternatively spliced mRNA in brain49. 42-13-12: The rat /32 eDNA probe detects mRNA species of 6, 4 and 3.5 kb in rat brain, heart and lung and in the insulin-secreting tumour cell lines derived from pancreatic /3 cells, HIT (hamster) and RIN5mF (rat)46.

/33

42-13-13: Northernt analysis and specific amplification of /33 (CaB3) specific cDNAs by polymerase chain reactions showed that CaB3 mRNA is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle52. 42-13-14: Northern blot: /34 mRNA is confined to neuronal tissues, with the highest levels being found in the cerebellum54. 42-13-15: The mRNA encoding the human skeletal muscle, subunit was detectable by RT-PCR only in skeletal muscle58 .

Mutations in the gene encoding paralysis

Q1S

cause hypokalaemic periodic

42-14-03: Hypokalaemic periodic paralysis (hypoPP) is an autosomalt dominantt disease characterized by episodic attacks of generalized muscular weakness. The disease gene has been mapped to chromosome lq31-32 and co-segregated in disease families with the gene for the (lIS subunit of the skeletal muscle Ca2+ channel125. Three missense mutations have been identified in this candidate gene, all affecting arginine (R) residues in the voltage-sensing S4 domains of repeats IT or IV: R528H, R1239H and RI239G126,127. The R528H mutation has been shown to shift the steady-state voltage dependence of the inactivation of the L-type Ca2+ current by 40 mV to

II

1......_e_n_t_ry_42

--'_

more negative potentials, but was without effect on the voltage dependence of activation128 . The reason for dominance of the hypoPP mutations and the pathophysiological basis of the disorder are not yet understood129.

Familial hemiplegic migraine and episodic ataxia type-2 due to mutations in the CACNL1A4 gene 42-14-04: Mutations in the human CACNL1A4 gene encoding an alA subunit of a P/Q-type Ca2+ channel have been identified in patients with familial hemiplegic migraine (FHM), a rare autosomal t .dominantt subtype of migraine associated with ictal hemiparesis and possible cerebellar atrophy. Systematic examination of all the 47 exons t of the gene, by single-strand conformational polymorphismt (SSCP) analysis following amplification by PCRt, identified four different missensel mutations that segregated with the disease in the corresponding families: R192Q in IS4 ('voltage sensor'), T666M in the pore region (SS-S6) of domain IT, V714A in ITS6 and I1811L in IVS6 105. The same analysis also revealed mutations in two out of four patients with episodic ataxia type 2 (EA-2), an autosomalt dominant l paroxysmal t disorder involving acetazolamide-responsive attacks of cerebellar ataxia and migraine-like symptoms: a one-nucleotide deletion in codon 1266, causing a frameshiftt and premature termination of translation at codon 1333, and a G to A transitiont at the conserved first nucleotide of intront 28, predicted to cause aberrant splicingt of the premRNAt 105.

Cerebellar ataxia associated with expansions in the subunit

Q1A

channel

42-14-05: Some splice variantst of the human QlA mRNA encode a subunit containing several consecutive glutamine residues in the intracellular Cterminal region 130. In the general population the number of consecutive glutamines varies in the range 4-16, but repeat lengths of 21-27 were found amongst patients with autosomal t dominantt cerebellar ataxia. The expanded allele t segregated with the disease phenotypet in affected families. These findings support the hypothesis that the polyglutamine expansion in the Ca2 + channel QlA subunit is the cause of the late-onset dominant ataxia. There is a correlation between the number of repeats and the age of onset of the disease. Post-mortem neuropathological examination shows that affected individuals show marked cerebellar atrophy, loss of cerebellar Purkinje cells, moderate loss of granule cells and dentate nucleus neurones and mild to moderate neuronal loss in the inferior 01ive13o. The locus on chromosome 19p13 responsible for the dominant §Pino~erebellar ~taxia has been designated SCA6 130. Note that familial hemiplegic migraine, episodic ataxia type 2 and cerebellar ataxia (SCA6) are allelic t disorders in which the nature of the mutation in the CACNL1A4 gene affects the clinical course of the disease (see paragraph 42-14-04).

Mutations in the mouse gene encoding the absence epilepsy

Q1A

subunit cause

42-14-06: Mutations at the mouse tottering (tg) locus cause a delayed-onset, recessive neurological disorder resulting in ataxia and behavioural seizures

III

_'--

e_n_try_4_2_

resembling petit mal epilepsy in humans. The gene that is altered in the tg mouse has been accurately mapped on mouse chromosome 8, isolated by cloning in a yeast artificial chromosome (YACt) vector and shown to encode the alA subunit of a P/Q-type Ca2 + channel116 . The mutation in tg mice is a C-T transitiont that results in a P601 L substitution close to the P region of repeat II of the QlA protein. The mutation in a more severe allele of tg, called leaner (tt a ), is a G-A transition t in a splice donor t site that results in aberrant splicing of the pre-mRNA and QlA variants with altered C-terminal sequences l16 .

The f31 subunit of L-type channels is essential for excitationcontraction coupling 42-14-07: The murine cchbl gene that encodes the Ca2+ channel {31 subunit has been inactivated by gene-targetingt. Mice heterozygous t for the cchbl mutation develop normally, but homozygotest die at birth from asphyxia because of the lack of excitation-contraction coupling in the skeletal muscles 131 . Myotubes can be isolated from homozygous 13l-null foetuses and used to study the behaviour of L-type Ca2+ channels lacking the 131 subunit. The L-type Ca2+ current density was reduced 13-fold by the absence of 131 subunits, was sensitive to dihydropyridines (5 JlM nifedipine reduced I Ba,L to background levels) and showed increased sensitivity to activation by Bay K 8644. Following activation by a depolarizing pulse (-40 m V holding potential to + lamV), the l,Bnull activated faster and inactivated more slowly than the l L of normal cells. Intramembranal charge movements induced by depolarizing pulses from a holding potential of -80mV were reduced '"'-'2.8-fold by the absence of the (31 subunit. The number of binding sites for radiolabelled dihydropyridines was reduced f'.J3.6-fold in muscle homogenates from. the (31 -null mice. Ca2+ transientst elicited by depolarizing pulses in the wild-type cells could not be detected in 13l-null cells, even with la-fold increases in pulse duration. The failure of the transduction mechanism in excitation-contraction coupling in 13l-null skeletal muscle could be due to the reduction in the density of QIS subunits in the membrane or to functional change in the voltage-sensor complex due to the absence of the 131 subunit132. f'.J

Voltage-gated Ca 2+ channels implicated in serotonin release 42-14-08: The small cell lung carcinoma cell line, GLC8, releases serotonin in a Ca2+ -dependent manner. Increase in intracellular Ca2+ concentration, induced by the Ca2+ ionophore, ionomycin, or the Ca2+ -ATPase antagonist thapsigargin, produced a parallel enhancement of serotonin release. Membrane depolarization resulting from increased external KCI concentration also produced a dose- and Ca2+ -dependent serotonin release that was inhibited by a range of Ca2+ channel antagonists and potentiated by the Ca2+ channel agonist, Bay K 8644. Anti-Ca2+ channel antibodies antagonized KCI- but not ionomycin-induced serotonin release. PCR analysis indicated that GLC8 cells express L-, N-, and P-type neuronal Ca2+ channel Ql subunits, and the presence of the three corresponding channels was confirmed by patch-clamp measurements133 .

II

l_e_n_try_4_2

_

Enhanced T-type currents in a rat model of absence epilepsy 42-14-09: The amplitudes of whole-cell L-type and T-type currents in rat reticular thalamic (RT) neurones gradually increase after birth until post-natal day 18. The increase in T-type but not L-type currents is exaggerated in RT neurones from the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), a rat model of absence epilepsy, which show significantly enhanced T-type currents compared with controls from day 11 onwards 134. It is suggested that the selective increase in T-type currents in RT neurones enhances the probability of recurrent intrathalamic burst activity, and might represent a primary neuronal dysfunction related to the epileptic phenotype 134.

Ca 2+ flow through a single channel can trigger neurotransmitter release 42-14-10: Simultaneous recording of Ca2+ currents and acetylcholine release at the release face of the pre-synaptic nerve terminal of the chick ciliary ganglion have shown that neurotransmitter release can be gated by very small Ca2+ transients. The time interval from the current peak to the release of a quantum of transmitter was 0.2-0.5 ms, and a flux of about 180 Ca2+ ions was sufficient to trigger release 135. This quantity of ions would enter the cell within a single flicker of an N-type Ca2+ channel in these cells (minimal open time constant rvO.15 ms 136,137). The observation that the opening of a single Ca2+ channel is sufficient to trigger neurotransmitter release supports the suggestion that the channel and the secretory vesicle are in close contact in the membrane of the pre-synaptic nerve terminal 135 .

Ca 2+ influx through L-type channels activates specific regulatory protein kinases 42-14-11: The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that phosphorylate a wide range of cellular proteins and play an important role in controlling cellular processes, including cytoskeletal dynamics, cytoplasmic signalling and gene expression. In the phaeochromocytoma cell line (pe12), membrane depolarization leads rapidly to activation of MAPK in a process that is sensitive to the L-type Ca2+ channel antagonist nifedipine (5 JlM). (Activation of MAPK by nerve growth factor is insensitive to Ca2+ channel blockers). The activation of MAPK is dependent on the small guanine nucleotide-binding protein p21 ras , the activation of which is Ca2+ dependent and nifedipine sensitive. Activation of Ras leads to phosphorylation and activation of a further protein kinase, the dual specificity (tyrosine and serine/threonine) MAPK/ERK kinase MEK1, which itself then phosphorylates the MAPK138.

N-type channels have a dominant role in release of noradrenaline from sympathetic neurones 42-14-12: Rat sympathetic neurones, which release noradrenaline, contain both N-type and L-type calcium channels. Potassium-evoked noradrenaline release was markedly reduced by Cd2+ and the cone snail peptide toxin wconotoxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine, a blocker of L-type but not N-type channels.

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e_n_try_4_2----J

N-type channels have the dominant role in the depolarization-evoked release of noradrenaline from sympathetic neurones 139. 42-14-13: Calcium influx through voltage-gated Ca2+ channels has been associated with induction of mRNA for expression of immediate-earlyt genes in dentate gyrus neurones (see this field under ELG CAT GLU NMDA, 08-14).

Ca 2+ channel activity associated with long-term potentiation 42-14-14: The induction of long-term potentiation (LTP) in developing kitten visual cortex is associated with low-threshold Ca2+ channel activity and sensitive to external Ni2+ (100 JlM) and an intracellular Ca2+ chelator. It is concluded that Ca2+ influx into post-synaptic cells through low-threshold Ca2+ channels induces the LTP response140. Ca2+ conduction through NMDA receptor-channels is commonly associated with events underlying learning and memory (see ELG CAT GLU NMDA, entry 08). Ca2+ entry via post-synaptic voltage-sensitive Ca2 + channels in the CAl region of the hippocampus has also been implicated in mediating LTp141 .

Protein distribution Complementary distributions of L- and N-type channels in rat brain 42-15-01': Protein subunit-specific immunostaining patterns show neuronal cell type specificity, including support for complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals and cell bodies of most central neurones of the rat brain39?142?143. Strong staining due to clusters of N-type channels in the pre-synaptic terminals of the mossy fibres of the hippocampus can be seen. The immunostaining of sections of rat brain with antibodies specific for the N-type Ca2+ channel 143 al subunit are shown and discussed in ref. . The cell-type specificity of Land N-type channels may underlie distinct functional roles in calciumdependent electrical activity, intracellular calcium regulation and neurotransmitter release for these two channel types 143 . Q1D

L-type channels are concentrated at the bases of major dendrites

42-15-02: A polyclonal antiserum generated against rat brain aID sequences immunoprecipitatedt "-120% of the L-type calcium channels (assessed as 3H-PN200-ll0-binding sites) from rat cerebral cortex. Immunoblottingt revealed two size forms of the aID subunit, with apparent molecular masses of "-1200 kDa and 180 kDa. Im:munocytochemical studies revealed strong staining in most central neurones, including those in layers I-VI of the cerebral cortex, the hippocampal CAI-CA3 pyramidal and dentate gyrus, the granule layer of the cerebellum, and on cerebellar Purkinje cell bodies. The strongest immunolabeling was found on cell bodies and proximal dendrites, with an even distribution over the cell bodies and accumulations at the base of major dendrites 144. Note that the distribution of ale and aID L-type channels on cell bodies and proximal dendrites is complementary to that of alB N-type channels, which are concentrated along the length of dendrites and at much lower levels on cell bodies143.

II

l_e_n_try_4_2

The

_

0lS, 02

and f3 subunits co-localize in skeletal muscle triads

42-15-03: Immunofluorescence t and immunogoldt labelling techniques have allowed the localization of the rat skeletal muscle O:IS, 0:2 and {3 subunits of the L-type voltage-activated Ca2+ channel to the T-tubule membranes of the triad junction formed between the T-tubules and the sarcoplasmic reticulum t 145,146.

Distribution of N-type Ca 2+ channels 42-15-04: Antibody CNB-l raised against rat brain class B Ca2+ channels (rbB) immunoprecipitate 430/0 of the N-type Ca2+ channels labelled by [l 25 I]wconotoxin16,143. CNB-l recognizes proteins of 240 and 2l0kDa, suggesting the presence of two size forms of this 0:1 subunit. CNB-l affinity sites are localized predominantly in dendrites: both dendritic shafts and punctate synaptic structures upon the dendrites are labelled with CNB-l. The large terminals of the mossy fibres of the dentate gyrus granule neurones are also heavily labelled, suggesting that the punctate labelling pattern represents Ca2+ channels in nerve terminals. The cell bodies of some pyramidal cells in layers II, ill and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain are also labelled at a low level with CNB_1 143 .

Distribution of P-type Ca 2+ channel proteins 42-15-05: Immunolabellingt with a polyclonal antiserum generated against a bovine cerebellar protein capable of forming P-type channels in lipid bilayers showed strong reaction throughout the molecular layer of the cerebellar cortex, especially at bifurcations of Purkinje cell dendrites. Strong labelling was also evident in the periglomerular cells of the olfactory bulb, scattered neurones in the deep layer of the entorhinal and pyriform cortices, the brainstem, habenula, nucleus of the trapezoid body and inferior olive and along the floor of the fourth ventricle. Lower intensity of labelling was

found in layer II pyramidal cells of the frontal cortex, the CAl cells of the hippocampus, the lateral nucleus of the substantia nigra, lateral reticular nucleus and spinal fifth nucleus. Light labeling was seen in the neocortex, striatum and some brainstem neurones 147.

Subcellular locations Ca 2+ channels show differential localization within neurones 42-16-01: Within individual neurones, calcium channels can be localized to cell bodies, dendrites t and growth cones t, but excluded from axons t. The spatial localization of channels in neurones begins early in development and distribution can change markedly during embryogenesis through to adult (reviewed in re!s.107,148). Dendrites of hippocampal neurones contain T-type, L-type and R-type Ca 2+ channels 42-16-02: The dendrites t of CAl pyramidal neurones contain T-type Ca2+ channels (conductance rv9 pS), R-type channels (conductance rv 15 pS) and a

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Table 4. Sizes of the mRNA species corresponding to the Ca 2+ channel subunits (From 42-17-01) Gene/subunit Species

Transcript size and commentary

CaChl/alS

Mouse Rabbit

6.5 kb38,80 6.4, 4.4 kb: the shorter form predominates in newborn skeletal muscle, but the longer species is prevalent: in adult tissue 109

CaCh2/a1C

Mouse

rv1S.5, rv8.9 kb (major) plus rv20 kb (minor) in mouse heart153 9.4kb 154 12 kb and 8 kb, (rat brain)2. The 8 kb species ('rbcC-IT') is the more prevalent in total brain and in most other rat tissues, though the 12 kb species ('rb(::-I') predominates in the pituitary gland2. The product derived from the mRNA of the rat adrenal gland by RT-PCR is slightly smaller than the corresponding product from mRNA of other tissues, suggesting that the alC mRNA in the adrenal gland undergoes a different splicing event2

Human Rat

CaCh3/a1D

Rat

rv11 kb 27

CaCh4/a1A

Human Rhesus monkey

8.5 kb 130 9.8 kb in cerebellum, cerebral cortex, thalamus and hypothalamus105

CaCh5/a1B

Rat

rv10kb in all regions of the rat CNS, including the spinal cord16

CaCh6/a1E

Rabbit Rat

rv11 and 10.5 kb 30 12kb29

Mouse

8.5 kb (most tissues), with second species at rv6 kb in brain and skeletal muscle l18 rv8 kb in heart, aorta, brain hippocampus, skeletal muscle and smooth muscle: an additional minor species of 7 kb is also detectable in brain, hippocampus and ileum38

Rabbit

r-J

CaB1/,BI

Human

Rabbit

II

rv 1.9 kb in skeletal muscle (,BIM) and brain (,BIBI): the skeletal muscle mRNA is slightly larger because of the inclusion of a 154 bp exon compared with a 19bp exon in ,BIBI 51 . The alternately spliced ,BIB2 mRNA from human hippocampus is rv3.0 kb 51 1.9, 1.6kb in skeletal muscle, but 3.0kb in brain48

l_e_n_t_ry_4_2

_

typical L-type channel. The T-type and R-type currents were recorded at distances> 100 Jlm from the soma, while the L-type currents were primarily recorded in patches within 100 Jlm of the soma149. Whole-cell voltageclamp measurements with isolated dendritic segments ('dendrosomes') from rat hippocampal neurones confirmed these data and showed that T-type channels provide a larger fraction of the Ca2+ influx in dendrites than in cell bodies 15o. At 2 mM Ca2 +, approximating physiological ionic conditions, the maximal amplitude of the T-type tail currents exceeded the combined amplitude of all high voltage activated (HVA) channels. The use of specific channel blockers allowed the high voltage activated Ca2+ currents to be dissected into L-type (1"V20%), N-type (1"V39%), P-type (1"V28%) and Q-type (1"V13%). A low contribution from R-type currents could be detected after inhibiting L-type, N-type and P/Q-type currents with w-conotoxin MVIIC (5 JlM) plus nimodipine (10 JlM)150. (For observations on the activation of dendritic Ca2+ channels by excitatory post-synaptic potentials, see Activation, 42-33.)

L-type Ca 2+ channels are localized in neuronal cell bodies and bases of proximal dendrites 42-16-03: Immunocytochemical studies have shown that L-type calcium channels are localized in neuronal somatat and the bases of proximal dendrites t in rat brain, spinal cord and retina, leading to the suggestion that the neuronal L-type channels may link summed electrical activity in dendrites with biochemical regulatory processes governed by intracellular Ca2+ in the soma39,142.

N-type channels are clustered in tactive zones' on peripheral nerve terminals 42-16-04: N-type Ca2+ channels, labelled with fluorescently tagged w-conotoxin GVIA, were revealed by confocal microscopy to be localized exclusively at the 'active zones' of the frog neuromuscular junction. (They co-localized with the nicotinic acetylcholine receptor, detected with fluorescent a-bungarotoxin.) Cross-sections of the junctions showed that the N-type channels are clustered on the pre-synaptic membrane adjacent to the post-synaptic membrane151 . Note that autoantibodies from patients with the myasthenic disorder Lambert-Eaton syndrome show similar staining patterns152.

Transcript size 42-17-01: The sizes of the mRNA species corresponding to the various Ca2+ channel subunits are summarized in Table 4.

SEQUENCE ANALYSES

11

The symbol {PDTM} denotes an illustrated feature on the channel protein domain topology model (Fig. 4).

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Chromosomal location Summary of locations of human genes encoding Ca 2+ channel subunits 42-18-01: The chromosomal locations of human genes encoding subunits of the voltage-gated Ca2+ channels are shown in Table 5.

The gene encoding the 0.2/8 subunit is linked to the malignant hyperthermia locus 42-18-02: Malignant hyperthermia susceptibility (MHS) is an autosomal t dominantt disorder affecting skeletal muscle, manifested as potentially fatal hypermetabolic crises triggered by commonly used anaesthetics and involving a breakdown in the mechanisms regulating sarcoplasmic Ca2+ fluxes. The gene (CACNL2A) encoding the human 0.2/6 subunit of voltagegated Ca2+ channels co-segregatest with the MHS locus in some families and remains a candidate for one of the genes affected in disease families 162. The CACNL2A gene is linked to a polymorphic t dinucleotide-repeat t marker on chromosome 7q162 and has been mapped to 7q21-q22 by somatic cell hybridt analysis 161 .

The human gene encoding

o.1S

maps to a disease locus, HypoPP

42-18-03: Hypokalaemic periodic paralysis (hypoPP) is an autosomal t dominantt disorder involving muscular weakness and paralysis that can be triggered by insulin, adrenaline and ingestion of carbohydrate. Patients have low levels of serum K+ «3 mM) during an episode. The hypoPP locus has been mapped in three independent disease families to chromosome 1q3132. The gene (CACNL1A3) encoding the muscle calcium channel o.lS subunit maps to the same region, and co-segregrates with hypoPP without recombinants in two informative £amilies125. Point mutations in the CACNL1A3 gene have been identified in hypoPP patients (see paragraph 42-14-03).

Some Ca 2+ channel subunit genes are mapped in the mouse 42-18-04: The mouse chromosomal locations of genes encoding the N-type Ca2+ channel al subunit (Cchnal gene) and three neuroneal /3 subunits (the Cchb2, Cchb3 and Cchb4 genes) have been determined by linkage analysis. The N-type aI, /32 and /34 subunits are specified by genes at separate sites on proximal chromosome 2 and the /.~3 subunit gene maps to chromosome IS, within 1.3 cMt of the Wntl marker167. Note that this location places the mouse Cchb3 gene within a large block of syntenyt to human chromosome 12q, and that the human /33 subunit gene maps to 12q13165. The Cch11a2 gene encoding the L-type Ca2+ channel al subunit from mouse brain is located on chromosome 14157 and the Cch11a3 gene specifying the skeletal muscle type alS subunit maps to mouse chromosome 1168 .

I II

Encoding 42-19-01: For subunit-specific data, see Gene family, 42-05 and Domain functions, 42-29.

g t"'t-

~ ~

t:--J

Table 5. Cmomosomallocations of human genes encoding subunits of voltage-gated Ca 2+ channels (From 42-18-01)

Gene CaChl (CACNL1A3) CaCh2 (CACNL1Al) CaCh3 (CACNL1A2) CaCh4 (CACNL1A4) CaCh5 (CACNL1A5) CaCh6 (CACNL1A6) CaAl (CACNL2A) CaBl CaB3 CaGl

II

Subunit encoded

Channel type

Location

Reference

°IB

N

lq32 12p13.3 3p14.3. 19p13.1-p13.2 9q32-q34 lq25-q31 7q21-q22 17q21-q22 12q13 17q24

1257 1557 156

°IA

L L L P/Q

°IS Ole OlD

OlE

02/8 {31 {33

,

R/(T?} all all all all

154 1577 158 1057 159 1597 160 159 1617 162 1637 164 165 587 1637 166

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_

Gene organization Multiple genes and alternative splicing generates variety of structure and function 42-20-01: The existence of different classes of calcium channel subunits, encoded by separate genes, together with alternatively spliced variants of those classes, may help account for the wide range of kinetic and pharmacological properties observed for Ca2+ currents in different types of native cells.

Variants of rbC

Q1C

subunit arise by alternative splicing

42-20-02: The rat rbC gene encodes two variants of the alC Ca2+ channel subunit, rbC-I and rbC-II, by alternative splicingt. The major sequence difference between the two, lying within a region of 28 amino acids representing the S3 segment of domain ~ arises from alternative exonst. The isolation and sequencing of rat genomict DNA clones covering this region of the rbC gene shows the 84 bf rbC-I exont lies upstream of a 84 bp rbC-II exon, separated by an intron of 632 bp2. Although the mRNAs encoding the rbC-I and rbC-II variants are found in all regions of the CNS, the relative amounts of the two mature mRNA species vary between brain regions 2 . (See mRNA distribution, 42-13, for more detail).

Alternative Q1C transcription starts in mouse erythroid leukaemia cells 42-20-03: The mouse gene specifying the cardiac ale subunit is transcribed from a different transcription start in the mouse erythroleukaemia cell line, MELC l53 . The MELC transcription-start site is within an intron of the alC gene, and gives rise to a shortened mRNA that encodes a protein lacking the first four transmembrane segments of the alC subunit. Transcription factor t -recogn,ition elements upstream of the MELC start site include three 'GATA boxes t , at -81, -350 and -434 and two 'CACCC boxes t , at -58 and -127 (with reference to the putative cap-site t of the MELC mRNA}l53. (The protein translated from the MELC alC mRNA in Xenopus oocytes does not result in active Ca2+ channels l53 .)

The human CACNL1A4 gene has 47 exons

42-20-04: The exon-intron t structure of the human CACNL1A4 gene, encoding the alA subunit of the P/Q-type Ca2+ channel, has been determined by sequencing all the DNA fragments (average size rv2 kb) containing exons that were subclonedt from 10 cosmidt clones t constituting a 'contigl ' covering the gene. The gene contains 47 exons t, with the untranslated 5' region of the mRNA and the translation-start codon in exon 1 and the stop codon in exon 47. The exons are distributed over rv300 kb of genomic DNA of human chromosome 19. Intron 7 contains a highly polymorphic t (CA}-repeat t sequence (D19S1150) with an observed heterozygosityt of 0.82. The exon-intron organization of the CACNL1A4 gene and a list of exon-specific primert pairs are given in ref. los .

II

l_e_n_t_ry_42

The

_

02-8

transcript is alternatively spliced

42-20-05: The 02 -6 subunit is encoded by a single gene in several mammalian species, but different isoforms of the protein are produced by alternative splicingt of the primary transcript (see Domain functions, 42-29).

Skeletal muscle and brain isoforms of human {3l subunit are encoded by a single gene 42-20-06: Two cDNAs (,81Bl and ,81B2) isolated from human hippocampal libraries and the human skeletal muscle ,81M cDNA are derived from splicet variants of the transcript from a single gene51 . The {lIM and {liBI cDNA sequences are identical except for a region of 154np encoding 52 amino acids in {lIM that is replaced by 19 np encoding seven amino acids in both {liBI and {lIB2. The sequence of {lIB2 is identical to that of {liBI for the first 1482 nucleotides, but {lIB2 contains an elongated 3' end encoding 152 amino acids. The corresponding genomic sequence contains two exons, the 5' one of 154 np specific to {lIM and the 3' exon of 19 np that is present in both {liBI and {lIB2. The,81 transcript undergoes a novel splicing event in {lIB2 that excises a translation termination codon and a poly(A)-additiont signal that are used in both {lIM and {liBI. The mature {lIB2 mRNA is 1.98 kb longer than the {liBI mRNA and encodes an alternative C-terminus t that differs from that of {liBI over the last 34 amino acids and is elongated by a further 118 amino acids.

The human gene encoding the 'Y subunit has four coding exons 42-20-07: The human gene encoding the skeletal muscle Ca2+ channel 'r subunit contains four coding exons, separated by introns of 9.4, 1.0 and 1.3 kb 58 .

Homologous isoforms 0lS

subunits make functional Ca 2 + channels in dysgenic myotubes

42-21-01: The alS subunit of skeletal muscle does not give active Ca2+ channels when produced by heterologous expression in Xenopus oocytes169. Skeletal muscle myotubes from mice with muscular dysgenesis (mdg) have provided a valuable host cell type for heterologous expression of cDNAs encoding (}IS subunits 80. Production of the (}IS subunit by heterologous expression in mdg myotubes results in restoration of dihydropyridinesensitive L-type Ca2+ channels and excitation-contraction (E-C) coupling8o. The ale subunit of cardiac muscle L-type Ca2+ channel also restores E-C coupling, but in this case it does not require Ca2+ entry, reflecting the situation in cardiac cells and contrasting with the (}ISdependent coupling121 . Heterologous expression in mouse L-cells of sequences encoding the skeletal muscle QIS subunit also produces functional L-type Ca2 + channels 170.

Subtypes of the rat brain splicing

OlD

subunit are formed by alternative

42-21-02: The rat brain aID subunit isoform RBol' which contains 1634 amino acids, has 71 % and 76% identity with the rabbit skeletal muscle

II

_L...-

.

e_n_try_4_2_

(aIS) and cardiac (ale) isoforms, respective1r6. Variant cDNA clones isolated from a rat brain library reveal RBal subtypes due to alternative splicingt. Two versions of a 28 amino acid region spanning the S3 segment of domain IV differ in 11 amino acids, all conservative t replacements26 . Note that this same region is alternatively spliced in the rbC-I and rbC-II variants expressed in the rat CNS2 (see 42-20-02). In addition, three variations of the sequence encoding the intracellular region between domains 1 and II were isolated. One predicted product lacks 12 and a second lacks an adjacent 20 amino acids in this region compared with the longest of the three variants26 .

Alternative splicing of the

Q1B

transcript in rat brain

42-21-03: Detailed analysis of the alB sequences expressed in rat superior cervical ganglia (rSCG) revealed four discrete sites where individual cDNA sequences differed from the rat brain-derived clone, rbB-I: (i) a single base change (A to G) resulting in the amino acid change E177G in IS3; (ii) a three-base deletion removing amino acid A415 from the intracellular IS6IISI loop; (iii) a 12-base deletion of sequences encoding a tetrapeptide, SFMG, in the putative extracellular IIIS3-IIIS4 loop, and (iv) a six-base insertion creating an insertion of two amino acids (ET) in the putative extracellular IVS3-IVS4100p171. The G177 form was invariant amongst 11 clones analysed, but both alternatives at the other three positions were found. There was a marked difference in the frequency of both of the SFMG- and ETcontaining variants between brain (+SFMG == 76 %, + ET == 5%) and rSCG (+SFMG==39%, +ET==87%). The dominant forms of alB in ganglia, rnalB-b (LlSFMG, +ET), and brain, rnalB-d (+SFMG, LlET), when produced by heterologous t expression in Xenopus oocytes in the presence of rat brain /33 subunit, show significant differences in the macroscopic rates of channel activation and inactivation and in the voltage dependence of activation. The rna 1B-d currents, on average, activate 1.7-fold faster, inactivate fourfold faster and activate at potentials 5 mV more negative than the rnalB-b currents171 . The injection of rnalB-d eRNA into Xenopus oocytes, in the absence of co-expressed /3 subunit cRNA, produced Ba2+ currents with gating kinetics 'similar to those of the native N channel'. Co-expression of the rba3 subunit enhanced the peak current amplitude by rv3.5-fold, without significantly affecting the gating kinetics 171 .

Marine ray proteins are highly honl0logous to mammalian brain subunits

0'.1

42-21-04: The doe-I cDNA isolated from the forebrain of the marine ray, Discopyge ommata, encodes an al subunit with the following amino acid identities to mammalian Ca2+ channel al subunits: BII (alE subunit of

rabbit brain), 68%; rbB (alB subunit of rat brain), 600/0; BI (alA subunit rabbit brain), 63%. Heterologous co-expressiont of doe-l coding sequences with those encoding mammalian a2 and /3 subunits produced high voltage activated, rapidly inactivating Ba2+ currents that were very sensitive to block by Ni2+ 14. The doe-4 sequence, which is strongly expressed in the electric lobe of D. ommata, encodes a protein with 61 % identity to doe-I, but more closely related to rbB (72~1 identity) and BI (68% identity)172. D. ommata also encodes a Ca2+ channel, doe-2, with high homology to the

II

entry 42

_

A B C (a)

SETLKPD 627 ..

a2a

., 50oHPNLQPKPIGVGIPTINLRKRRPrNQNPKSQEPvTLDFLD539 . . . 610KLEETITQAR~

a2b

.. 50oHPNLQPK

NPKSQEPVTLDFLD 520

591 KLEETITQARSKKGKMKDSETLKPD 615 •.

a2c

.. 50oHPNLQPK

EPVTLDFLD 515

586KLEETITQARSKKGKMKDSETLKPD610 ••

a2d

.. 50oHPNLQPK . . . • . . . . . . . . . . . . . . . . . . . . EPVTLDFLD 515 . . . 586 KLEETITQARY . . . . • . . SETLKPD 603 ..

a2e

.. 500HPNLQPK . • . . . . . . . . . . . . . . . . . NPKSQEPVTLDFLD 520

(b)

.. CCA

591KLEETITQARY

SETLKPD 608 ..

AAG CCT ATT GGT GTA GGT ATA CCG ACA ATT AAT TTA AGG

P

K

PIG

V

G

I

P

TIN

L

R

AAA AGG AGA CCC AAC GTT CAG AAC CCC AAA TCT CAG GAG CCA .. K

(c)

R

a2a/d/e

R

P

V

Q

N

P

K

S

Q

E

P

.. CAG GCC AGA TAT TCA GAA ACC .. Q

a2b/ c

N

A

R

Y

SET

.. CAG GCC AGA TCA AAA AAG GGA AAA ATG AAG GAT TCA GAA ACC

Q

A

R

S

K

KG

K

M

K

D

SET

Figure 2.#Variants of the mouse 02/8 subunit arising from alternative splicingt. (a) Amino acid sequences of the five variants: the alternatively spliced regions (A, B and C) are shown at the top. (b) cDNA sequence and encoded amino acids in regions A and B, with alternative splice acceptort sites (CAG) underlined. (c) cDNA sequence and encoded amino acids in region C. Alternatively spliced sequences and encoded amino acids are shown in bold type. (Reproduced with permission from Angelotti (1996) FEBS Lett 397: 331-7.) (From 42-21-07) mammalian L-type channels172. The separation of Ca2+ channels into L-type and 'non-L-type' must therefore predate the divergence of the cartilaginous marine rays from bony animals >4 x 108 years ago.

Homology between {3 subunit isoforms 42-21-05: The four isoforms of /3 subunit (rabbit) show the following sequence identities: CaBl/CaB2a, 71.0%; CaBl/CaB2b, 71.50/0; CaBl/CaB3, 66.60/0; CaB2a/CaB2b, 97.2%; CaB2a/CaB3, 64.7%; CaB2b/CaB3, 64.9%52.

Mouse and human a2/8 variants arising from alternative splicing (Fig. 2) 42-21-06: A single gene encoding 02/8 subunits in the mouse gives rise to five variants of the Ca2+ channel component by alternative splicing t of the premRNAt. RNAase-protection t assays show that the 02a variant is present in skeletal muscle and aorta, 02b is specific to brain, 02c is specific to heart, where 02d is also found at a lower level, and smooth muscle expresses the 02d and 02e mRNAs l18 . Corresponding human splice variants have been located to skeletal muscle (02a), brain (02b) and aorta (02c)15. The sizes of the various isoforms of the Ca2+ channel subunits and the corresponding mRNA species are shown in Table 6.

II

11

Table 6. Sizes of the different Ca 2 + channel subunits and their mRNAs (From 42-21-07) Class

cDNA

CSkm

alB

ale

aID

Number of aa

1873 1191 a Carp (Cyprinus carpio) skeletal muscle 1852

Rabbit skeletal muscle

alS

alA

Source

BI-l BI-2 rbA-I hBI-l malA

Rabbit brain Rat brain Human brain Mouse brain

2273 2424 2212

Molecular mass (kDa) mRNA size (kb) 6.4 4.4

257 273 252

9.4

5

8.8 and 8.3 8.5 8.6 and 8.2

4

2164 262 252 262 261 265

Rat aorta Mouse brain Mouse erythroleukaemic cell line

2171 2166 2140 2143 2169 2139 1864

243 242 240 240 244 240 211

Human neuroblastoma cell line Human pancreatic islet Rat hippocampus Hamster insulin-secreting cell line

2161 2181 1634 1610

245 248 187 182

Human neuroblastoma cell line

pCARD3 pSCaL rbC-I rbC-IT VSMal mbC MELC

Rabbit heart Rabbit lung Rat brain

aID CACN4 RBal HCa3a

Rat brain Rabbit brain Marine ray, Discopyge ommata

32

212 147 210

2239 2237 2336 2339 2326b

alB-I alB-2 rbB-I Bill doe-4

Reference 109

173

130

116 15 16 18

12-13

174

19 20

ca. 12 and 8

2 21 104

22,9.5 and 7.5

153

ca. 11 8.6 and 6.5 c

27

15 26 28

(1)

~

~ ~ ~

OlE

Rat brain Marine ray, Discopyge ommata

2259 2178 2222 2223

254 245 252 252

a2a a2a a2b a2b a2c a2d a2e rB-a 2

Rabbit skeletal muscle Mouse skeletal muscle Human brain Mouse brain Mouse heart Mouse heart Mouse, smooth muscle Rat brain

1080 + 26 d 1067 + 24 d 1072 + 24 d 1060 + 24 d 1055 + 24d 1048 + 24 d 1053 + 24 d 1080 + 24d

CaB-I (3c (3IM

Rabbit skeletal muscle Human heart Human skeletal muscle

(3lb (3a (3IB2

Rabbit brain

11 and 10.5

30 (t)

~

t'+

29

8

174

124 123 123 122 122 121 122 124

/"V8 and 3.8 e

38

524 522 523

58 58 58

1.9,1.61

Rat brain Human heart Human hippocampus

597 597 596

65 65 65

(32 (3b (3IBI

Rat brain Human heart Human hippocampus

478 477 478

53 53 53

{32a

CaB2a

Rabbit heart

606

68

52

{32b

CaB2b

Rabbit heart

632

71

52

02

{31a

{31b

{31c

II

BIT-1 BIT-2 rbE-IT doe-1

118 15 118 118 118 118 40

48 50 51

49

3.4 3.0g

50 51

15 50 51

~ ~ ~

II

Table 6. Continued Class

cDNA

Source

/32c /32d

CaB2c CaB2d

Rabbit brain Rat brain

{33

{33

{34 f

Number of aa

Molecular mass (kDa) mRNA size (kb)

Reference

604

68

46

CaB3

Rat brain Rabbit brain

484 477

55 54

52

{34

Rat brain

519

58

Rabbit skeletal muscle Human skeletal muscle

222

25

52

53

54

1.2

57 58

Notes: 1. For use of rbA-rbD nomenclature see, for example, refs. 2,4,175,176. 2. The L-type Ca2+ channel is composed of five subunits (aI, a2, (3, 8 and f). a The mRNA encoding this variant of the skeletal muscle Ca2 + channel al subunit is 2 kb shorter than the alS mRNA that predominates in adult tissue, due to an internal deletion109. b A variant doe-4 cDNA encodes a product with a block of 20 extra amino acids inserted after residue 406, due to alternative splicing172. C The rat brain aID sequence is expressed via an 8.6 kb transcript in brain, heart, aorta, uterus and lung, and as a 6.5 kb mRNA in aorta and skeletal muscle26 . dThe rabbit skeletal muscle a2 subunit is encoded as a protein of 1106 amino acids that includes a signal sequence of 26 residues38 . The human15 and mouse l18 brain homologues have similar structures, but with 24 amino acid leader peptides. e The 8 kb mRNA species was present in rat brain, skeletal muscle, heart and lung; the 3.8 kb mRNA was present as a minor species in rat skeletal muscle40. fThe 1.9 and 1.6kb species were present in rabbit skeletal muscle, but the {31a mRNA in rabbit brain was a 3.0kb species48 . 48 g 3.0 kb is the size of the cognate mRNA species in rabbit brain .

(I)

Ia ~ ~ ~

II...-__ _ _ry_42

_

en t

.....

... u

1S

(Carp Sk)

.....- - - - -.. u 1s(Sk)

DHP-sensitive

. - - - - - -.. u 1c(rbC-II)

....- - - - - - u 1A(rbA-1) ........- - - - - u 1s (rbB-1) ....- - - - - u 1s(doe-4) , . . . - - - - - u 1E(doe-1)

DHP-resistant ....-

I

20

I

40

....- - - - - - u1E(rbE-II)

i

60

80

i

100

Percentage identity

Figure 3. Similarity tree of primary sequences of cloned voltage-gated Ca 2+ channel al subunits. The predicted amino acid sequences of the following major classes of al subunits are compared: Carp Sk 173, Sk32, rbc-II2 , alD 15, rbA-I4 , rbB-I16, doe-1, doe_4 172, rbE-II29• (Reproduced with permission from Stea et al. (1995) In Handbook of Receptors and Channels (ed. R. A. North), pp.113-51. CRC Press, Cleveland, Ohio.) (From 42-21-08)

Comparison of 0'.1 subunit sequences 42-21-07: Sequences encoding six distinguishable classes of voltage-activated Ca2+ channel al subunits have been characterized by molecular cloning. The primary sequences of these subunits have been compared by means of a 'similarity tree', as shown in Fig. 3.

An alternative O'.le transcript produced in a mouse erythroleukaemic cell line 42-21-08: Molecular cloning of cDNAs derived from mRNA of a mouse erythroleukaemia cell line (MELe) revealed a truncated ale transcript, produced by transcription of the mouse alC gene from a start site downstream of the one used in heart. The MELC mRNA encodes an alC subunit variant that is missing the first four putative transmembrane regions of motif I of the cardiac alC subunit. The AUG codon that specifies Met299 of the cardiac al subunit is used as the translation-initiation signal in the MELC mRNA, which encodes a polypeptide of 1864 amino acids. Messenger RNA species containing the 5' sequence of the mouse cardiac alC mRNA were not detectable in MELC cells. Injection of MELC alC cRNA into Xenopus oocytes failed to produce functional Ca2+ channels153 .

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2__1

The trp gene of Drosophila has homology with

al

subunit genes

42-21-09: The trp ('!ransient !eceptor I!otential') gene of Drosophila melanogaster, mutations in which are affected in phototransduction such that the light response quickly declines to baseline during prolonged intense illumination, encodes a light-activated Ca2+ channel subunit177 with sequence homology to the al subunit of vertebrate Ca2+ channels, particularly in the membrane-spanning regions S3_S6 178 . Three of the four basic amino acids in S4 that constitute the voltage sensor of the al subunits are absent from the trp S4 region, though the overall homology can still be discerned178. A cDNA isolated from a Drosophila head cDNA library encodes a calmodulint -binding protein that has 39% overall identity with the trp protein, including 76% identity in the region spanning S3_S6 178. The gene encoding this protein has been named the !.ransient receptor Qotential-like (trpl) gene178.

A modulator of pre-synaptic N-type Ca 2+ channel activity identified

by tsuppression cloning' 42-21-10: On injection into Xenopus oocytes, the mRNA from the electric lobe of the electric ray, Torpedo californica, gives rise to a characteristic Ca2+ current that resembles N-type currents of mammalian neurones. A novel cDNA sequence has been isolated on the basis that its in vitro transcript inhibits the ability of the electric lobe mRNA to generate Ca2+ currents in Xenopus oocytes. (This approach, involving screening for clones via the inhibition of expression of known mRNAs, has been called 'suppression cloning'.) This in vitro transcript is an antisense RNA that is complementary to the sequence of part of the mRNA encoding a subunit of the Torpedo Ca2+ channel, or an associated protein that stimulates channel activity. The expression of the corresponding sense RNA selectively potentiates the N-type Ca2+ currents obtained from translation of electric lobe mRNA in Xenopus oocytes. The full-length cDNA encodes a predicted ~a2+ ~hannel ~ubunit l' (CCCS1), containing 195 amino protein, '~andidate acids, that is cysteine-rich and has "V70% identity to 'cysteine string proteins' (CSP) of Drosophila 179 . These CSP proteins have been immunochemically detected at nerve-ending membranes at synapses throughout the Drosophila nervous system (quoted in ref.179).

Protein molecular weight (purified) Two isoforms of a subunit in skeletal muscle L-type channels 42-22-01: The al subunits of the L-type Ca2+ channel of rabbit skeletal muscle occur as two isoforms t : a 212kI)a species that comprises about 5% of the total, and a major (95%) 190kI)a form, derived from the 212kDa form by proteolytict cleavage near amino acid residue 16908 .

Complexes containing the shorteneti form of the functional channels

alS

subunit are

42-22-02: It was proposed that the smaller form of alS could act as the 'voltage sensor' working in conjunction with the ryanodine receptor (see ILG Ca

II

i_e_n_ _ry_42

_

t

Ca RyR-Caf, entry 17), whereas the larger form acts as an L-channel. Countering this suggestion, L-type channel activity has been observed following exclusive reconstitution of solubilized low molecular weight form into phospholipid bilayers25 . In addition, a shortened form of the rabbit skeletal muscle Ql subunit, missing the final 211 amino acids from the C-terminust and therefore similar to the naturally occurring truncated form, obtained by expression of an appropriately deleted eDNA, has been shown to function as both voltage sensor and calcium channel in dysgenic myotubes 180. The L-type calcium channel purified from skeletal muscle also contains the {3 subunit of 57 kDa, the '"Y subunit of 25 kDa and the disulphide-linked Q2/8 subunits of 125 kDa 589 .

L-type Ca 2+ channels can be purified as an w-conotoxin GVIA-binding protein

42-22-03: L-type Ca2+ channels from rat brain have been purified by

monitoring the binding of 12sI-labelled w-conotoxin GVIA and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-[12SI]-w-conotoxin GVIA. The purified w-conotoxin receptor complex comprised a 230 kDa Ql subunit, a 140 kDa Q2 subunit, and additional proteins of apparent molecular masses of 110, 70 and 60kDa62 . Photoaffinityt labelling of partially purified protein fractions from bovine 181 , porcine182, guinea pig183 and chick184 heart showed that the Ql subunit of the cardiac L-type channel has an apparent molecular mass of 195 kDa. I"V

Disulphide linkage to the {; subunit associates the the membrane-bound channel

Q2

subunit with

42-22-04: The 0.2/6 subunit from skeletal muscle is a glycoproteint with an

observed molecular weight of 175000 under non-reducing conditions. After reduction, the Q2 subunit has an observed size of 150 kDa, and 8-related species of 25 kDa, 22 kDa and 17 kDa are produced. The stoichiometric ratios of these species in the Q2/8 complex are 1.0 (Q2), 0.31, 0.47 and 0.08 in order of decreasing size. All three 8-related species have the same Nterminust, starting at Ala935 of the encoded propeptide, and differ from each other only in the degree of N-linked glycosylation t . After deglycosylationt with endoglycosylasest the Q2-peptide has a molecular weight of 105kDa and the 8 peptide one of 17kDa. Under reducing conditions, the Q2-peptide can be solubilized from skeletal muscle membrane preparations by extraction at pH II, a procedure that removes peripheral but not integral membrane proteins. The 8 subunit remains in the membrane fraction under these conditions. The Q2 subunit is thus associated with the Ca2+ channel in the membrane through S-S linkage to the 8 subunit36 .

Purified N-type channels contain an additional glycoprotein subunit

42-22-05: A functional N-type Ca2+ channel complex purified from digitonin-

solubilized rabbit brain membranes consisted of a 230 kDa subunit, QIB, tightly associated with a 160kDa subunit, Q2/8, a 57kDa subunit, {33, and an additional 95 kDa glycoprotein subunit. Affinity-purified antibodies prepared against the 95 kDa subunit precipitated 740/0 of the 12sI-labelled

II

_L-...

e_n_try_4_2_1

w-conotoxin-binding sites from rabbit brain, without precipitating the brain L-type channels. The purified complex formed a functional Ca2+ channel with the same pharmacological and electrophysiological properties as those of the native w-conotoxin-sensitive N-type channel in neurones 63 .

Molecular weights of native (3 subunits from Western blots

42-22-06: Antibodies against specific Ca2+ channel /3 subunit subtypes recognize {31 subunits of 78 and 80 kDa, a {32 subunit of 74 kDa, a {33 subunit of 58 kDa and {34 subunits of 55 and 59 kDa in extracts from rat brain membranes. The /32 antibody detected subunits of 70, 74 and 87kDa in rat cardiac microsome membranes. The rat phaeochromocytoma cell line, PC12, produced readily detectable /33 subunit, and a /32 subunit of 70kDa could be detected only after "J10-fold enrichment on an immunoaffinity column106. Immunoprecipitation with anti-/3 subunit antibodies shows that the 0lB subunits of N-type channels are associated with /32 and /33 subunits in PC12 cells 106 (see Protein interactions, 42-31). The /3 subunit from skeletal muscle Ca2+ channels has a reduction-insensitive apparent molecular mass of 52-65 kDa as measured on SDS_PAGEt185-187.

The skeletal muscle channel, subunit 42-22-07: The purified, subunit from the skeletal muscle dihydropyridine (DHP) receptor is a heavily glycosylated protein with an apparent molecular mass of 30-33 kDa as measured on SDS_PAGEt186-188.

Protein molecular weight (calc.) 42-23-01: See Table 6, 42-21-07.

Sequence motifs 42-24-01: For subunit-specific data, see Gene family, 42-05. All six of the conserved consensus sites for cAMP-dependent protein phosphorylation on the 01(212) subunit are located in the C-terminal tail (see {PDTM}, Fig. 4). Note: The truncated 01(175) form (see paragraph 42-29-01) lacks at least three of these motifs.

N-glycosylation (Asn-X-Ser/Thr) balD: 44 (NSS), 96 (NSS), 155 (NST), 225 (NHS), 329 (NGT), 463 (NTS), 478 (NVS), 1547 (NAT), 1635 (NTT), 1705 (NTT), 1762 (NMS), 2013 (NGS)15. PKA {K/R-K/R-X-S/T} biD: 464 (KRNT), 687 (KRST), 1700 (RRDS), 1773 (KRPS), 1922 (RRSS), 1932 (RRQS)15. PKC {S/T-X-K/R} balD: 45 (SSK), 81 (SQR), 91 (SKK), 228 (SGK), 502 (SRR), 646 (SMK), 683 (TKR), 913 (SFR), 1310 (SNR), 1670 (TKR), 1695 (SDR), 1707 (THR), 1724 (TEK), 1788 (SHK), 1878 (SER), 1902 (SRR), 1905 (SPR), 1917 (SHR), 1966 (SSK), 1977 (STR), 2032 (SYR), 2046 (SFR), 2052 (SDK), 2123 (SHR)15.

II

l_e_n_t_ry_42

----'_

Southerns The

QIS

subunit is encoded by a single gene in the mouse

42-25-01: Southern blots of mouse (strain 129/ReJ) liver DNA, cut with restriction enzymes Apal, BamHI, EcoR! and Kpnl and probed with different regions of the rabbit skeletal muscle DHP receptor cDNA, are consistent with a single gene encoding the mouse DHP receptor80. Comparison of the blots of DNAs from wild-type and mutant (mdg) mice with muscular dysgenesis, probed with cDNA segments corresponding to different sections of the rabbit coding sequence, revealed differences between +/+ and mdg/mdg DNAs in at least two regions of the mouse gene encoding the skeletal muscle DHP receptor80 . Southern blots of rabbit and human DNAs cut with the restriction enzymes EcoR! and BamHI and probed with a rabbit alS cDNA at high stringencyt are consistent with a single gene. Probing at reduced stringency reveals other sequences related to the alS gene38 .

A single gene encodes the rbC-I and rbC-II Ql subunits in rat 42-25-02: Southern blots of digests of rat genomic DNA with the EcoRI, Pstl, HindllI and EcoRV restrictiont endonucleases gives identical patterns of single bands when probed with oligonucleotides specific for the rbC-I and rbC-II al subunit coding sequences. The conclusion that a single gene encodes the two subunits by differential splicing was confirmed by PCR analysis and by direct sequencing of genomic clones2 . The exons t encoding the rbC-I and rbC-II domain IV S3 segments are separated by an intront of 632 bp, with the rbC-I-specific exon located upstreamt of the intron2 .

Restriction map of the human CACNLIA4 gene 42-25-03: The human CACNL1A4 gene that encodes the alA subunit of the P/Q-type Ca2 + channel has been isolated as a set of ten overlapping cosmidt clonest. The DNA cloned in each cosmid has been restriction mapped and the positions of the BcoRI sites presented105. The collection of ten cosmids, covering >300 kb of genomic DNA, is available on request (see 42-10-02).

Southerns of the mouse

Qle

gene

42-25-04: Southern blots of mouse (DBA/2) genomic DNA, digested with BgID, Ndel, Pstl and Xhol and developed with an alC cDNA probe, are shown in ref. 153 . These blots also show the genomic rearrangement in both alleles of the alC locus in the mouse MELC erythroleukaemic cellline153 (see paragraph 42-21-09).

STRUCTURE AND FUNCTIONS

Domain arrangement 42-27-01: See Subtype classifications, 42-06.

_"--

e_n_try_4_2__1

Domain conservation Mutations affecting the pore region alter cation selectivity 42-28-01: Certain deletion mutants of the Shaker K+ channel display functional similarities to voltage-gated Ca2+ channels (see Selectivity, 42-40 and ref. 189). This may be due to a Gly-Asp pair in the K+ channel deletion mutant matching a Gly-Glu pair in three of the four pore-region domains of a Ca2 + channel. Mutation of the acidic Asp to Glu alters the Ca2+-blocking affinity rvl0-fold (see Selectivity, 42-40). Mutations changing the equivalent residues (K1422 and A1714) to glutamates in Na+ channels result in Ca2+ channel-like ion conduction properties190 (see Domain conservation under VLC Na, 55-28 and Selectivity under VLC Na, 55-40).

Domain functions (predicted) The multimeric nature of L-type channels 42-29-01: L-type channels: Native L-type channels are a complex of five protein subunits, aI, a2, {3, 'Y and 8 (for reviews, see refs. 34,35,191-193). The a1 subunit constitutes the central functional component of the complex, contains the receptor sites for calcium channel antagonists (see below) and can function independently as a voltage-gated ion channel in heterologous expression systems (see [PDTMj, Fig. 4). The alS subunit of skeletal muscle is present as a minor, full-length species of 214 kDa and a major, processed form sometimes referred to as ad 175), but shown to have an Mr of 190 kDa by Fergusont plot analysis8 .

The mode of excitation-contractiol1 coupling is determined by the subunit

0'.1

42-29-02: L-type channels: The putative intracellular loop connecting homologous domains IT and III (see [PDTM), Fig. 4) was found to be sufficient to induce direct calcium release through E-C coupling t in skeletal muscle 194 . The IT-III loop domain is likely to interact with the SR Ca2+ release channel or may be coupled through an additional 95 kDa protein195. Chimaerast of the skeletal and cardiac L-type channels expressed in dysgenic myotubes (see Phenotypic expression, 42-14) have shown that if the IT-III cytoplasmic loop region derives from skeletal muscle, induction of Ca2 + release does p.ot require extracellular Ca2 +194 .

The DHP-binding site on L-type channels is external 42-29-03: L-type channels: Most DHP derivatives used in electrophysiological experiments are uncharged molecules at physiological pH and their access to the DHP-binding site is not limited by the lipid bilayer of the cell membrane. When a fully charged, quaternary DHP derivative, SDZ 207-180 (1 ~M), was used, external but not internal application blocked L-type channel current in a voltage-dependent manner196. Note: that dihydropyridines bind strongly to lipid bilayers and concentrate along the surface of the bilayer197.

Sites of DHP and phenyalkylamine binding to 0'.1 subunits identified 42-29-04: L-type channels: The first repeat of the putative transmembrane helices is required for fast-activation kinetics of the Ca2 + current as seen in

II

l_e_n_t_ry_4_2

---'_

cardiac muscle198 (see Activation, 42-33). The DHP-binding site of the QlS subunit of L-type channels has been localized by photoaffinityt labelling followed by limited proteolysis and peptide mapping. Diazipine and azidopine label peptides spanning 989-1022, within the extracellular loop N-terminal to the sixth membrane-spanning segment of the third repeat (IIIS6)10,11. Isradipine, whose photoreactive group is intrinsic to the binding centre of the antagonist, covalently labels a peptide corresponding to residues 1023-1088, including transmembrane segment IIIS6 and adjacent extracellular and intracellular residues 199. Dihydropyridines also photoaffinity label a sequence in the region of IVS6 11,200. The latter sequence, extending from E1349 to W1391, is also involved in phenyalkylamine binding11 . A model for dihydropyridine binding to the L-type Ca2+ channels proposes a hydrophobic binding cleft at the extracellular end of the interface between domains III and ~ allowing DHP binding to affect domain interactions that are important in channel gating199. (See 42-29-05 for the determination of sites that are crucial for DHP action).

Mapping of critical sites for dihydropyridine action 42-29-05: L-type channels: Critical sites for the action of dihydropyridines (DHPs) on the Ql subunits of L-type Ca2+ channels have been mapped by construction of chimaerict subuits in which segments of the DHP-insensitive rat brain alA subunit, BI-2, were introduced into the cardiac ale subunit. Currents produced by chimaeric channel subunits were analysed after heterologous co-expression with Q2 and 13a subunits in Xenopus oocytes. Currents produced by chimaeric channels containing the S3-S6 region of motif IV of the BI-2 subunit were insensitive to the DHP agonist, (+)-(S)-202-791, and the antagonist, (-)-(S)-202-791 (both at 1 JlM). A chimaera containing the S3-S5 region of motif IV of the BI-2 subunit showed normal sensitivity to both DHPs, but one in which the substitution covered the region S2-S6 of region IV showed a dramatic decrease in the sensitivity to the DHP agonist, (+)-(8)-202-791 (ECso 213 OM, compared with ECso == 89 OM for the control QlC channel), and to other DHP agonists, while retaining normal sensitivity to antagonists (ECso 787 OM). These data show that the important area for DHP action on QlC subunits is the S5-S6linker of motif I~ and that there are distinct sites of interaction for the agonist and antagonist enantiomers of 202-791 201 • Note that these findings contradict the model based on photoaffinity labeling studies (see paragraph 42-29-04), suggesting that the sites labelled by photoaffinity ligands are not important for functional effects of DHPs.

The Ole subunit contains a Ca 2+ -binding motif 42-29-06: L-type channels: The site required for Ca2+-dependent inactivation of L-type cardiac Ca2+ channels is a Ca2+ -binding EF-handt motif at residues 1499-1533 in the C-terminus of the rat alC subunit202,203 (see Inactivation, 42-37). A conserved motif in the I-II linker of the

01

subunit binds to the {3

subunit 42-29-07: L-type channels: 3sS-labelled rat Ca2+ channel 13la, 13lh and 133 subunits prepared by coupled in vitro transcription and translationt of the

II

_ entry 42

1.-.---

_

cognate cDNAs bind to the QIS subunit of rabbit skeletal muscle after separation by SDS-PAGEt and transfer to nitrocelluloset 2 0 4 . Cloning of small fragments of the QIS eDNAt into an expression vectort allowed the identification of peptide 'epitopes' that were capable of interacting with the radiolabelled I3tb subunit probe. All interacting peptides shared a 45 amino acid sequence extending from amino acid 341 to 385 of the atS subunit204 • This region is located within the putative cytoplasmic linker between repeats I and II of the QIS subunit (see {PDTM}, Fig. 4). Similar regions of the rabbit QlC-a, rabbit QlA and rat QlB subunits also allowed in vitro interaction with the labelled ,BIb subunit, even though the I to II linker sequences of the four Ql subunits show only 19% overall identity. Sequence comparisons of the interacting Ql subunit peptides reveal an interaction motif that can be minimally described by QQ-E-L-GY-WI-E, which is always found 24 amino acids downstream of the S6 transmembrane element of repeat I. Site-directedt mutagenesis and directed deletion of the codonst for residues in this motif confirms their importance in the interaction with the,B subunit and in the in vivo modulation of channel properties204 . A glutamate in SS2 repeat III is a determinant of ion selectivity 42-29-08: L-type channels: The peptide loop joining transmembrane regions S5 and S6 in all four repeats of the Ql subunit is hypothesized to fold back into the membrane to form the lining of the channel. These SS2 segments of the four repeats of the at subunits each contain a similarly placed glutamic acid (E) residue. Mutational alteration of these E residues in repeats III and N changes the ion permeation properties of the channel. The channels containing the QlA subunit with the change E1469Q, produced by transient co-expression of cRNAs encoding QlA, Q2 and ,B subunits in Xenopus oocytes, shows a shift in reversal potential t from the 57.6mV of the wild-type channel to 37.5mV. Oocytes expressing E1469Q channels also pass much higher ratios of outward currents to inward currents than those expressing wild-type channels. The E1469Q change also reduced the sensitivity of the channel to block by Cd2+, changing the ICso from rv 1 J.lM to rv200 J.lM. The mutation had no significant effect on the sensitivity of the channel to C02+ or Ni2+, and marginally decreased the sensitivity to Li3+. The corresponding mutation in repeat ~ E1765Q, had a smaller effect on the reversal potential, changing it to 49.8 mV, but no significant effect on the sensitivity to the inorganic cations205 • (Note that the corresponding amino acids to the E1469 and E1765 of QlA in the rat Na+ channel II are K1422 and A1714: changing either of these Na+ channel residues to E altered the permeation properties of the Na+ channel to resemble those of a Ca2+ channel190 (see VLC Na, entry 55).

The C-terminal tail of the cardiac opening

Gle

subunit affects channel

42-29-09: L-type channels: The cardiac QlC subunit has an intracellular cterminal ltail' of 665 amino acids. The expression in Xenopus oocytes of cDNAs containing deletions, producing QlC subunits lacking from 307 to 472 amino acids at the C-terminus, led to Ba2+ currents that were 4- to 6fold higher than those obtained with the wild-type QlC. There was no

II

lL.....-.._ _ _ry_4_2

_

en t

change in the amount of charge movement during voltage-dependent gating, or in the unitary conductancer. Removal of up to 70% of the intracellular Cterminal sequence increased current density by facilitating the coupling between the voltage-dependent gating and channel opening, leading to an increased Popen for the mutant channels206 . The intracellular action of the proteolytic enzyme trypsin in ventricular myocytes can also cleave the Cterminal tail of the al subunit and generate increased currents207 (see Inactivation, 42-37-13).

Determinants of voltage-dependent inactivation kinetics 42-29-10: L-type channels: Analysis of the properties of channels containing

chimaeric t al subunits showed that the key determinants of the kinetic differences in voltage-dependent inactivation are localized to a region of <200 residues, extending from the middle of IHS to the beginning of motif II of the al subunit (see 42-37-12).

The w-conotoxin-binding site in N-type channels 42-29-11: N-type channels: The N-type Ca2 + channels are sensitive to the

peptide toxin w-conotoxin GVIA from the cone snail, Conus geographicus. Each of the four domains of the alB subunit contributes to the binding of the toxin, but al subunit chimaeras and mutational analysis show the importance of the region of domain ill that lies between illSS and illHS, close to the outside of the putative pore208 (see Blockers, 42-43-14).

Transmembrane segment 84 acts as the voltage sensor 42-29-12: N-type channels: The 84 regions of each internal repeat of the

various al subunits contain five or six positively charged amino acids (arginine or lysine), generally located at every third position and interrupted primarily by non-polar residues. By analogy with the voltage-gated Na+ 209 and K+ 210 channels, these positively charged amino acids in the 54 segments (see [PDTMj, Fig. 4) serve as voltage sensors. The outward movement of these positively charged residues in response to depolarization causes the conformational changes associated with gating of the voltagesensitive Ca2 + channels.

Predicted protein topography The skeletal muscle L-type channel visualized in the electron microscope 42-30-01: Electron micrographs of rotary shadowed, freeze-dried preparations

show the T-tubule DHP receptor to be an ovoid particle of dimensions rv16 x 22nm, with two halves separated by a small central clefr11 . The findings are consistent with the presence of one of each of the five polypeptide chains (aI, a2, 8, (3 and ,) in a pentameric complex of rv43S kDa211-213.

Linkage of other subunits to the

al

subunit

42-30-02: az-8 subunit complex: Encoded by a single gene whose primary

product is cleaved proteolytically to form the a2 and 8 polypeptides, linked by a disulphide bond t, the a2-8 complex is non-covalently associated with

11

_L.....

•

e_n_try_4_2_1

the central 0:1 subunit (see [PDTMj, Fig. 4). The, and f3 subunits are noncovalently associated with the 0:1 subunit (see [PDTMj, Fig. 4).

Protein interactions Link between VLC Ca 2+ channels and Ca 2+ -release channels in E-C coupling 42-31-01: Some subtypes of voltage-gated Ca2+ channels make direct proteinprotein interactions with calcium-release channels in E-C couplingt (see ILG Ca Ca RyR-Caf, entry 17). DHPR 0:1 and 0:2 subunits are co-localized in the junctional T-tubule membrane of rat skeletal muscle, but ankyrin label in the triad shows a distribution different to that of DHPR subunits 145. To balance the Ca2+ influx that accompanies electrical or hormonal-based opening of calcium channels, the Ca2+ ATPase and Na+ /Ca2+ exchanger in the plasma membrane terminate the Ca2+ signal by moving Ca2+ into the extracellular fluid (cf. this field under ILG Ca InsPa, 19-31 and ILG Ca Ca RyR-Caf, 17-31). See also ILG key facts, entry 14 for activating effects of elevated [Ca2+h on enzymes such as phospholipase Az and protein kinase C. Specific G protein isoform interactions have also been described for voltage-gated Ca2+ channels (see Receptor/transducer interactions, 42-49).

Ca 2+ channels and Kca are co-localized and functionally associated 42-31-02: Active Ca2+ channels and Kca channels have been co-localized within Caz+ domains in Helix neurones214. There is a functional association of these channels in the regulation of firing, since Kca channels located in areas devoid of Ca2+ channels remain quiescent during cell firing214.

Voltage-gated Ca 2+ channels can be activated by cation influx through other channels 42-31-03: In excitatory synapses, voltage-gated calcium channels can be activated when the cells are sufficiently depolarized by Na+ or Ca2+ influx derived from another channel. For example, extracellular ligand-gated (ELG) cationic influx channels such as the ATP-gated channels in muscle cells have a dual excitatory function: Na+ entry depolarizes cells to the firing thresholdt for action potentialst and stimulates voltage-gated Ca2+ entry in

Figure 4. Generalized monomeric protein domain topography model (PDTM) for a voltage-gated calcium channel 0:1 subunit. The amino acid changes in

mutant proteins are shown in the correct relative position for each variant of the 0:1 subunit, with the associated number of the amino acid in the relevant primary sequence being shown (e.g., R1920, alA, R52BHin alB, etc.). The insert shows PDTM for the 0:2, f3 and b accessory subunits. Note that only the fJ subunit is believed to be membrane-spanning. The f3 subunit is noncovalently associated with 0:1 on the cytoplasmic face of the plasma membrane, and the largely extracellular 0:2 subunit is associated with fJ by disulphide bonding. AID and BID are the mapped protein-protein subunit interactive domains on the 0:1 and f3 subunits respectively.

II

Ia1-su bunit

r---------r-----.--,

1---.------------.----------,

(D

a

~ ~

t-J

I

Repeat I

Repeat II

Repeat III

Repeat IV

54 voltage sensor in U1ll! repeat

'E-F hand' responsible for Ca-dependent inactivation of a1C-containing channel

Key

GJ3y - Mapped protein domain interaction sites with G~y DHP, S-S

subunits

- Dihydropyridine(s), Cys-Cys intersubunit covalent (sulphur-sulphur) bonds

P - Pore-forming

(P) domain by analogy to K+ channel monomers (see entry 49)

Ext, Memb, Int - Extracellular, membrane, intracellular, regions

II

NOTE: All relative positions of motifs, domain shapes and sizes are diagrammatic and are subject to re-interpretation

Ca

Channel symbol

I

111~:rn allill

_ entry 42

- - - - - - - addition to the direct entry of Ca2+ through the integral ATP receptorchannels21S,216.

N-type and Q-type channels are modulated by interaction with syntaxin 42-31-04: Co-expressiont in Xenopus oocytes of cDNAs encoding the subunits of N-type or Q-type Ca2+ channels with a sequence encoding syntaxin, a 35 kDa pre-synaptic membrane protein that is involved in synaptic vesicle docking and fusion, decreases the availability of functional Ca2+ channels via stabilization of channel inactivation. The phenomenon results in markedly reduced currents at holding potentials of -60 and -80mV in the presence of syntaxin, with the effect being overcome by hyperpolarizationt to -120mV. The presence of syntaxin shifted the prepulse voltage dependence of the normalized currents by -20mV, greatly reducing channel activity at a neuronal resting potential near -75 mY. (The mid-point voltage (Vl /2) was -83.9mV:in the control and -104.6mV in the presence of syntaxin.) A quantitatively similar effect was seen on coexpression of syntaxin eDNA with cI)NAs encoding the 0lA, {33 and 02 subunits of Q-type channels, but syntaxin was without effect on L-type channels. Syntaxin missing the C-terrrLinal 99 amino acids failed to exert the suppressive effect on N-type and Q-type channel function. It is suggested that syntaxin inhibits pre-synaptic Ca2+ entry by direct interaction with the 01 subunits of N-type and Q-type Ca2+ channels, and that this inhibition is overcome by the docking of a synaptic vesicle to syntaxin217•

N-type channels are associated with proteins of the synaptic vesicle

42-31-05: The N-type Ca2+ channel solubilized from rat brain synaptic membranes copurifies and co-immunoprecipitates218 with syntaxin, a 35 kDa plasma membrane protein that binds synaptic vesicle t proteins, and synaptotagmin, a 58 kDa Ca2+ -binding membrane protein present in neuronal axonal terminals219. The syntaxin-binding site on the alB subunit of the rat N-type Ca2+ channel has been localized to an 87 amino acid sequence, 773-859, in the intracellular loop connecting domains IT and m220• There is no such interaction with the 0lA subunit of Q-type or the 0lS subunit of L-type Ca2+ channels. The binding of 0lB to syntaxin is Ca2+ dependent, with a sharp maximal binding at '"'-120 JJM Ca2+. The synaptic membrane protein SNAP25, another component of the synaptic core complex, also interacts with the same region of 0lB subunit in a Ca2+dependent manner, with optimal binding at 18 JJM Ca2+, but the synaptic vesicle-associated membrane protein VAMP/synaptobrevin, does not bind to 01B 221 • Native N-type Ca2+ channels, purified from rat brain synaptic membranes and labelled with l25I-(v-conotoxin GVIA, showed Ca2+dependent binding to a complex of syntaxin, SNAP25 and VAMP/synaptobrevin, with maximal binding at 20 JJM Ca2+ 221 . It is suggested that synaptic vesicles are docked near pre-synaptic Ca2+ channels through the interaction between the 0lB subunit of the N-type channel and the synaptic core complex. At low, resting Ca2+ concentrations the complex between the channels and the synaptic vesicles is of low affinity, but Ca2+ influx

II

1L..-__ _ _ry_42 en t

_

greatly increases the affinity and triggers initiation of the membrane fusion process. As the concentration of free Ca2+ reaches the threshold for release (20-50 JlM), the channel-core complex binding affinity is reduced, causing syntaxin and SNAP25 to dissociate from the channel to allow membrane fusion to occur21 .

N-type channels associate with synaptotagmin and syntaxin in small cell lung cancer 42-31-06: The synaptic vesicle proteins synaptotagmin and syntaxin and Ntype Ca2+ channels are co-produced by several independent small cell lung cancer (SCLC) cell lines. Synaptotagmin and N-type channel proteins are co-precipitated with syntaxin from solubilized SCLC membrane preparations by antisyntaxin monoclonal antibodies222 .

Syntaxin and SNAP25 interact functionally with L- and N-type channels 42-31-07: Co-expression of cRNAs encoding syntaxin lA with those encoding the subunits of L-type (alc, a2/8 and 132A) or N-type (alB, a2/8 and 132A) Ca2+ channels in Xenopus oocytes produced a strong (>600/0) inhibition of wholecell Ba2+ currents and reduced the rate of inactivation in both cases. The inhibitory action of syntaxin 1A on ICa,L involves interaction with the alC subunit, since it obtains in the absence of the auxiliary a2/8 and 13 subunits. A truncated form of syntaxin, containing only amino acids 1-267 and missing the C-terminal membrane-anchor sequence, was ineffective in inhibiting currents generated by the full complement of L-type subunits, suggesting that the interaction depends on association of syntaxin with the membrane. Co-expression of cRNAs encoding SNAP25 and N-type channel subunits reduces the current amplitude (rv 29%) and shifts the steady-state voltage-dependence of inactivation by rv 10mV towards positive potentials. SNAP25 is also able to reverse the stronger, syntaxin-generated inhibition, without affecting the syntaxin-reduced rate of inactivation. SNAP25 also reduces (by rv21 0/0) the current amplitude obtained with L-type channels and partially reverses the strong inhibitory effect of syntaxin 1A. These data are consistent with the formation of complexes involving L-type or Ntype channels, syntaxin 1A and SNAP25 that are likely to be important in synaptic vesicle t docking and neurotransmitter release223 .

Interaction of N-type channel with synaptic core complex is important for neurotransmitter release 42-31-08: N-type Ca2+ channels can be extracted from rat brain membranes and partially purified in association with the synaptic core complex, containing syntaxin, SNAP25 and synaptobrevin. Syntaxin is co-immunoprecipitated from this complex by an antibody (CNB3) against a C-terminal peptide of the N-type Ca2+ channel alB subunit. This co-precipitation is blocked by incubation with recombinant peptide 'Ln_m(718-963)' (5.5 JlM), containing the synaptic protein interaction ('synprint') site from the intracellular loop connecting domains II and ill of alB. A control fusion protein, Ln_m(670-800) from the alS subunit of an L-type Ca2+ channel, has no blocking effect224 . Microinjection of Ln_m(718-963) (1.6 JlM in cell soma)

II

_ 1.....---

entry42

I ,

into pre-synaptic superior cervical ganglion neurones (SCGNs) reversibly inhibited synaptic transmission (",24% decrease in excitatory post-synaptic potentials by ",15 min, recovering to control level by 30-40 min). The Ln_m(718-963) peptide was not inhibitory to Ca2+ currents through N-type channels in SCGNs, measured by whole-cell patch-clamp recording. These results support the hypothesis that binding of the synaptic core complex to pre-synaptic N-type Ca2+ channels is necessary for Ca2+ influx to trigger the release of neurotransmitter224.

Protein phosphorylation Phosphorylation by protein kinase A activates L-type Ca 2+ channels 42-32-01: L-type: Phos/PKA: The activity of L-type Ca2+ channels can be regulated by cAMP-dependent protein phosphorylation. Skeletal muscle contractile force and L-type Ca2+ currents are increased by agents that increase intracellular cAMP. Phosphorylation of purified channels increases the number that are active in ion conductance186 (see also Sequence motifs, 42-24). Purified skeletal muscle Ca2+ channels do not form functional channels in lipid bilayers unless phosphorylated by PKA225 and their activity is linearly related to the extent of phosphorylation226 . Under basal conditions, 30-40% of the PKA phosphorylatable sites of rat skeletal muscle Ca2+ channels were phosphorylated: additional phosphorylation occurred when myocyte cAMP was elevated by 8-bromo-cAMP, forskolin, ,B-adrenergic agonists or calcitonin gene-related peptide227. Phosphorylation following stimulation by ,a-adrenergic agonists can increase macroscopic t lca by "'tenfold228, but is blocked by PKI (1 JlM), a specific inhibitor of PKA229 . Single-channel conductance is !lot affected by phosphorylation, but channels shift to gating modes with repeated or long channel openings, and more 'dormant-state' channels are activated230,231 (see also Rundown, 4239). Note that in intact cardiac myocytes, elevation of cAMP leads to phosphorylation primarily of the ,B subunit of the voltage-gated Ca2+ channel232 . The L-type channels of dorsal root ganglion233, hippocampal234 and cerebellar granule235 neurones are also activated by PKA. Facilitationt of Ltype Ca2+ channels by depolarizing voltage steps requires a protein phosphorylation event and is blocked by inhibitors of PKA236,237 (see Activation, 42-33).

The

alS

subunit is phosphorylated in vitro by PKA

42-32-02: Both the 'short' (190kDa) and the 'long' (212kDa) forms of the rabbit skeletal muscle (tIS subunit are rapidly phosphorylated in vitro by PKA, at Ser687, located in the intracellular loop between repeats IT and Ill, and slowly phosphorylated at Ser1617, within the intracellular C-terminal domain238 . The longer form is also phosphorylated by PKA on Ser1854239.

cAMP-dependent phosphorylation is ineffective following heterologous expression 42-32-03: Although the Ca2+ current (lca) of rabbit cardiac myocytes increases threefold during internal dialysis with 5 mM forskolin plus 50 mM ffiMX, Ca2+ channels obtained by expression of cDNAs encoding the QIC-a and QIC-b

II

lL....-e_n_t_ry_42

-----I_

subunits in stably transfected Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cell lines were not affected by treatments designed to stimulate cAMP-dependent phosphorylation24o . In similar experiments in stably transfected human embryonic kidney (HEK-293) cells producing cardiac alC and f32 subunits, although basal Ca2+ current was not increased by forskolin, inhibitors of PKA did decrease the basal current. This decrease was reversed by either forskolin or okadaic acid241 . It is therefore apparent that heterologously expressed Ca2+ channel subunits can be fully phosphorylated under basal conditions.

Phosphatase inhibitors increase L-type calcium currents in frog cardiac myocytes 42-32-04: The phosphatase inhibitors okadaic acid (OA) and microcystin (MC) caused large increases in L-type calcium currents (ICaL) in frog cardiac myocytes in the absence of f3-adrenergic agonists. The dose-response curves for ventricular cell lCaL fitted with a single-site relationship and K l / 2 values of 1.58 JlM for OA and 0.81 JlM for MC. These data suggest that the predominant form of phosphatase active on the L-type channels in this cell type is protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. Reducing intracellular ATP concentrations did not affect the basal ICa, but ATP-depletion completely prevented the increase in lca induced by OA or MC, demonstrating that the stimulatory effects of OA and MC on lca depend on a phosphorylation reaction. Internal perfusion of PKI(S-22), a peptide inhibitor of PKA, was without effect on basal levels of ICa, suggesting that this kinase is not phosphorylating these channels under basal conditions. Although PKI is capable of completely blocking the response of lca to isoprenaline or forskolin, it does not affect the increase in ICa induced by MC or OA. The increased lca obtained in the presence of saturating concentrations of OA or MC could be further elevated by application of a l3-adrenergic t agonist, forskolin or cAMP, showing that PKA does not mediate the OA response and that phosphatase inhibition does not result in complete phosphorylation of PKA sites242 . Comparative note: The effects of phosphatase inhibitors on L-type Ca2+ channels are generally opposite to those (i.e. decreased current amplitude) on the delayed rectifier K+ channel, lK. (For details, see Protein phosphorylation under VLC K DR [native], 45-32).

A-kinase anchoring protein required for phosphorylation of L-type channel subunits by PKA

42-32-05: Voltage-dependent potentiation of skeletal muscle L-type Ca2+ channels requires phosphorylation by PKA. Potentiation by endogenous PKA is prevented by a peptide derived from the conserved kinase-binding domain of a PKA-anchoring protein (AKAP). This peptide does not inhibit potentiation in the presence of exogenous catalytic subunit of PKA (2 JlM), suggesting that kinase anchoring is specifically blocked by the AKAP peptide. The AKAP peptide did not affect the level or voltage dependence of basal Ca2+ channel activity before potentiation, suggesting that proximity between the skeletal muscle Ca2+ channel and PKA is critical

II

_L..-

e_n_try_4_2_1

for voltage-dependent potentiation of Ca2+ channel activity but not for basal activitr43,244. In HEK-293 cells transiently expressing cardiac L-type Ca2+ channel O:le and f32a subunits, phosphorylation of the 0:1 subunit by activated PKA, but not that of the f32a subunit, depended on co-expression of functional AKAP79244. The in vivo phosphorylation of the 0:1 subunit was reflected in increased currents in response to forskolin or cell-permeant cyclic AMP analogues244. The crucial site for PKA-dependent regulation of the cardiac L-type Ca2+ channel appears to be Serl928 in the 0:1 subunit: channels containing 0:1 subunits with an Sl928A substitution do not show increased phosphorylation or higher peak currents after PKA activation244. 42-32-06: N-type and P-type: Phos/PKA: The al subunit of N-type Ca2+ channels from rabbit brain is phosphorylated by PKA245, and cAMP enhances P-type currents obtained by expression of rat cerebellar mRNA in Xenopus oocytes246 .

Activation of PKC potentiates synaptic transmission 42-32-07: Phos/PKC: The activation of PKC by phorbol esters enhances neurotransmitter release from sympathetic neurones247 and potentiates synaptic transmission in the hippocampus248 and neuromuscular junctions249. These effects are associated with activation of voltagedependent Ca2+ channels and increased Ca2+ influx.

Activation of PKC inhibits some Ca 2+ currents but stimulates others 42-32-08: In some neuronal preparations, activation of PKC can lead to inhibition of N-type Ca2 + channel activity as well as occluding the inhibitory effect of neurotransmitters250-252. Observations with other neuronal preparations contrast with this and show stimulatory effects of PKC activation on Ca2+ currents in frog sympathetic253,254, rat255,256 and guinea-pig hippocampa1257 neurones. Activation of PKC by phorbol esters (0.2 JlM phorbol 12,13-dibutyrate) increased T-type Ca2+ currents by 30% in cultured neonatal rat ventricular cells l14.

Activation of PKC stimulates L-type channel activity in skeletal and smooth muscle cells 42-32-09: Activation of PKC with phorbol esters or diacylglycerol analogues stimulated DHP-sensitive Ca2+ currents in cultured embryonic chick muscle cells258. In vitro phosphorylation of purified rabbit T tubule membrane by PKC stimulated Ca2 + currents after reconstitution into liposomes259 and treatment of planar lipid bilayers containing T tubule membranes with purified PKC increased the average Popen of L-type Ca2+ channels by 50%260. Phorbol esters also stimulated L-type Ca2+ channel activity in rat aortic rings261, cultured aortic A7r5 cells262, rat portal vein cells263 and rabbit saphenous artery cells264.

Activation of PKC reduces G protein-mediated inhibition of Ca 2+ currents in a variety of neurones 42-32-10: External application of phorbol-12-myristate-13-acetate (PMA) (500 nM) enhances the basal IBa in cerebral cortical, CAl pyramidal, CA3

II

1'--_e_n_t_ry_42

_

pyramidal, superior cervical ganglion and dorsal root ganglion neurones of the rat and bullfrog sympathetic neurones265 . The enhancement is voltage dependent: in cerebral cortical pyramidal neurones, for example, maximal enhancement of IBa (1500/0) was obtained at a test potential of -25mV and the strength of the enhancement declined with increasing depolarization beyond this value. In the same set of neuronal preparations, the PMA application also greatly reduced the inhibitory effects of a neurotransmitter receptor agonists, including baclofen (a GABAB agonist), 2-chloroadenosine (an adenosine receptor agonist), oxotremorine methiodide (a muscarinic receptor agonist) and luteinizing hormone-releasing hormone, all at saturating concentrations. The disruptive effects of PMA were eliminated by including 200 JlM PKC(19-36), a pseudosubstrate t peptide inhibitor of PKC, in the patch pipette. Note that the PKC inhibitor did not block the inhibitory effects of the neurotransmitters themselves, only the ability of PMA to interfere with that inhibition, showing that PKC is not part of the pathway coupling transmitter receptors to Ca2+ channels, but has the ability to regulate these pathways265.

Activation of PKC in sympathetic neurones increases N-type Ca 2+ currents 42-32-11: The application of the phorbol ester PMA (500nM), to acutely dissociated adult rat superior cervical ganglion neurones increases the amplitude of voltage-gated Ca2+ currents. The stimulatory effect is voltage dependent, maximal stimulation being obtained at a test potential of ",0 mV, and is blocked by PKC(19-36) (200 JlM), a pseudosubstrate inhibitor of PKC activity. This activation of Ca2+ currents involves both N-type channels and a channel type that is insensitive to w-conotoxin GVIA. The phosphatase inhibitor okadaic acid (1 JlM) accelerates the activating effect of PKA266 .

The nature of the Ca 2 + channel a subunit is crucial in determining modulation by PKC 42-32-12: Following heterologous t co-expression of rat Ql and 13lB cDNAs in

Xenopus oocytes, whole-cell Ba2+ currents from channels containing alB and alE subunits were increased by treatment with phorbol-12-myristate-13acetate (PMA; 100nM), while those from cells expressing QlA and Qle subunits were unaffected267. Pre-incubation of the injected oocytes with the protein kinase inhibitor staurosporine (5-10 JlM), eliminated the inducing effect of PMA. The stimulation of PKC activity through the phospholipase C-dependent second messengert pathway, via activation of the metabotropic glutamate receptor, mGluR1a, also up-regulates the currents obtained from QlE + 13lb channels in Xenopus oocytes267. Staurosporine (5 JlM) blocks this glutamate-induced up-regulation267.

{3 subunits are required for PKC-dependent up-regulation 42-32-13: The IBa through Ca2+ channels produced in Xenopus oocytes by expression of QlB or QlE cRNA are not affected by the stimulation of PKC activity, unless a (3 subunit is present. Stimulation of currents by PKC activation occurred when sequences encoding (3lb (140%), (32a (126%), (33

III

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2---J

(146%) or {34 (184%) were co-expressed with those encoding the alB subunit. These effects are unaltered by co-expression of sequences encoding the 02 subunit267.

The domain I-II linker is crucial for PKC-dependent up-regulation

42-32-14: The Ca2+ channel /3 subunit binds to the domain I-II linker of 01 subunits204 (see Domain functions, 42-29). This same region is also the

crucial determinant of PKC-dependent modulation of Ca2+ channels. A chimaerict 01 subunit, 0lA/B, consisting of the entire 0lA subunit except for substitution of the 0lB domain I-II linker, produces currents that are sensitive to PKC-dependent stimulation in the presence of /3lb subunits in Xenopus oocytes, in contrast to the currents through 0lA + {3lb channels267.

Phosphorylation by PKC affects voltage dependence and kinetics

42-32-15: The action of PKC in increasing whole-cell currents through Ca2+

channels containing 0lB and OlE subunits in Xenopus oocytes is accompanied by differential effects on the voltage dependence of the currents. The I-V relationship for currents elicited by alB//3lb channels are shifted about 4 mV to the left after treatment of the oocytes with the phorbol ester PMA (100nM), while those from alE//3lb channels show a 4 mV shift to the right. In both cases, the voltage dependence of inactivation was shifted about 10mV rightwards after PMA treatment267. The rates of activation and inactivation of the currents shown by oocytes producing the OIE//3lb subunits were also significantly slowed by incubation with PMA, but those of cells making OIB/{3lb channels were unaffected267.

PKC enhances L-type and N-type Ca 2+ currents in frog sympathetic neurones 42-32-16: Frog sympathetic neurones possess both w-conotoxin GVIA (w-

CTx)-sensitive N-type and dihydropyridine-sensitive L-type Ca2+ channels. Isolation of the individual types using 'v-CTx (3 JlM) and nimodipine (1 JlM) shows that 76% of the IBa in these preparations is carried by N-type channels254 . Treatment of the cells with phorbol dibutyrate (1 JlM) enhances whole-cell currents by an average of 35C}6, with roughly similar quantitative effects on both L-type and N-type channels. The stimulatory effects of the phorbol estert were eliminated by pretreatment with staurosporine (500nM), a protein kinase inhibitor, and with PKC(19-3l) (5 JlM), a specific pseudosubstratet inhibitor of PKC. When G protein activation was varied by the use of GTP analogues, the extent of enhancement of IBa by PKC activation was unaffected, indicating that stimulation of IBa was not the result of removing tonic G protein inhibition in this case (see Channel modulation, 42-44, for contrasting examples). Studies of single-channelt currents in isolated patches showed that both L-type and N-type channels opened more frequently after application of phorbol dibutyrate, with the increased Popen being due to a decrease in the closed-time interval between adjacent openings from 68 ms to 32 ms. 'The unitaryt currents at -10 mV of N-type channels (0.72pA) were not affected by stimulation of PKC activitr54.

II

l_e_n_t_ry_42

--'_

PKC enhances neurotransmitter release triggered by Q-type Ca 2+ channels

42-32-17: Synaptic transmissiont between hippocampal CA3 and CAl neurones relies on the activities of N-type and Q-type Ca2+ channels. In the presence of w-conotoxin GVIA (1 JlM) to block N-type channels, application of the phorbol ester phorbol-12, 13-dibutyrate (1 mM), an activator of PKC, produces a delayed but sustained potentiation (222 0/0) of the synaptic response. This potentiation was eliminated by w-conotoxin MVIIC (5 JlM), a blocker of Qtype Ca2+ channels61 . PKC activation also diminishes the inhibition of Q-type channel activity caused by the interaction of neuromodulators with their receptors61 (for details see Channel modulation, 42-44).

Caveat on Ca 2+ channels in Xenopus oocytes 42-32-18: Note that Xenopus oocytes possess a small (ca. -5 nA) endogenous Ca2+ current that is insensitive to dihydropyridines, w-conotoxin and Agelenopsis aperta venom, and is blocked by the divalent cations C02+, Cd2+ and Ni2+. These currents are increased by intracellular injection of cAMP and by application of phorbol ester, consistent with regulation by PKA and PKC268 . These endogenous currents are also enhanced about fourfold by the heterologous expression of cDNA encoding the rat ,BIb subunit267. cis-Fatty acids attenuate Ca 2+ currents via activation of PKC 42-32-19: cis-Fatty acids, which activate protein kinase C, attenuate Ca2+ currents in mouse neuroblastoma cells269 . Arachidonic acid (AA) depresses hippocampal Ca2+ current in a dose- and time-dependent manner, similar to the effects of phorbol esters. A similar depression of Ca2+ currents has been observed using a xanthine-based free-radical-generating system270. The specific PKC inhibitor PKCI19-36, the protein kinase inhibitor H-7, and the superoxide free-radical scavenger superoxide dismutase (SOD) each blocked ICa depression by 70-800/0. Complete block of the AA response occurred when SOD was used simultaneously with a PKC inhibitor, suggesting that both PKC and free radicals t playa role in AA-induced suppression of lca.

The L-type Ca 2+ current in rat pinealocytes is inhibited by cGMP 42-32-20: Phos/PKG: Administration of dibutyryl-cGMP (100 JlM), or elevation of cGMP by nitroprusside or noradrenaline, caused inhibition of the L-type Ca2+ channel current in rat pinealocytes, as measured in wholecell patch-clamp t determinations. This action of cGMP was independent of cAMP elevation, since cAMP antagonists had no effect on the inhibition. The protein kinase inhibitors (1-(S-isoquinolinesulphonyl}-2-methylpiperazine (H-7) and N(2-guanidinoethyl}-S-isoquinolinesulphonamide (HAI004), blocked the dibutyryl-cGMP effect on the L-type Ca2+ channel current, suggesting the involvement of cGMP-dependent protein kinase in the inhibition by cGMp271 .

PKA phosphorylates Ca 2+ channel Ql subunits in rat hippocampus 42-32-21: Antipeptide antibodies specific for the 01 subunits of the class B, C or E calcium channels from rat brain specifically recognize pairs of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa,

II

_ entry42

I

'------------_.

respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on pre-synaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurones. Both size-forms of alB and alE and the full-length form of alC can be phosphorylated by PKA after solubilization and immunoprecipitationt. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25-50% of the PKA sites on class B N-type channels, class C L-type channels and class E Ca2+ channels. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking voltage-gated K+ channels, also stimulated phosphorylation of class B, C and E al subunits, suggesting that phosphoryation of these three classes of channels by PKA is responsive to endogenous electrical activity in the hippocampus272.

Two

Q1B

species as substrates for pl'otein kinases

42-32-22: The rat brain N-type Ca2+ channel al subunit exists as two size variants, 220 and 250kDa143 . Immunoblotting with antibodies directed against C-terminal peptides of the longer species showed that the two proteins have different C-termini273 . Both the 220 and 250 kDa forms of the alB subunit acted as in vitro substrates for PKA, PKC and PKG. In contrast, calcium- and calmodulin-dependent protein kinase IT (CaM kinase II) phosphorylated only the long form of the alB subunit273 .

Two size variants of Qle subunits as substrates for protein kinases 42-32-23: The alC subunit of the neuroneal L-type Ca2+ channel from rat brain exists in two size forms, LC2, with an apparent molecular mass of 210-235 kDa, and a C-terminally truncated shorter form, LCl, with an

apparent molecular mass of 190-195 kI)a. The longer isoform, but not the shorter, can be phosphorylated in vitro by PKA274 . Both LCI and LC2 are substrates for PKC, CaM kinase II and PKG274 .

Phosphatases inhibit PKA-dependerzt stimulation of Ca 2+ channel activity 42-32-24: Dephosphorylation: Phosphatases I, 2A and 2B have all been shown to be capable of the in vitro dephosphorylation of L-type Ca2+ channels of skeletal muscle275. Phosphatase 1 (2 flM) completely inhibits the ,B-adrenoreceptormediated increase in cardiac Ca2+ currents276, and a specific peptide inhibitor of phosphatase 1 enhances Ca2+ currents in cerebellar granule neurones235 and dorsal root ganglia277. Note that the endogenous inhibitor of phosphatase 1 is itseH dependent on phosphorylation by PKA for its activity (reviewed in ref. 278) and that the calcium-activated phosphatase 2B ('calcineurin') is implicated in the Ca2+ -dependent inhibition of Ca2+ currents279,280

ELECTROPHYSIOLOGY Note on endogenous ion channels in Xenopus oocytes: Denudedt oocytes t from Xenopus laevis are frequently used as the host cells for transient t expression of cloned sequences encoding Ca 2 + channel subunits. The

II

1'--_e_n_t_ry_4_2

_

design and interpretation of such experiments is complicated by the presence of several voltage-dependent ion channels endogenous to the Xenopus oocyte. The most prominent such conductance is a Ca 2+-dependent Clcurrent. Interference from this is usually prevented by recording Ca 2+ channel activity with Ba 2+ as the charge carrier, in a barium-methanesulphonate solution: Ba 2+, in contrast with Ca 2+, does not activate the Clcurrent281 and methanesulphonate does not permeate the Cl- channe1282 . The use of Ba 2+ has the additional advantage that it tends to block endogenous K+ channels. Endogenous voltage-dependent Na+ channels can be blocked by including tetrodotoxin in the bath solution. Endogenous Ca2+ channels activating around -25 m V and giving peak currents at +10mV have also been described in non-injected oocytes282-284. Currents with these characteristics are activated when Xenopus oocytes are injected with human neuronal f32 subunit cRNA alone, or in combination with a2b cRNA 15. This behaviour is in contrast with that of oocytes injected with rabbit skeletal muscle a2a and f31 cRNAs, which apparently do not develop an IBa upon depolarization 5 . f",J

Activation Ca 2+ channels vary in their sensitivity to depolarization 42-33-01: The different voltage-activated Ca2+ channels show marked differences in their sensitivity to depolarization t . The Ca2+ channels that are activated by small depolarizations, termed 'low voltage activated (LVA) Ca2+ channels', also tend to show rapid, voltage-dependent inactivation. The 'high voltage activated (HVA) Ca2+ channels' often lack rapid inactivation, and can therefore be detected without interference from LVA currents by starting from depolarized holding potentials (e.g. -30 mV).

Sequences in repeat I of the L-type Ql subunit are critical in determining activation kinetics 42-33-02: Heterologous t expression of eDNA constructs encoding chimaeras t of the skeletal (CaChl) and cardiac (CaCh2a) muscle a1 subunits in dysgenic mouse myotubes shows that refeat I determines the activation kinetics of the channel following depolarizing steps from a holding potential of -80 mV. All functional chimaeric channels containing repeat I from skeletal muscle showed 'slow' activation (Tact 10-100ms), and all those with repeat I from the cardiac muscle subunit had 'rapid' activation (Tact 1-7ms)198. Analysis of tail currents t obtained after stepping from a strongly depolarizing potential gives the same conclusion: the region of repeat I determines the kinetics 198. More detailed mapping with chimaeric t channels shows that it is the structures of the 83 segment and the linker connecting 83 and 84 segments of repeat I that are critical for the difference in activation kinetics between cardiac and skeletal muscle L-type calcium channels285 .

Photolysis of caged InsP3 activates Ca 2+ channels in T cells 42-33-03: T-type: Flash photolysis of a caged InsP3 analogue which is not readily metabolized to InsP4 (l-(a-glycerophosphoryl) inositol 4,5bisphosphate) is sufficient to activate plasma membrane calcium current

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_

resembling T-type voltage-gated Ca2+ channels in human T cells286 . While elevation of internal Ca2+ inactivates the channel, internal perfusion with InsP4 does not affect it. This effect of caged InsPa may be indirect, through depletion of Ca2+ stores (see ILG Ca CSRC [native), entry 18), or direct, with a plasma membrane-associated InsPa receptor86 (see ILG Ca InsPa, entry 19).

L-type currents rapidly inactivate on repolarization 42-33-04: Repolarization of the membrane after brief activating pulses produces inward tailt currents that decay rapidly as the L-type Ca2+

channels close. The time constants for the L-type currents in various preparations, and for the more stable T-type currents, are shown in Table 7.

Depolarizing pre-pulses activate L·-type Ca 2+ channels 42-33-05: Currents through L-type Ca2+ channels can be reversibly enhanced

by manipulation of the membrane potential by depolarizing pre-pulses, a phenomenon known as 'facilitation'. This behaviour of L-type channels has been extensively studied in bovine chromaffin cells297-299, where wholecell currents tripled after facilitation by depolarizing pre-pulses. The increase in current was brought about by dramatically increased opening probabilities of an L-type channel, due to long-lived openings299 . The same channels can also be activated by by repetitive depolarizations in the physiological range, such as by increased splanchnic nerve activitr98. It has been suggested that the physiological role of this increase in Ca2+ current is the stimulation of rapid catecholamine secretion in response to danger or stress298 . Voltage-dependent facilitation of L-type Ca2+ currents has also been studied in skeletal, smooth and cardiac muscle (reviewed in ref. 300), and with 01 subunits produced by heterologous expression in mammalian cell lines or Xenopus oocytes.

Facilitation of L-type Ca 2+ currents is associated with long channel openings 42-33-06: The smooth muscle Ca2+ channel ale subunit, produced by

heterologoust expression in stably transformed Chinese hamster ovary cells, generated Ca2+ currents that were potentiated two- to three-fold by strongly depolarizing pre-pulses. Analysis of single-channel behaviour showed that pre-pulse facilitation involved the induction of 'mode 2-like gating', characterized by long openings and high Popen• In this system, there was no evidence for the involvement of channel phosphorylation in the facilitation process301 . The induction of mode-2 gating by depolarizing prepulses has also been observed in chromaffin302 and cardiac303 cells.

Facilitation of L-type currents involves voltage-dependent phosphorylation 42-33-07: Facilitationt of L-type currents by depolarization can be suppressed

by inhibitors of protein phosphorylation or by the intracellular activity of phosphatase 2A. The facilitation process is normally reversible, but reversibility is blocked by phosphatase inhibitors. The voltage- and

II

(D

=' ~ ~ ~

Table 7. Time constants for L-type and T-type currents (From 42-33-04) Current type

Cardiomyocytes

L-type

0.4-0.8 (bovine ventricular cardiomyocytes: -50 mV; 35°C)287 1.7-1.9 (rat and guinea-pig ventricular cardiomyocytes: -50mV; 22°C)288 0.2-0.4 (guinea pig atrial myocytes: -45mV; 21°C)289 1.5 (rat ventricular myocytes: -50mV; 22°C)290 5 (guinea-pig atrial myocytes: -45 mV; 21°C)289

T-type

II

t

(ms)

Smooth muscle

t

(ms)

Skeletal muscle

t

(ms)

0.4 (rat smooth muscle cell 9 (tfast) and 220 (tslow ) line, A7r5: -40mV; 22°C)291 (frog skeletal muscle 0.25 (guinea-pig basilar artery fibres: -90mV; 16°C)293 cells: -55 mV; 22°C)292 5-10 (rat skeletal muscle fibres: -60 to -90mV; 17°C)294

Other cell types

t

(ms)

0.2 (chick sensory neurones: -60mV; 20°C)295

1.1 (chick sensory neurones: -60 m V; 20°C)295 6 (mouse fibroblast 3T3 cell line: -60 m V; 22°C)296

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_1

frequency-dependent facilitation ('potentiation') of skeletal muscle L-type Ca2+ currents by high-frequency depolarizing pre-pulses requires phosphorylation by cAMP-dependent protein kinase (PKA) in a voltagedependent manner37• PKA also potentiates the voltage-dependent facilitation obtained when the cardiac al subunit is produced by heterologous t expression in Chinese hamster ovary cell lines304 and with the neuronal ale subunit in Xenopus oocytes305 . Reversal of the potentiated currents was blocked by the phosphatase inhibitor okadaic acid304. These findings suggest that facilitation of L-type Ca2 + currents by pre-pulses or repetitive depolarizations involves voltage-dependent phosphorylation of the L-type Ca2+ channel or a closely associated modulatory protein236 . Note that currents produced by co-expression of alA, alB or alE subunits with (3 subunits in oocytes do not show voltage-dependent facilitation305. Frequency-dependent facilitation of L-type Ca2 + currents in cardiac myocytes and skeletal muscle fibres and of L-type and T-type currents in smooth muscle myocytes have been reviewed98 .

Ca 2+ channels in dendrites activated by post-synaptic potentials

42-33-08: The dendrites t of CAl pyramidal neurones have been shown to contain T-type, R-type and L-type Ca2+ channels, in addition to voltageactivated Na+ channels149. The T-type channels and Na+ channels were opened by subthreshold excitatory post-synaptic potentials (EPSPs t) of 10mVat the site of recording, but opening of the high voltage activated (Rtype) channels required somatically generated action potentials or trains of suprathreshold synaptic stimulation. The EPSP-activated T-type channel activity was infrequent at holding potentials of 10-15 mV depolarized from resting potential, but a 4 s hyperpolarizing pre-pulse 400 ms before synaptic stimulation increased the fractional open time in a voltage-dependent manner. This suggests that the contribution of T-type channels to EPSP amplitude and Ca2+ influx would be maximal for EPSPs occurring after hyperpolarizing inhibitory post-synaptic potentials (IPSPs t) or spike-mediated after-hyperpolarizations t 149.

Current type L-type channels in transverse tubules as voltage sensors for E-C coupling 42-34-01: Transverse tubule Ca2+ currents are small and slow, underlying the role of L-type Ca2+ channel components as voltage sensors: E-C couplingt in skeletal muscle does not depend on extracellular Ca2+ influx, nor a diffusible second messenger (see Abstract/general description under ILG Ca Ca RyRCaf, 17-01).

Current-voltage relation Voltage-dependent opening involves the movement of three to five gating charges 42-35-01: The different types of voltage-dependent Ca2+ channels all open with depolarization in a highly voltage-dependent manner. Peak Popen rises

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1'--_e_n_t_ry_42

----"_

to a maximum at large depolarizations and its dependence on voltage can be described by a Boltzmann relation t equivalent to three to five gating charges t moving across the membrane during the activation process 71 .

Voltage-dependent gating of skeletal muscle Ca 2+ channels 42-35-02: Native skeletal muscle SR channels in excised patches or incorporated into planar bilayers show a macroscopic I-V relation consistent with voltage-dependent gating. The macroscopic conductance is significantly increased at positive voltages, due to an increase in channel open time, with T remaining unchanged (linear between -lOOmV and +lOOmY, Popen rvO.l; dramatically increasing between +60mV and +120mV Popen --t rvl.0)306-309.

1- V relationships for L-type and T-type channels compared 42-35-03: The macroscopic I-V relation for both L-type and T-type Ca2+ channels are 'bell-shaped', with near zero amplitude at threshold t, maximal current at Vpeak and smaller amplitude at more positive potentials310. The I-V relation for T-type currents has its maximum about 30mV negative to that of L-type currents96, due to the higher Popen of T-type channels at the more negative potentials310.

The 1- V relations of L- and T-type currents are sensitive to shielding effects 42-35-04: The position of peak Ca2+ channel current on the voltage axis of the 1-V plot is dependent on the external cation species and its concentration,

due to shielding t effects of the cations. At concentrations in the range 2 to 25 mM, equimolar substitution of external Ca2+ by Ba2+ or Sr2+ shifts the 1-V relation of L-type currents towards more negative voltages by ca. 10mV in cardiac311 and smooth muscle 91,96 myocytes, and by as much as 20mV in frog skeletal muscle312 . Very much smaller shifts in the I-V relation are seen with T-type currents313 . Raising the external Ca2+ concentration shifts the I-V curve to the right by l-2mV/mM in cardiac, skeletal muscle312 and smooth muscle314 cells. The T-type 1-V relation shows a similar response 96 . The magnitude of the shifts caused by Ca2+, Ba2 +, Sr2 + and other divalent cations saturates at higher concentrations, with shifts of 30-40mV for both L_type 90,91,96,311,315 and T-type currents 96,313 when millimolar extracellular Ca2+ or Ba2+ is raised to 100mM. The I-V relationships for L-type and T-type Ca2+ channels are compared in Fig. 5.

Dose-response Current amplitudes plateau at high external ion concentrations 42-36-01: The amplitude of the whole-cell current through voltage-gated Ca2+ channels depends on the external Ca2+ concentration. With the latter in the range between 0.1 and 3 mM the current amplitude response is approximately linear in cardiac myocytes316 and frog skeletal muscle317, but the response decreases above this range and the current amplitude plateaus at Ca2+ concentrations of 30-50 mM. The concentration giving half-saturation was

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e_n_try_4_2----1

5 Ca//145 Cs +100

Figure 5. I-V relationship for peak L-type and T-type currents in dog atrial myocytes bathed in 5 mM Ca 2+ solution. (Reproduced with permission from Bean (1985) J Gen Physiol 86: 1-30.) (From 42-35-04)

13.8 mM in frog atrial myocytes318, 20--30 mM in frog skeletal muscle317 and only 1.2mM in guinea-pig taenia caeci cells319• The currents obtained with monovalent cations as charge carriers also saturate, but at much higher ionic concentrations. Half-saturation of the Ca2+ channels derived from rat skeletal muscle incorporated into lipid bilayers was achieved at 200300mM Na+, when I Ba half-saturated at 40mM Ba2+32o.

Inactivation Inactivation of L-type currents is voltage dependent 42-37-01: The relation between membrane potential and steady-state inactivation of L-type Ca2+ channels can be assessed by applying short ( rv 300 ms) depolarizations t to a constant potential from varying holding potentials. The sigmoidal relation fits a Boltzmannt function: for cardiac tissue321 and myocytes322 bathed in millimolar concentrations of Ca2+, Vh is rv30mV and k is rv7mV (i.e. Vh is 10-15mV positive to threshold and rv30mV negative to Vpeak) (see {PDTM}, Fig. 4). In smooth muscle myocytes, the slope factor of the Boltzmann relation is again rv7mV but the Vh is commonly 35-50mV negative to Vpeak31S,323,324. The Vh in skeletal muscle cells can be even more negative than that in smooth muscle cells312. Subthreshold depolarizations can induce significant inactivation of L-type currents in cardiac42, smooth muscle 91 and skeletal muscle32s preparations, suggesting that channel opening is not necessary for inactivation.

II

1L....-__ _ _ry_4_2

en t

_

L-type currents decline during maintained depolarization 42-37-02: Macroscopic L-type currents activated by depolarizing steps decline during maintained depolarization of cells from cardiac tissue311, smooth muscle326-328 and skeletal muscle325 . The kinetics of the decline are complex and variable, with fits to one or two exponentials321 and a slow (seconds) component329 being described for cardiac tissue preparations. For guinea-pig ventricular myocytes, the faster exponential has a time constant of 3-7 ms and amplitude 0.6, and the slower component 30-80 ms and amplitude 0.4 at rv35°C 33o . Similarly complex time courses have been described for the decay of lea,L in smooth muscle cells328~331. For guinea-pig urinary bladder myocytes, the estimated time constants for lea,L inactivation near Vpeak at 35°C were 5, 40 and 200ms332. Estimates for the inactivation time constants for lea,L from rabbit portal vein myocytes at 22°C were 1525, 80-215 and> 1000 ms314.

L-type Ca 2+ currents in skeletal muscle decay slowly during maintained depolarization 42-37-03: The L-type currents in skeletal muscle decay very slowly, by a voltage-dependent inactivation, during maintained depolarizationt 312. Single channels from rabbit T tubular membrane, incorporated into lipid bilayers and activated by treatment with BAY K 8644, showed voltagedependent inactivation with T = 3.7s at OmV333 .

Inactivation of L-type channels in neuronal cells is Ca 2+ dependent 42-37-04: L-type Ca2+ channels in a wide variety of neuronal and muscle cell preparations undergo calcium-dependent inactivation, where Ca2+ ions entering the cell during depolarizing test pulses cause or accelerate inactivation. In neurones of the mollusc Aplysia califarnica injection of the Ca2+ chelator EGTA (2-10mM), slows the rate of inactivation of lea during a depolarizing pulse from a holding potential of -40 mV. The substitution of 100 mM Ba2+ for 100 mM Ca2 + in the extracellular solution also retards inactivation334 . Similar observations have been made with guinea-pig ventricular myocytes335, rat ventricular myocytes288 and guinea-pig atrial cardioballs336 . The extent of inactivation of lea in a pituitary tumour cell line, GH3, during a 60ms pre-pulse at various depolarizing voltages correlates well with the amount of Ca2+ entering the cell during the prepulse. The inactivating effect of the conditioning pulse is eliminated by replacement of the extracellular Ca2+ by .Ba2 +337 and reduced by concentrations of external C 0 2+ or Cd2+ that give partial block of lea,L 327. Intracellular Ca 2+ -release closes L-type Ca 2+ channels 42-37-05: Intracellular release of Ca2+ from the photolabilet Icaged,t Ca2+ chelator DM-nitrophen causes inactivation of the L-type Ca2 + current in isolated guinea-pig ventricular heart cells. This Ca2+ photorelease inactivates the Ca2+ current without affecting the inactivation-associated intramembrane charge movement, or 'gating current't, whereas voltage-induced inactivation reduces the Ca2 + current and the gating current proportionally. The Ca2 +dependent inactivation apparendy closes the L-type Ca2+ channel through a mechanism that does not involve its voltage-sensing region338 .

II

_'--

e_n_try_4_2-----J

Ca 2+ -dependent inactivation of L-type currents is a localized event 42-37-06: The kinetics of IBa,L and INa,L in embryonic chick ventricular cells are unaffected by simultaneous entry of Ca2+ into the cell through Ca2+ channels that are outside the membrane patch339. This observation supports the hypothesis that Ca2+ -dependent inactivation of L-type currents involves Ca2+ ions localized within or near those open channels that have transported the ions into the ce11340.

Ca 2+ -dependent inactivation requires a Ca 2+ -binding motif on the subunit

(}1

42-37-07: L-type Ca2+ channels produced by heterologous t expression in HEK-293 cells of cDNAs encoding alC and (32a subunits show Ca2+dependent inactivation. In contrast, channels produced by co-expression of cDNAs encoding (32a and alE, the pore-forming component of a mediumthreshold, neuronal Ca2+ channel, lack the Ca2+ -dependent process. Manipulation of the alC and alE cDNAs to produce chimaerict channels showed that Ca2+ -dependent inactivation depends on a Ca2+ -binding motif (an EF-hand t ) located at amino acids 1510-1560 on the alC subunit203 .

T-type channels do not undergo calcium-dependent inactivation 42-37-08: T-type Ca2 + channels do not undergo Ca2+ -dependent inactivation: the time course of their inactivation is insensitive to the replacement of Ca2+ by Ba2+ 96,313 or Sr+ 341, and to increases in external Ca2+ concentration322. The inactivation time constants at 22°C are 1".J50ms at 20mV negative to 96 Vpeak and 1".J20 ms at Vpeak and more positive potentials ,325,342.

T-type currents inactivate at more negative potentials than L-type currents 42-37-09: The steady-state inactivation of T-type currents in a variety of muscle cell types occurs at more negative potentials than that of L-type currents. In cardiomyocytes, for example, T-type current is fully inactivated at potentials positive to -45 mV when the bathing solution contains millimolar concentrations of divalent cations322. The inactivation-voltage relation fits with a Boltzmannnt distribution function with a slope of 46 mV in cardiomyocytes313,322,341, smooth muscle myocytes 96 and skeletal muscle myotubes325 . The Vh is 1".J-70mV at millimolar concentrations of external Ca2+ for the T-type channels in cardiomyocytes313,322 and 1".J-80mV in skeletal muscle cells325 and smooth muscle myocytes 96 .

Inactivation rates depend on (3-subunits 42-37-10: The kinetics of inactivation of Ca2+ channels formed by heterologousteo-expressiont of cRNAst depend on the identity of the (3 subunit. For channels containing the rabbit alA subunit produced in Xenopus oocytes, inactivation was :most rapid for the alA + a2/ 8 + (33 combination (7 = 23 S-l), less rapid for alA + a;/8 + (31 (7 = 16 S-l) and slowest for alA + a2/8 + (32b complexes (7 = 9 S-l )13. The same three (3 subunit isoforms impose the same relative order of inactivation rates on the co-expressed alC subunit52 . For any particular (3 subunit, the rate of decay of alA current was at least an order of magnitude faster than that of

II

1'--_e_n_t_ry_4_2

_

the corresponding ale current13. In other studies, the rat brain {32a subunit dramatically slowed the inactivation of the alA current obtained by transient expression in Xenopus oocytes 7 .

Molecular determinants of voltage-dependent inactivation 42-37-12: The difference in the rates of voltage-dependent inactivation of Ca2+ channels containing the alA and doe-1 (see paragraph 42-21-04) al subunits have been exploited to localize the determinants of the inactivation process. Following co-expression of cRNAs encoding the aI, a2 and {31 subunits in Xenopus oocytes, channels containing the doe-1 al subunit inactivated two- to threefold faster than those contining alA subunits. (These recordings were carried out with Ba2+ as the charge carrier, to minimize the effects of Ca2+ -dependent inactivation; see paragraphs 42-3705 to 42-37-07). Analysis of the properties of chimaeric t channels showed that the key determinants of the kinetic differences are localized to a region of <200 residues, extending from the middle of IH5 to the beginning of motif IT of the al subunit. Translocation of this region from the doe-1 into the alA subunit resulted in a channel with kinetic behaviour closely resembling that of the channel containing the doe-1 subunit itself343 . These kinetic effects due to differences in the al subunit were equally manifest in the presence of {3I, {32b and {33 subunits. In addition, the small region of the I-IT loop that is responsible for the interaction of al with the {3 subunits (see paragraph 42-29-06) has no effect on the inactivation rate of the Ca2+ channe1343 .

Intracellular proteolysis slows inactivation of L-type channels

42-37-13: Intracellular dialysis of trypsin t (1 mg/ml) into guinea-pig ventricular myocytes increased Iea,L threefold and slowed inactivation: tl/2 for inactivation was increased from 25 ms to >250 ms207. Carboxypeptidase At (1 mg/ml) had a similar action on the current, but leucineaminopeptidase t was without effect. Inactivation rates obtained with Ba2+ as charge carrier were not affected by proteolysis, suggesting that the slowing of Iea,L was due to loss of Ca2+ -dependent inactivation. In porcine coronary artery smooth muscle cells, trypsin (1 mg/ml) and carboxypeptidase A (1 mg/ml) produced a fourfold increase in Iea,L without affecting the kinetics344 . Lower concentrations of trypsin (0.05-0.1 mg/ml) dialysed into A7r5 smooth muscle cells increased Iea,L with no change of inactivation rate, but the enhanced IBa,L was associated with a marked slowing of inactivation345 •

Kinetic model Opening of L-type channels modelled to involve two voltagedependent steps 42-38-01: For the voltage-dependent activation of L-type Ca2+ channels, opening has been modelled as a two-step process involving the transition from one closed state to another before the open state is reached: both these steps are assumed to be voltage dependent297. Appropriate choices for the rate constants governing the reversible transitions between the closed

II

_ _ _ _ _ _ _ _ _ _ _ _ _.

en_t_ry_4_2----11

and open states describes the behaviour of single L-type channels during depolarization. The model has been embellished to describe the activation of each of the Ca2+ channel types in sensory neurones (see Kostyuk et al., 1988, under related sources and reviews, 42-56).

Rundown Rundown involves dephosphorylation

42-39-01: The HVA Ca2+ channels of vertebrates and molluscs display rapid rundown in the absence of an A kinase phosphorylation system346,347. The intracellular agents which slow this process, including ATP, Mg2+, the catalytic subunit of protein kinase At and cAMP, indicate that rundown, which has three distinct phases, is due in part to loss of phosphorylation348 . Channel inhibition could also be due to endogenous calcium-independent phosphatases349, calcium-dependent proteases350 and calcineurin, a calcium-dependent phosphatase that dephosphorylates the channel causing inactivation. The L-type Ca2+ channe:ls in patches excised from smooth muscle cells show rundown, but their activity can be retained for several hours by bathing the inside surfaces with buffer solutions containing mM EGTA351-353. f"'.J5

ATP or ADP plus Mg2+ inhibit rundown of L-type Ca 2+ channels 42-39-02: In isolated chromaffin cells, the rundown of HVA Ca2+ channels was abolished by ATP at concentrations above 0.4 mM. Inosine 5'trisphosphate (2mM) could not replace ATP, whereas ·GTP could, but at higher concentrations. The effect of .ATP in blocking rundown required MgCl2 and the liberation of a phosphate group, since the ATP analogue 5'adenylyl-imido-bisphosphate (AMP-PNP) could not substitute for ATP. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range, even when the pathways converting ADP into ATP were blocked; GDP was ineffective. The inhibition of rundown by ATP or ADP was abolished by increasing the internal Ca2+ concentration (from pCa?? to pCa6.0, where pCa=-loglo[Ca2+]). Mg-ADP (ImM) in the bathing solution did not prevent rundown of the Ca2+ channels in the inside-out patch recording configuration354.

Ca 2+ -dependent proteases can be a major cause of rundown 42-39-03: In molluscan (Helix) neurones, leupeptin, a general inhibitor of Ca2+ -dependent proteases, protects against rundown, especially in the presence of ATp355. The rundown of Ica,L in guinea-pig ventricular myocytes was accelerated by dialysis with calpain, a cysteine endopeptidase, and inhibited by dialysis of calpastatin, a specific inhibitor of calpain activity348.

Rundown is aggravated at depolarized holding potentials 42-39-04: The whole-cell Ica,L in frog ventricular myocytes declines in two phases during pulsing from a holding potential of -40 m V: a faster phase (t 40s), reversible by shifting the holding potential to -90 mY, and a slower (t 15 min), irreversible phase of rundown. The biphasic decay was f"'.J

f"'.J

f"'.J

II

i_

e_n_t _ry_4_2

-----'_

absent at a holding potential of -90mV, but strongly accelerated at holding potentials positive to -60mV356 . Enhancement of rundown at holding potentials positive to -80mV has also been seen with rat 357 and rabbit358 ventricular myocytes and with frog atrial359 and ventricular313 cells. Note that rundown results in functional uncoupling of voltage-dependent charge movement from ion-permeation, since the Ca2+ channel gating current remains unchanged as the ionic current runs down360-362.

Note: The LVA or T-type Ca2+ channels rundown much less rapidly under whole-cell recording conditions.

Selectivity Voltage-gated Ca 2+ channels are permeable to several divalent cations 42-40-01: The voltage-gated Ca2+ channels are generally highly permeable to Ca2+, Sr2+ and Ba2+, though the relative order of permeability depends on the channel type. For L-type and N-type channels, higher currents are obtained with Ba2+ than with Ca2+, whereas T-type channels give similar conductances for these two ions. In addition to giving elevated currents in L-type channels, Ba2+ ions also block K+ currents, making them preferable to Ca2+ ions for the electrophysiological study of L-type channels. Note that Mg2 + ions do not permeate the L-type Ca2 + channels of skeletal muscle fibres 363, cardiac tissue364 or smooth muscle myocytes365.

Ca 2+ channels show anomalous mole-fraction dependence 42-40-02: Like several other ion-channel types, including inwardly rectifying K+ channels, delayed rectifier K+ channels and KCa channels, voltage-gated Ca2+ channels show a behaviour called 'anomalous mole-fraction dependence'. The conductance of L-type Ca2+ channels, for example, is high with external Ca2 +, t increased with Ca2 +-free Ba2 + solutions, but decreased when Ba2 + ions are added to Ca2 +-containing solutions316,317. These observations have been interpreted to support a single-file, multi-ion pore modeI316,317,366,367, in which the Ca2+ channel pore is capable of holding two or more divalent cations at the same time. Ca2+ binds more tightly to the channel than Ba2+, and therefore dominates in the binding competion, but leaves the channel to pass through more slowly than Ba2+. A more extreme example of the same phenomenon can be seen when Ca2 + is added to divalent-free solutions containing monovalent cations. At very low Ca2+ concentrations «7 nM), L-type Ca2+ channels from frog skeletal muscle are highly permeable to Na+. As Ca2+ is added, the Na+ current is titrated away, Ca2+ behaving as a channel blocker with a K D of 0.7 JlM. Current carried by Ca2 + ions returns as the [Ca2+] rises into the millimolar range (see Hille, 1992, under Related sources and reviews, 42-56).

L-type channels are highly permeable to several monovalent cations 42-40-03: In the absence of divalent cations, external monovalent cations can carry inward currents through Ca2 + channels. External Na+ can support large currents in cardiac myocytes316, skeletal muscle fibres 317, urinary bladder

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entry 42

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smooth muscle368 and from single channels incorporated into lipid bilayers369. (Compared with the single-channel conductance of rv20pS obtained with 110mM Ba2 +, that measured in the presence of 110-150mM Na+ was 75-110pS in guinea-pig cardiac myocytes370.) External Li+, Cs+ and K+ can also carry inward currents in cardiac tissue371,372. Organic cations such as the tetramethylammonium ion can permeate through Ltype Ca2 + channels in skeletal muscle and cardiac ventricular myocytes373,374. Permeability to tetramethylammonium ion determines that the minimum pore diameter is 6A374, a value that was confirmed by studies of permeability to monovalent cations in Ca2 + channels from rat skeletal muscle transverse tubules incorporated into planar lipid bilayers375 . In these in vitro preparations, NHt ions produced a higher single-channel conductance than Na+, Li+, K+ or CS+ 375. The reconstituted T-tubule channels had a fourfold lower conductance for hydrazinium ions than for ammonium ions, although these ions are amost identical in size375.

T-type channels are permeable to monovalent cations 42-40-04: The T-type Ca2 + channels of chick dorsal root ganglion (DRG) cells are permeable to monovalent cations at low external Ca2+ concentrations «100 JlM). The permeability ratios for monovalent ions with reference to internal Na+(1.0) were Li+(1.0), K+(0.45), Rb+(0.45) and Cs+(0.33)376.

Reversal potential for Ca 2+ channels is strongly influenced by K+ permeability 42-40-05: Voltage-gated Ca2+ channels are highly selective for Ca2 +, i.e. they generally show rv1000-fold preference for Ca2 + over K+ and Na+ {e.g. see ref. 377 for cardiac Ca 2+ channels}. Note that the internal K+ concentration is usually 106 times higher than that of Ca2+ ions so that, despite their lower permeability, K+ ions still carry the majority of the outward current through Ca2 + channels under strong depolarization. This means that the reversal potentialt (Erev ) for Ca2 + channels is not at the thermodynamic Eca (+124mV), but at a much less positive value. The measured value of Erev in the presence of known extracellular and intracellular concentrations of two competing ions allows calculation of the relative permeabilities of the two ions. Using this approach, PCa/PNa and PCa/PK are in the range 1000-11000 in frog atrial myocytes318 and guinea-pig ventricular myocytes370,377. The value of PCa/PCs in guinea-pig ventricular myocytes has been estimated as 4200-10000370,378, and PBa/Pcs as 1356_1700311,370,379. Similar values of PCa/PCs and PBa/Pcs have been derived for skeletal muscle and smooth muscle Ca2 + channels. The general order of ion selectivity for L-type channels is Ca > Sr > Ba > Li > Na > K > Cs (reviewed in ref. 380). Note that this order is directly opposite that determined on the basis of currentcarrying ability, and reflects the relative mobilities of the ions in dilute solutions375 . Mutant K+ channels mimic Ca 2+ channels 42-40-06: Site-directed mutagenesist of cDNAs encoding the Shaker K+ channel189 has produced deletion mutant channels having biophysical properties reminiscent of voltage-gated Ca2+ channels. In the absence of

II

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---'_

divalent cations, the mutant K+ channels conduct monovalent cations nonselectively, and divalent cations inhibit this process. Divalent cations at low concentrations block conduction through Ca2+ channels: only at millimolar concentrations of Ca2+ does Ca2 + conduction occur (see also Domain conservation, 42-28).

Changes of glutamate to lysine affect cation selectivity of L-type channels 42-40-07: Replacement of conserved glutamic acid (E) residues in the SSI-SS2 (Ipore-lining') regions of each of the first three repeats of the L-type (}:1 subunits by positively charged lysine (K) residues results in channels that carry Li+ currents, but negligible Ba2+ currents. The mutant channels effectively become selective for monovalent cations. Current-voltage relationships are also affected by these mutations, and the permeability ratio PLi/PK increased about three-fold for E736K (repeat II) relative to wild-type. The potency of Ca2 +-block of Li+ currents is reduced from 100- (E1446K: repeat IV) to 1000-fold (El145K: repeat ill). The conserved glutamic acid residues in all four repeats each make significant but different contributions to ion binding, selectivity and permeation381 .

Model for Ca 2+ selectivity and permeation involves simultaneous binding of two Ca 2+ ions 42-40-08: Data obtained with (}:1 subunits carrying mutational replacements of the four conserved glutamic acid residues in the SSI-SS2 repeats suggest that all four glutamate carboxylatest contribute collectively but asymmetrically to high-affinity binding of a single divalent cation. This binding blocks the passage of monovalent cations in the narrow pore and generates strong selectivity for Ca2+ over Na+ or K+. It is suggested381 that the four glutamates can also simultaneously interact with two Ca2+ ions, but with a greatly reduced affinity for both. This doubly occupied state is attained when a Ca2 + ion enters from outside, to compete with a previously bound Ca2 + ion for the carboxylate groups. Sharing of the ligands and possible electrostatic repulsion between the two adjacent Ca2 + ions would reduce their binding affinity and promote net influx of Ca2+ ions into the cell.

Single-channel data Popen for L-type channels is voltage dependent 42-41-01: The single L-type channels of cardiac and smooth muscle cells are activated by depolarizationt, with Popen increasing to reach a maximum at about +40 mV. In cardiomyocytes, Popen has a sigmoidal dependence on voltage, with slopes of about 7mV382 . Slopes of 4-7mV have been reported for L-type channels from arterial383-385 and airway386,387 smooth muscle cells. Note that the current elicited on depolarization to a maximally activating potential does not arise from all the functional Ca2+ channels in the cell membrane: only a fraction of the channels which open will be open at any given time after depolarization, and functional channels rapidly enter and leave the activatable state380 .

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L-type channels show two kinds of gating 42-41-02: L-type Ca2+ channels show sudden changes of gating kinetics at the single-channel level. Channels commonly show frequent short «1 ms) openings during a 200 ms depolarization. In a series of consecutive sweeps, clusters of prolonged openings, mimicking the single-channel behaviour in the presence of a Ca2+ channel agonist, are occasionally found. Kinetic description of these long openings requires a rate constant for the channel closing that is 100 times lower than that in the more typical short opening behaviour388,389. The two types of gating behaviour have been termed 'mode I' (short opening) and 'mode 2' (long opening), and the phenomenon has been called 'mode-switching'389. It has been suggested that Ca2+ channel agonists bind more strongly to channels in mode 2 and thereby favour that gating mode389. The stimulation of L-type currents by dihydropyridine agonists involves an increase in Popen. Mean open times increased from 1-2 to 5-20ms in cardiac myocytes, smooth muscle myocytes, skeletal muscle cells and non-muscle cells (reviewed in reference 98).

Popen of T-type channels increases "With depolarization 42-41-03: Single-channel T-type current has a threshold 20-30mY more negative than that of L-type currents in cardiomyocytes 90,92 and smooth muscle cells310. The Popen increases with depolarization, with the slopes of the Boltzmannt relationship in the range 4-11 my 92,310. The latency t of Ttype currents is voltage sensitive92,310: in guinea-pig ventricular cells, for example, mean first latency decreased from 11 ms at -35 mY to 3.5 ms at _10my39o.

Shielding by extracellular cations is evident in single-channel 1-V curves

42-41-04: The shielding t effects of extracellular cations (see Current-voltage relation, 42-35) are observable at the single-channel level. For example, the raising of the external Ba2+ concentration from 10 to 110mM causes a 20 mY positive shift in the single-channel I-V curves for both L-type and Ttype Ca2+ channels in guinea-pig coronary artery myocytes310.

L-type and T-type currents show multiple single-channel conductance levels 42-41-05: L-type channels in cell-attached patches can switch between different conductance levels. With cell-attached patches of guinea-pig ventricular myocytes, in addition to the common rv25pS (90-110mM Ba2+) conductance, transitions to and from a rv 17 pS subconductance are seen391,392. Five distinct sublevels, 8-9, 12-13, 16-18, 23-24 and 28 pS, have been detected in cell-attached patches of GH3 pituitary cells393 . Ttype currents in cell-attached patches of guinea-pig ventricular myocytes show a subconductance at about one-half the conductance of the predominant 7pS level92.

L-type channels show distinct modes of gating 42-41-06: Single-channel recordings of L-type currents from cardiomyocytes show two types of channel opening behaviour: 'short-opening', in which

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l_e_n_t_ry_42

_

channel openings last about 1 ms, and 'long-opening', in which opening times are 5-20 times longer388 . Long-openings represent about 100/0 of the total Ltype openings in cardiac cells303,339,388. They are also apparent in smooth muscle cells from the pig urinary bladder394 and airway395 and in bovine chromaffin cells298,396. Channels produced by expression of cRNAs encoding rabbit Qle, Q2/8 and {31 subunits in Xenopus oocytes had mean opening times of rvO.5ms and a unitary conductance of 21.4pS13.

Unitary currents through L-type channels saturate at extreme membrane potentials 42-41-07: Unitary outward currents t through L-type Ca2+ channels in membrane patches from undifferentiated PC12 cells increase linearly with membrane potential in the range +20 to +220mV. The response at higher test potentials begins to diminish and the unitary current reaches a plateau at about 240mV. At this saturating level the current is diffusion-limited, as shown by the response to changes of viscosity t (produced by adding glycerol to the test solution) and changes in the concentration of the charge-carrying cation397. Unitary inward currents carried by 170 mM external K+, an effective permeant ion in the absence of divalent cations, are linearly related to voltage in the range -10 to -340mV. Decreasing the diffusion coefficientt, by using 20-40% glycerol solutions, produces saturation of the unitary inward current, with a linear relationship between relative diffusion coefficient and the current amplitude at saturation. The different saturation values of the outward and inward currents have been used as the basis of calculations demonstrating that the functionally defined external pore entrance is almost twice as wide as the internal mouth (radii of 5.29 and 2.70 A respectively)397. Under physiological conditions, where the external Ca2+ concentration is about 1 mM, inward flow of Ca2+ is not likely to be diffusion-limited397. alA

channels produced in Xenopus oocytes show two open times

42-41-08: The Ca2+ channels produced by heterologous t co-expressiont of cRNAs t encoding rabbit alA, a2/6 and {31 subunits in Xenopus oocytes open briefly in response to depolarization from a holding potential of -80mV. The distribution of opening times was fbi-exponential', with T values of rvO.S ms and rv2.6 ms 13 . The mean single-channel conductance t was 15.9pS, and the unitary current at OmV was 0.8pA13.

N-type channels show three gating modes 42-41-09: Single-channel recordings of N-type Ca2+ channels in frog sympathetic neurones show three distinct patterns of gating, termed high-, medium- and low-Po, at test potentials of -10mV. In the high-Po mode, openings are relatively long-lasting (mean open duration rv3 ms) and closings are short (mean closed time rv2 ms): Po is typically rvO.5. The medium-Po mode is characterized by briefer openings (mean open duration rv1.6ms) and longer closings (mean closed time rv10ms), giving a Po of rvO.1S. In the low-Po mode, openings are even shorter (rv O.5ms) and closings more prolonged (rv 50 ms). Within a single sweep, direct transitions between any two gating modes are found. Noradrenaline (100)lM) in the

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.

e_n_try_4_2_

patch pipette inhibits the current by about 50% and markedly reduces the prevalence of high-Po gating39B .

Voltage sensitivity The rate of activation of L-type currents is voltage-dependent 42-42-01: The rate at which cardiac L-type Ca2+ currents reach peak activation following a depolarizing voltage-switch is increased at more positive potentials. For guinea-pig ventricular myocytes, the time to peak is but only about ams at +20m~ measured at 35°C in about 9ms at -20m~ 3.6 mM Ca2+ bath solutions 9B . The L-type currents of smooth muscle myocytes behave similarly314. The same voltage-sensitive phenomenon is seen with the L-type currents of skeletal muscle cells, but the times to reach full activation are about 10-fold elongated399.

The f3 subunit increases peak currents and shifts voltage sensitivity 42-42-02: Co-expressiont in Xenopus oocytes of cRNAs encoding /3 subunits with those specifying the QIC subunit leads to a large increase in peak currents, the magnitude of which is voltage dependent. This effect arises from a change in the voltage sensitivity of the QI subunits, with /32 causing a 9 mV displacement of the 1-V relationship towards more negative potentials46 . Co-expression of /32 coding sequences also led to a four-fold acceleration of channel activation kinetics and a 3.5-fold increase in specific binding of the DHP antagonist [3H] PN200-110. The latter observation argues for a role of the {3 subunit in increasing the number of channels at the cell surface46 .

The f3 subunit facilitates pore-opening in cardiac L-type channels 42-42-03: Gating currentst associated with the movement of charged elements in the ion channel protein have been measured for Ca2+ channels produced by expression of cDNAs encoding the rabbit cardiac QI subunit QIC-a in Xenopus oocytes. The co-expression of cDNA encoding the rabbit type 2a cardiac f3 subunit (f32a) had no effect on the magnitude or time course of the gating current, but did increase I Ba severalfold and shifted the G-V curves by 16mV towards more negative potentials. These observations imply that the f3 subunit does not modulate the movement of the voltage sensor, but interacts with the QI subunits to facilitate the opening of the poret after the voltage sensor charge has moved102.

The activation of T-type currents occurs at more negative potentials 42-42-04: The activation of T-type currents shows many similarities to that of L-type currents (see above), including the slope of the activation curve, time to peak current and increase of rate with progressive depolarization t. The activation thresholdt and Vh are generally 20-40mV more negative for Ttype than for L-type currents (reviewed in ref.9B). The rates of activation and inactivation of T-type channels are both voltage dependent over the range from -60 to -IOmV71 .

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PHARMACOLOGY

Blockers Voltage-gated Ca 2+ channels show differential sensitivity to multivalent cations 42-43-01: The sensitivities of the various types of voltage-gated Ca2+ channels to divalent cations are shown in Table 8.

Lanthanum ion is a powerful blocker of L-type channels 42-43-02: The lanthanum ion is a powerful blocker of ICa and lBa in L-type Ca2+ channels. In frog atrial myocytes, lca in the presence of 2.5 mM external Ca2 + was completely blocked by 10 JlM La2+ 400,406. Assuming a 1: 1 binding of La2+ to a channel site, the K o was estimated as < 1 JlM400 . Binding of La2+ to the channel was inhibited by increasing concentrations of Ca2 + 400 . The major effect of La2+ at the single-channel level is to reduce Popen by prolonging interburst closures371 . La2+ ions are also powerful blockers of L-type Ca2+ channels in smooth muscle407,408.

Magnesium ions are weak blockers of L-type channels 42-43-03: M g2+ is a weak blocker of ICa through L-type Ca2+ channels. In frog atrial cells, for example, 5 mM Mg2+ failed to reduce lca in the presence of 2.5 mM external Ca2 + 406 . The EC so of Mg2 + for lca (2.5 mM Ca2 +) in guineapig taenia caeci myocytes is 10mM319, and that in skeletal muscle (10mM external Ca2 +) is ",33 mM317. The effects of Mg2 + on lBa are more striking: in frog atrial myocytes, lBa at 2.5 mM Ba2+ was almost completely inhibited by 5 roM Mg2 + 406 . The blocking effect of Mg2+ is increased at more negative membrane potentials371 . Given the weakness of the Mg2 +_ dependent block in the presence of Ca2+, it is unlikely that external Mg2+ gives any significant inhibition of Ca2+ currents under physiological conditions in vivo. Note that elevated intracellular Mg2+ concentrations obtainable during metabolic inhibition can significantly inhibit ICa,L in frog 409 and guinea_pig410 cardiomyocytes.

Three main structural classes of Ca 2+ channel blockers 42-43-04: Three structurally unrelated classes of drugs, the dihydropyridines, phenylalkylamines and the benzothiazepines, have been valuable in defining different types of Ca2+ channel and evaluating their physiological roles. The structures of representative members of the three classes are shown in Fig. 6.

Blockage of cardiac L-type channels by dihydropyridines is voltage sensitive 42-43-05: L-type Ca2+ channels are characterized and defined by their sensitivity to dihydropyridines, such as nifedipine, nitrendipine and nimodipine. These drugs block L-type channels more potently if the current is elicited from depolarizedl potentials, when the channels are partially inactivated, than from hyperpolarizing t holding potentials, when the channels are in the non-inactivated 'resting' state411 . Nitrendipine at ",0.4nM causes 500/0 block of cardiac L-type channels for currents elicited

II

iii Table 8. Sensitivities of various types of voltage-gated Ca 2+ channels to divalent cations (From 42-43-01) Channel type

ICso (J,1M) for block by

Tissue Cd2+

L-type

N-type P-type Q-type T-type

Atrial myocytes (frog) Dorsal root ganglia (chick) Skeletal muscle (frog) Ventricular myocytes (guinea-pig) Dorsal root ganglia (chick) Heterologous expression alA, a218, {3l, 'Y (rabbit) in . J. (enopus oocytes Cerebellar granule cells (rat) Pituitary cell line (GH3) Atria (canine) Sinoatrial node (rabbit) Dorsal root ganglia (chick)

C0 2+

Ni2 +

Reference La2+ 1

10 300 20 10 1

401

1280 2S0

402

680

371 401

13

1 188

66 777

14 2.4

403 404

2000 40 20

400

74 401

The N-type currents in the rat neuroblastoma x glioma hybrid cell line NGI08-1S are particularly sensitive to block by gadolinium (Gd3+) ions (Kn 0.7J,1M)405.

g t"+

~ ~ ~

---l_

II-._e_n_t_ry_42 DIHYDR()PYRIDINES

PHENYLALKYLAMINES

Nitrendipine

Verapamil

0600 Nifedipine BENZOTHIAZEPINES

~FJ O,N6~OOCHJ CH)

N H

CH)

Bay K 8644 Diltiazenl Figure 6. Structures of members of the three major classes of Ca 2+ channel antagonists dihydropyridines, phenylalkamines and benzothiazepines. (From 42-43-04)

from a depolarized holding potential, but rv 700 nM is required for the same inhibition of currents elicited from a negative holding potential411 . Nimodipine is more potent than nitrendipine or nifedipine in blocking at negative holding potentials: 1 mM nimodipine blocks cardiac L-type current completely even at negative holding potentials412 . At this concentration, nimodipine has very little effect on P-type Ca2+ channels in rat cerebellar Purkinje neurones or N-type Ca2+ channels in rat sympathetic neurones 68 • Note that nimodipine (5 ~M) augments the currents through Ca2+ channels containing QlA subunits produced by heterologous co-expression of cRNAs

_'--

e_n_try_4_2_1

in Xenopus oocytes13. Dihydropyridines, including nifedipine, are used clinically for the treatment of angina, hypertension, cerebral and cardiac ischaemia and Raynaud's phenomenon. Nimodipine has been approved for the treatment of ischaemic neurological deficits following subarachnoid haemorrhage.

High concentrations of dihydropyridines have non-specific blocking effects 42.-43-06: Although micromolar concentrations of dihydropyridines are specific blockers of L-type channels, concentrations above 10 JlM can result

in non-specific blocking effects on non-L-type Ca2+ channels and on Na+ and K+ channels. Nitrendipine at 10 JlM inhibits cardiac T-type current by rv50% 404, neuronal N-type current by 10_25%413 and cardiac Na+ current by 10_50%414. This concentration of nitrendipine is without effect on Ptype Ca2+ channels in cerebellar Purkinje neurones413 . (Beware that apparent blocking effects can be influenced by the solvent in which the dihydropyridine is dissolved. Bay K 8644 dissolved in dimethyl sulphoxide stimulated Ica,L and inhibited Ica,T in neuroblastoma cells, but the inhibitory effect disappeared when the solvent was ethanol or polyethylene glycol415.)

Phenylalkylamines and benzothiazepines are blockers of L-type Ca 2+ channels 42.-43-07: The clinically useful Ca2+ channel blockers, the phenylalkylamines,

such as verapamil, and the benzothiazepines, such as diltiazem, are effective blockers of L-type Ca2+ channels. Both classes of compound have highaffinity binding sites on L-type Ca2+ channels that are distinct from the high-affinity dihydropyridine-binding sites. Verapamil and diltiazem show use-dependent block, so that it is difficult to obtain steady-state block on a reasonable time scale. The phenylalkylamine-binding site in the L-type Ca2+ channel 01 subunit has been mapped by photoaffinity labellingt and immunoprecipitationt to helix S6 of domain IV and the beginning of the intracellular C-terminal region416. These findings are consistent with the observations that D800, a quaternary phenylalkylamine, blocked cardiac Ltype Ca2 + channels from the intracellular surface of the membrane417, and that inhibition of Ca2+ currents by phenylalkylamines is greatly accelerated by depolarizations that open the channels418 . A model placing the phenylalkylamine receptor site within the intracellular opening of the channel pore, accessible for high-affinity phenylalkylamine binding only when the pore is open, has been suggestedl99. Binding of phenylalkylamines at a receptor site in such a position would directly block ion movement through the pore. Note: Diltiazem at concentrations between ~ 12 and 200 JlM has been shown to give rise to non-specific increases in ionic permeability in biological membranes419.

The binding of phenylalkylamines to the L-type channel is inhibited by divalent cations 42.-43-08: Cd2 + and other divalent cations at concentrations > 100 JlM reversibly inhibit the binding of radiolabelled phenylalkylamines to L-type

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Ca2+ channels in skeletal muscle membranes42o. Note that this behaviour is in complete contrast to the high-affinity binding of dihydropyridines, which depends on the presence of divalent cations42o .

Different classes of blocker can bind co-operatively to L-type channels 42-43-09: Binding studies using homogenates of various tissues containing Ltype Ca2+ channels have shown that dihydropyridines, phenylalkylamines and benzothiazepines occupy distinct, allostericallyt coupled sites on the Ltype channel protein (reviewed in ref.421). These allosteric t interactions between different classes of blockers are voltage dependent422,423.

Diphenylalkylamines are non-specific blockers of L-type and T-type channels 42-43-10: A number of diphenylalkylamines, including flunarizine, cinnarizine, bepridil and fendiline, act as relatively non-specific Ca2+ channel blockers. (They are also calmodulin antagonists and can block other cation channels.) Fendiline block of ICa,L in guinea-pig ventricular myocytes involves acceleration of inactivation and a negative shift in steady-state inactivation424 . In the presence of fendiline, Bay K 8644 caused a further reduction of ICa,L, explained as involving an allosterict interaction between fendiline and Bay K 8644424. In guinea-pig atrial and ventricular myocytes, cinnarizine, flunarizine 425 and cinnarizine426 were more effective at blocking T-type (KD rv 1 JlM for inactivated channels) than L-type channels.

Some sodium channel blockers can also block L-type Ca 2+ channels 42-43-11: A number of lipophilic compounds that block Na+ channels, including quinidine, flecainide, propafenone, phenytoin, amiodarone, amiloride and prajmalium, can also block L-type and T-type Ca2 + channels when used at relatively high concentrations. The actions of these compounds on L-type and T-type Ca2+ channels have been reviewed98 •

Miscellaneous synthetic blockers of L-type Ca 2+ channels 42-43-12: Because of the potential value of pharmacological modulation of Ltype Ca2+ channel activity, a wide range of synthetic organic ligands have been investigated. Relevant observations on some of these L-type channel ligands are presented in Table 9.

L-type and T-type channels are blocked by extracellular protons 42-43-13: The current through L-type channels in cardiac ventricular and atrial tissue is blocked by reduction of the external pH by 1.5-3 units 433,434. Blockage of lBa and lsr was more severe than that of lCa 434. Protonationt of a site at the external surface of the L-type channel reduced single-channel currents threefold with Na+ as the charge carrier, without affecting currents obtained in the presence of 110 mM external Ba2+. The apparent pK of the external protonation site was affected by the nature of the charge-carrying ion: the pK of 8.6 obtained in the presence of Cs2+ was reduced to 8.2 and 7.4 with K+ and Na+, respectively, as charge carriers. It

II

_ entry42

I

"'-------------

Table 9. Miscellaneous antagonists of L-type Ca 2+ channel activity (From 42-43-11)

Compound (type)

Observations and references

McN5691 (ethynylbenzenealkanamine)

2 JlM McN5691 abolishes L-type Ca2+ currents in neonatal rat ventricular myocytes without affecting those in rat anterior pituitary (GH3) cells. The blockade is voltage-dependent, and binding of [3H]-DHP is competitively inhibited. The ICso for inhibition of Ca2+ influx into heart cells by McN569l is 7.6nM, 100-fold lower than that obtained. with GH3 cells427.

HOE166 (benzolactam)

Binds to skeletal muscle channels with a K D of 0.27 nM: gives voltage-dependent block of L-type current in smooth muscle cells (A7r5) at 10-lOOnM428 .

MDL1233A (lactamimide)

Inhibits L-type current in rat anterior pituitary (GH3) cells at micromolar concentrations, without affecting T-type currents429.

BMY20064 (DHP derivative)

A hybrid molecule that is as potent as nifedipine as an L-type Ca2+ channel antagonist in smooth muscle, but also acts as a potent Ql adrenoreceptor antagonist43o.

Niguldipine (DHPdiphenylpiperidyl hybrid)

Has a Ki of 0.1 nM for Ca2+ channels of skeletal muscle, heart and brain, with the (+)-enantiomer being 40 times more active than the (- )-enantiomer. Niguldipine also binds non-stereoselectively (Ki == 0.05 nM) to brain QlA adrenoreceptors431 .

Information taken from a review432 on Dliscellaneous ligands for L-type Ca2+ channels.

has been concluded that the interaction between protons and permeant cations is allosterict 372,435. T-type channels in guinea-pig ventricular patches has also been shown to be sensitive to external H+ ions: whole-cell T-type conductance was maximal at plfo 9.0, reduced to 35% maximal at pHo 7.4 and 'near zero' at pHo 6.0436 .

Taicatoxin blocks L-type channels 42-43-14: The peptide toxin taicatoxin, from the Australian taipan snake, is a voltage-dependent blocker of L-type Ca2+ channels effective at nanomolar

II

l_e_n_try_4_2

_

concentrations437. Binding of the toxin reduces the Popen of L-type channels from cardiac cells and influences drug-binding at the dihydropyridine site through an allosterict interaction437.

Blockage of L-type channels by an alkaloid 42-43-15: Tetrandine, an alkaloidt isolated from the Chinese medicinal herb, Stephania tetranda, is vasodilatory in vivo and reversibly inhibits L-type Ca2+ currents in cardiac membranes and GH3 cells, competing directly for the diltiazem-binding site438 .

The peptide toxin w-conotoxin is a selective blocker of N-type Ca 2+ channels 42-43-16: A peptide isolated from the venom of the sea snail, Conus geographicus, called w-conotoxin GVIA (w-CTx), is a highly selective blocker of N-type Ca2+ channels. The high voltage activated Ca2+ currents in chick sensory neurones have single-channel conductances of 13 pS and 25pS in 110mM BaCl2, characteristic of N-type and L-type currents respectively. The large-conductance channels are selectively eliminated by dihydropyridines, while w-CTx (5 JlM) irreversibly blocks only the smallconductance channels. In whole-cell recordings, the macroscopicthigh voltage activated Ca2+ current was completely blocked by w-CTx and insensitive to DHPs in 600/0 of the cells. The remaining cells expressed both a DHP-sensitive Ca2+ current and a DHP-insensitive, w-CTx-sensitive, N-type current439. In rat dorsal root ganglion neurones, saturating concentrations of w-CTx (>1 JlM) block about 50% of the high voltage activated Ca2+ current. The dose of toxin giving half-maximal block is ~30nM413. Note that w-CTx (2 JlM) does not block L-type channels in these preparations413 . The Ca2+ entry associated with neurotransmitter release from rat Purkinje cell neurones is 500/0 blocked by w-CTx, with an IC so of ~100nM84.

Heterologously expressed N-type channels are sensitive to w-conotoxin

42-43-17: When the cDNA encoding the human neuronal Ca2+ channel alB-I subunit was transiently co-expressed with sequences encoding human {32 and a2b subunits in the human embryonic kidney cell line HEK-293, an N-type Ca2+ current, sensitive to w-CTx but insensitive to dihydropyridines, was obtained17. The transfected cells showed a single class of saturable binding sites for w-CTx, with a KD of 55 pM 17.

Structural determinants of blockade of N-type channels by w-conotoxin GVIA 42-43-18: Following heterologous expression of cRNAs in Xenopus oocytes (N-type) calcium channels containing alB subunits, together with a2 and {31 subunits, are sensitive to w-conotoxin GVIA (w-CTxj 5 JlM), while those comprising alA, a2 and {31 subunits are unaffected by this peptide. Channels containing chimaerict subunits, in which single domains of the alB subunit have been replaced by the corresponding domain from the alA subunit, all show reduced sensitivity to w-CTx, with subunit ill from alA

_L...-

e_n_try_4_2_

having the largest effect (nine-fold reduction of rate of onset of blockade). Analysis of the effects of a series of mutations in the mSS-llIHS region of the alB subunit showed the crucial importance of residues immediately Nterminal to llIHS, consistent with blockage of the pore by binding of w-CTx at this position2 0 8 . At least some of the mutations that reduced sensitivity to w-CTx-GVIA also reduced the inhibitory effects of w-CTx-MVIIA, suggesting that the two w-conotoxins interact with similar sites on the alB subunit208 .

The peptide blocker w-agatoxin IVA is specific for P-type channels 42-43-19: The peptide toxin w-agatoxin IVA (w-Aga-IVA), a 48 amino acid peptide originally purified from Agelenopsis aperia spider venom, blocks Ptype Ca2+ channel current in rat Purkinje neurones (KD rv 2 nM), but has no effect on identified T-type, L-type or N-type currents in a variety of central and peripheral neurones68 . This block by w-Aga-IVA is reversible by a brief regime of strong depolarizations. The rate of dissociation of toxin from the channel is increased by a factor of about 104 at +90mV compared with -90mV68 . w-Aga-IVA and the related peptide w-Aga-IIIA block glutamate release from pre-synaptic nerve terminals prepared from rat frontal cortex with ICso values of 30nM and O.74nM, respectively, suggesting that P-type channels are important in excitation-secretion coupling in these synapses69. The inhibitory effect of w-.Aga-IVA in rat Purkinje cell axons, measured by depression of post-synaptic currents, had an ICso of rvlOnM 84 .

Funnel-web spider toxin (FTx) blocks P-type channels 42-43-20: The low molecular weight toxin FTx purified from the funnel-web spider, Agelenopsis aperta, specifically blocks the P-type Ca2+ channels in cerebellar Purkinje cells67. The naturally occurring FTx and a synthetic analogue, sFTx, inhibited pre-synaptic 'Ca2+ currents in nerves innervating the mouse levator auris muscle, and blocked muscle contraction and neurotransmitter release evoked by nerve stimulation in an isolated nervemuscle preparation, indicating that the: P-type channel is the predominant voltage-dependent Ca2 + channel in the :motor nerve terminals440.

Neuroleptic drugs can block L-type Ca 2+ channels

42-43-21: Neuroleptic t drugs of the diphenylbutylpiperidine series, usually considered as blockers of the dopamine receptor, also block L-type Ca2+ channels in neuronal and muscle cells. One such compound, fluspirilene, blocks L-type channels in skeletal muscle with an ICso of O.1-0.2nM, independently of membrane voltage441 . These compounds are much less potent in neuronal tissue and smooth muscle, where ICso values of rvlOOnM have been reported442,443. Diphenylbutylpiperidines have been shown to inhibit the binding of dihydropyridines and devapamil to L-type channels noncompetitively, probably via allosteric interactions444,445.

An oxime inhibitor of L-type channels

42-43-22: The nucleophilicr oxime, 2,3-butanedione monoxime (BDM), inhibits muscular contraction and depresses action potentials in muscle fibres. Voltage-clamp measurements show that BDM (rvlOmM) depresses

II

i_

e_n_t_ry_42

---l_

in guinea-pig taenia caeci cells446, rat447 and guinea-pig ventricular myocytes (IC so = 6 mM)448.

[Ca,L

Phenytoin blocks T-type but not L-type channels

42-43-23: Phenytoin (3-100 JlM) blocks T-type Ca2+ channels in NIE-II5 neuroblastoma cells without affecting the L-type channels in these cells. The blockage of T-type curents is use- and voltage-dependent, increasing at higher holding potentials449.

Calciseptine is a specific blocker of L-type Ca 2+ channels 42-43-24: Calciseptine, a 60 amino acid peptide isolated from the venom of the black mamba, Dendroasopis polylepis polylepis, reversibly inhibited contractile activity (IC so = 15 nM) and [Ca,L, but not [Ca,T, in A7R5 rat aortic smooth muscle cells and cardiac preparations45o . The L-type currents in rat insulinoma cell lines (RINm5F and HIT) and in chicken dorsal root ganglion cells were 500/0 inhibited by 1 JlM calciseptine. T-type calcium currents in undifferentiated rat NIE-II5 neuroblastoma cells, N-type currents in chick dorsal root ganglion cells and L-type currents in skeletal muscle cells were insensitive to the toxin (1 JlM)450. SKetJF 96365 is a selective Ca 2+ channel blocker 42-43-25: SK&.F 96365 {1-{,8-[3-{4-methoxy-phenyl}propoxy]-4-methoxyphenethyl}-lH-imidazole hydrochloride}, structurally distinct from the known 'calcium antagonists,' shows selectivity in blocking receptormediated calcium entry (RMCE) compared with receptor-mediated internal Ca2+ release. The IC so for inhibition of RMCE by SK&F 96365 in human platelets stimulated with ADP or thrombin was 8.5 JlM or 11.7 JlM respectively: such concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth muscle cells held under voltage-clamp was also inhibited by SK&F 96365, but the ATP-gated Ca2+-permeable channel of rabbit ear-artery smooth muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2 + entry and RMCE, although its lack of effect on ATP-gated channels shows that it can discriminate different types of RMCE451 .

Dextromethorphan blocks L- and N-type Ca 2+ channels 42-43-26: The morphinan dextromethorphan blocks voltage-operated inward Ca2+ and Na+ currents and NMDA-induced currents in cultured cortical neurones and PCI2 cells452 . Dextromethorphan blocks Ba2 + current through L- and N-type Ca2+ channels with similar potencies (52-71 JlM) in both types of cells. The effect is not voltage-dependent, in contrast to that of amlodipine (a dihydropyridine). Dextromethorphan blocks Ba2 + current completely, unlike amlodipine and w-conotoxin, which produce only partial inhibition. Note: The voltage-activated Na+ and Ca2+ channels in cortical

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_1

neurones were inhibited by similar concentrations of dextromethorphan (ICso rv 80 JlM)452 but this is rv100-fold less potent than its effect on NMDA receptors (IC so 0.55 JlM) (see Blockers under ELG CAT GLU NMDA, 08-43).

T-type currents in pituitary GH3 cells are resistant to Ni 2+ and ethosuximide 42-43-27: T-type Ca2 + current in pituitary (GH3) cells is relatively resistant to blockade by Ni2+. The concentrations of inorganic cations producing 50% block of GH3 T-type currents are: La3+ 2.4 JlMj Cd2+, 188 JlMj Ni2+, 777 JlM. The T-type currents in GH3 pituita.ry cells are also blocked by the following compounds: nifedipine (rv 50 JlM), D600 (51 JlM), diltiazem (131 JlM), octanol (244 JlM), pentobarbital (985 JlM), methoxyflurane (1.41 mM) and amiloride (1.55mM). Phenytoin and ethosuximide produce 36 and 10% block at 100 JlM and 2.5 JlM, respectively403. This lack of sensitivity to ethosuximide contrasts with the block of T-type currents in thalamic relay neurones obtained with this petit mal anticonvulsant453 . J,

Diphenylbutylpiperidine-based antipsychotics preferentially block T-type currents 42-43-28: Diphenylbutylpiperidine (I)PBP)-based antipsychotic agents, including penfluridol and fluspirilene)' preferentially block T-type Ca2 + currents in the rat medullary thyroid carcinoma 6-23 (clone 6) cell line. Penfluridol inhibited T-type current, with 10mM Ca2 + as charge carrier, with an ICso of 224 OM. The high voltage activated Land N currents in these cells were less sensitive to inhibition by penfluridol. Block of T-type currents by penfluridol was enhanced by depolarizing test pulses. T-type Ca2+ currents in the human TT C cell line were also blocked by penfluridol, and the potency was enhanced by reduction of the extracellular Ca2+ concentration. Non-DPBP antipsychotics, including haloperidol, clozapine and thioridazine, also blocked T-type channels, but these were 20-100 times less potent than the DPBPS454.

Na+, Mg2+ and Ca 2+ compete for tIle same binding site in T-type channels 42-43-29: The T-type Ca2 + channels of chick dorsal root ganglion (DRG) cells are permeable to monovalent cations at low concentrations of external Ca2 + (see paragraph 42-40-04). External Ca2+ ions block Na+ currents with an apparent Kn of 1.8 JlM at -20 mV. Mg2+ ions are also potent blockers of the Na+ current, but much less effective against Ca2 + currents, consistent with the idea that Na+, Mg2+ and Ca2+ compete for the same binding site in Ttype channels376.

Channel modulation Ca 2+ channel activity is modulated via G protein-linked receptors 42-44-01: The activities of voltage-dependent Ca2 + channels are subject to modulation via a very wide range of neurotransmitter receptors. In many cell types different subtypes of channel are modulated independently (see Resource A-G protein-linked receptors). Modulation of Ca2 + channels by

II

1__e_n_t_ry_42

--'_

protein kinase C (PKC) activity in many different tissue preparations has been reviewed98,455. PKC activity results in either down- or up-modulation of Ca2 + channel currents262 with the precise effect showing a strong cell-type dependency. Most reports of PKC modulation show a stimulatory (e.g. smooth muscle) or inhibitory (e.g. sensory neurones, PC12 cells) response on Ca2+ currents (cf. the majority of observations on K+ and CI- channels, which show PKC phosphorylation to have an inhibitory effect, although the lK(v) current of cardiac ventricle is a notable exception to this 455 ). The inhibition of N-type and P/Q-type currents via G protein-linked- receptors usually involves direct, membrane-limited interaction of the activated G protein f3T subunits with the a1 subunit of the Ca2+ channel (reviewed in re!s456,457), though there are examples in which a diffusible second messenger is implicated458-46o.

Inhibition of Ca 2+ currents via G protein-linked receptors 42-44-02: Examples of inhibition of calcium currents by neurotransmitters and neuromodulators acting at G protein-linked receptors are shown in Table 10. Ginsenoside Rf inhibits Ca 2+ channels via a pertussis-sensitive G protein 42-44-03: A saponint, ginsenoside Rf (Rf), isolated from the oriental 'folk medicine' ginseng t , inhibits N-type and other unidentified types of highthreshold Ca2+ channels in rat dorsal root ganglion sensory neurones. Halfmaximal inhibition was achieved at 40 J.1M Rf and the inhibitory effect was eliminated by pre-treatment of the neurones with pertussis toxin (250 ng/ ml for 16h). The receptor responsible for mediating the effects of Rf has not been identified. Similar inhibition of lea by Rf was obtained with differentiated F-11 cells, a cell line that is a hybrid between rat dorsal root ganglia and mouse neuroblastoma cells467.

Purified

GsQ

subunit stimulates L-type channels in vitro

42-44-04: Purified Gsa subunit, activated with guanosine 5'-O-(3-thiotrisphosphate), increases the activity and delays the rundown of porcine cardiac L-type Ca2+ channels incorporated into planar lipid bilayers. This effect is specific for activated Gsa and is inhibited by activated Gil a. The actions of PKA and Gsa are additive, suggesting different pathways for channel stimulation468 . Similarly, the activated as subunit from G s purified from human erythrocytes stimulated Ca2+ currents through single L-type channels in membrane patches from guinea-pig ventricular myocytes469 and bovine cardiac sarcolemmal vesicles incorporated into lipid bilayers47o. In the presence of Bay K 8644 (1 J.1M), activated as increased Popen fivefold, via a decrease in activation, without changing the unitary conductance47o. Evidence for the direct interaction of G s and L-type channels is provided by their co-purification from skeletal muscle471 .

(3-adrenoreceptor agonists increase cardiac Ca 2+ currents 42-44-05: Noradrenaline increases cardiac Ca2+ currents via activation of the ,B-adrenoreceptor, linking to G s and the activation of adenylate cyclase to

II

II

Table 10. Examples of inhibition of Ca 2+ currents by neurotransmitters and neuromodulators acting at G protein-linked receptors

(From 42-44-02) Receptor agonist

Receptor

Tissue

Channel

Acetylcholine

MI

Sympathetic neurones (rat) Superior cervical ganglion neurones (rat) Neuroblastoma x glioma hybrid cell line (NG 108-15) Sensory neurones (rat) Superior cervical ganglion neurones (rat) Ciliary ganglion (chick) Dorsal root ganglion (rat) Hippocampal pyramidal neurones (rat) Adrenal cortical cell line, Y1 (mouse) Superior cervical ganglion neurones (rat) Neuroblastoma x glioma hybrid cell line (NG 108-15) Ventricular myocytes (rat) Sensory neurones (rat) Cerebellar Purkinje cells sympathetic ganglion neurones (bullfrog)

N-type

Acetylcholine

M2/~

Adenosine Al Adenosine Al Adenosine Angiotensin IT (see note 1) All AT-1 Cannabinoids Endothelin-1 GABA GABAB GABA GABAB Luteinizing hormonereleasing hormone (LHRH) Neuropeptide Y NPY receptor Dorsal root ganglion (rat) Myenteric plexus Cerebellar granule neurones Noradrenaline Sensory neurone (chick) 02 Sympathetic neurones (frog) Noradrenaline (see note 2) {3 Hippocampal granular neurone (rat) Opiates Dorsal root ganglion J-t (mouse) 8 Parathyroid hormone Tail artery smooth muscle (rat) (see note 3)

References 598 460 599 600 460

N-type N-type N-type L-type N-type N-type T-type N-type P-type N-type

591 602 603 461 458 502 114 596~597

601 509

N- and T-type

478

Q-type N-type

609

608~139

592 593

N-type N-type

234 479 594

'"

595

L-type

464

('b

~

f""t

~ ~

t:..J

Prostaglandin £2 Somatostatin Substance P Vasoactive intestinal polypeptide (VIP) (see note 4) Vasopressin

SSTR4

VI

Superior cervical ganglion neurones (rat) Pituitary cell line (AtT-20) Heterologous expression in Xenopus oocytes Superior cervical ganglion neurones (rat) Sympathetic neurones (rat)

N-type L- or N-type N-type N-type N-type

A7rS smooth muscle cells

L-type

604 605,606

552 607

466

610

Notes: 1. Angiotensin II stimulates Ca2+ currents in the adrenal cortical cellline461 and in bovine adrenal glomerulosa cells462,463. 2. The N-type currents in rat hippocampal granular neurones are elevated by noradrenaline234 . 3. Bovine PTH depresses L-type Ca2+ currents in smooth muscle cells from rat tail artery464, but synthetic rat PTH fragment (1-34) stimulates ICa,L in snail neurones, probably via activation of PKC465 . 4. The inhibitory action of VIP is pertussis toxin insensitive, but reduced by cholera toxin and anti-Gs antibodies, suggesting that it involves G s 466.

II

('b

= ~ t"t-

~ ~

_'--

e_n_try_4_2

----J

increase cAMP levels and potentiate protein kinase A (PKA}230,472. The effect of phosphorylation by PKA is to increase Popen 472 and the mean open time of the cardiac channel231 (see Protein phosphorylation, 42-32). The stimulation of cardiac L-type currents via the ,B-adrenoreceptor, Gs , PKA pathway has been reviewed98 . Studies of ,B-adrenergi.c stimulation of L-type channels in smooth muscle cells have produced highly variable results, with inhibition, stimulation and no effect all being reported for different preparations (reviewed in ref.98).

Facilitation of Ca 2+ channel activity involves relief of G proteinmediated inhibition 42-44-06: Depolarizing pre-pulses (e.g. +80 mV for SO ms) transiently increase, or 'facilitate l ', Ca2+ currents by reversing the G protein-mediated inhibition induced by a neurotransmitter, GTP,S473-475 or under basal conditions in the absence of a transmitter474,476. In rat cerebral cortical neurones, maximally effective pre-pulses (+80mV for SOms) facilitated Ca2+ current by 23% with internal GTP (300 JlM) and by SOC~ with internal GTP,S (300 IlM)265. The pre-pulse facilitation was greatly reduced (6%) in the presence of internal guanosine S'-O-(2-thiodiphosphate) (GDP,BS) (2IlM), an inhibitor of G protein activation. In some, but not all, cortical neurones, pre-pulse facilitation was inhibited by w-conotoxin GVIA (10 IlM) block of N-type Ca2+ channels. Similar results were obtained in parallel experiments with rat superior cervical ganglion neurones~!65. Pre-pulse facilitation in cerebral cortical and superior cervical ganglion neurones was strongly reduced or prevented by activation of PKC265.

Neuronal Ca 2+ channels are subject to tonic inhibition by G proteins 42-44-07: In several (but not all) neuronal cell types, the Ca2+ current is subject to 'tonic inhibition' by intrinsic G protein activity in the absence of added agonist474,476,477. This inhibition is relieved by intracellular guanosine S'-[,B-thio]diphosphate, an i.nhibitor of G protein activation. Activation of G protein via a receptor agonist further inhibits Ca2+ channels that are already tonically inhibited. Following removal of an inhibitory agonist by washout, the tonie inhibition is temporarily removed. This 'rebound facilitation', which fades within a few minutes, is prevented by the inhibitory action of pertussis toxin on the G protein474.

The inhibitory G protein is often Go 42-44-08: In sensory neurones, inhibition. of calcium currents by neuropeptide Y can be prevented by blocking ao, and inhibition reappears following reperfusion with activated ao478. The activated Gao subunit is also able to restore opiate-mediated inhibition of Ca2+ currents in pertussis toxintreated NGI08-IS cells479 and dopamine-induced inhibition of Ca2+ currents in snail neurones480. Such experiments can be criticized because of the use of artificially high concentrations of non-physiological G proteins, which might non-specifically dominate the signal transduction process 41 . The use of a mutant NGI08-IS cell line in which the Go al subunit is resistant to pertussis toxin overcomes this criticism. In this mutant line,

II

lL...--_ _ _ry_4_2 en t

_

inhibition of Ca2+ currents by opioids and noradrenaline are resistant to pertussis toxin, in contrast to its sensitivity in the wild-type parental line, demonstrating that the transduction pathway involves Go a1 481 . The inhibitory effect of somatostatin remained toxin-sensitive in the mutant cell line, consistent with its involving a different Ga subunit481 . The use of anti-G protein antibodies and antisense DNA oligonucleotides to identify the species of G protein involved in modulating Ca2+ currents has been reviewed in ref. 41 (see also multiple effects of Go on K+ currents 482 and Resource A-G protein-linked receptors). The Gao subunit co-purifies with the N-type voltage-dependent Ca2+ channels from rat sympathetic neurones 483 .

The Ca 2+ channel {3 subunit is involved in the interaction with Go 42-44-09: The DHP agonist Bay K 8644 enhances GTPase activity in neuronal membranes, via an effect on the V max of the enzyme. This stimulation is blocked by antibodies against Gao, but not by anti-Gai antibodies484, and by antibodies against the Ca2+ channel f3 subunit (quoted in ref. 41). Depletion of the Ca2+ channel f3 subunits using antisense r oligonucleotides in dorsal root ganglion neurones enhances the ability of the GABAB agonist (- )-baclofen to inhibit the Ca2+ current. These observations have been interpreted to suggest that Gao binds to a site on the channel al subunit in such a way as to inhibit, sterically or allosterically, the association of the channel al and f3 subunits 41 . The modulation of N-type channels involves G protein {3, subunits 42-44-10: The whole-cell ICa in adult rat sympathetic neurones, largely carried by N-type Ca2+ channels, is subject to inhibition known as 'kinetic slowing' by 10 JlM noradrenaline or direct G protein activation by intracellular guanylyl-imidodiphosphate (GppNHpi 500 JlM), a non-hydrolysable GTP analogue. Intranuclear injection of eDNA constructs t encoding G{31 and G1'2 subunits in sympathetic neurones gave rise to kinetic slowing of lca 'virtually identical' to that obtained with noradrenaline or GppNHp. Similar inhibitions were observed in neurones injected with G{31 + G1'3 or G{31 + G1'7 cDNAs, but not in those injected with either Gf31 or G1'2 eDNA alone. The injection of a eDNA encoding a constitutively active GaDA subunit (Q20SL mutant) produced no apparent effect. Application of noradrenaline (10 JlM) to neurones previously injected with G{31 + G1'2 cDNAs produced little additional effect. Titration of endogenous Gf3l' subunits by overexpression of injected eDNA encoding Gao attenuated the ability of noradrenaline to inhibit lca. Very similar observations have been made by injection of purified bovine brain Gf3l' protein or RNA encoding Gf321'3 into rat superior cervical ganglion neurones which express N-type Ca2+ channels485 . These findings demonstrate that the voltage-dependent inhibition of neuronal N-type Ca2+ channels by noradrenaline is mediated by the G protein {3, 1 subunits48s ,486.

G protein {3, subunits modulate PIQ-type Ca 2+ channels 42-44-11: Co-transfection of cDNAs encoding alA, ,BIb and a28 subunits of PI Q-type Ca2+ channels into tsA-201 cells gives rise to Ba2+ currents that

II

_'--

e_n_try_4_2_1

respond to activation of endogenous G proteins by GTP,S and were facilitatedt by a large depolarizingt pre-pulse in the presence of the GTP analogue. Co-transfection of tsA-201 cells with cDNAs encoding the Ca2+ channel subunits and G protein subunits, G{32. plus G-r3' mimicked G protein activation, shifting the voltage dependence of channel activation to more positive potentials and allowing facilitation by a conditioning prepulse. The G{32 subunit alone was nearly as effective as the {32,3 combination in modulating Ca2+ channel behaviour, but the G'3 subunit alone was without effect. The pertussis toxin-sensitive Ga subunits, Gail, Gai2, Gai3 and GaOA, had very little effect on voltage dependence and none on facilitation of the heterologously produced P/Q-type channels485 .

G protein (3"'( complex binds to the I-II cytoplasmic loop of the subunit

alA

42-44-12: Radiolabelledt G{3112. complex:, prepared by in vitro translationt in the presence of [35S]-methionine, binds directly and specifically to fusiont proteins containing the cytoplasmic loop connecting repeats I and IT (amino acids 360-486) of the Ca2.+ channel alA subunit487. Similar binding is found to the analogous I-IT loop regions of alB and alE subunits, but not with those from alS or ale subunits. The binding of the {3, complex involves two regions within the I-IT loop of alA, the al interaction domain (AID), a sequence of 18 amino acids that is necessary and sufficient for binding of the Ca2+ channel {3 subunit (see paragraph 42-29-06), and a second region (D2) within amino acids 402-487. The apparent Kd of the in vitro binding of the G{3I,2 complex to the AID sequence of alA is 63llM487, suggesting a 10- to 20-fold lower affinity than that of the Ca2+ channel {3 subunit488 . Binding of {3, to the D2 region of alA occurs with an apparent Kd of 24llM. Within the AID region of alA, which has the sequence QQIEBE1NGYMEWISKAE, alteration of the underlined residues strongly reduced or eliminated in vitro {3, binding. (Note that Y-WI (10-14) has been shown to be critical for binding of the Ca2+ channel f3 subunit489.) Calcium channels containing the mutant alA subunit with a R387E replacement within the AID sequence are insensitive to activation of G proteins in the Xenopus oocyte487.

Phosphorylation in the I-II loop of :the inhibition by G(3"'(

al

subunit antagonizes

42-44-13: The inhibition of Ca2+ currents by activated G proteins can be studied during transient expression of cl)NAs encoding alA or alB subunits, together with those encoding a2 and f3lb' in human embryonic kidney (HEK) cells. Stimulation of an endogenoust somatostatin receptor with 100llM somatostatin gives 650/0 inhibition of alB currents and 23% inhibition of alA currents. This inhibition can be relieved by a strong (150 mY) depolarizing pre-pulse ('pre-pulse facilitation'). Purified Gf3r subunits (10nM) in the patch pipette also mediate an inhibition of the Ntype currents from alB-containing cha.nnels that is subject to pre-pulse facilitation. Synthetic peptides (2 JlM) containing sequences from the alA or alB I-IT loop region applied via the patch pipette were able to relieve the G{3,-imposed inhibition: peptides containing aIB(353-371), aIB(372-389), aIB(410-428), aIA(384-403) and aIA(416-434) were all effective in this

II

1....._e_n_t_ry_4_2

-----I_

assay. In vitro phosphorylation of the aIB(410-428) and aIA(416-434) peptides by protein kinase C (PKC) eliminated their ability to quench the G{3'Y inhibition. These data support a model in which inhibitory G{3'Y interaction with I-II regions of alA or alB subunits is affected by PKC-dependent phosphorylation of residues within the Gf3'Y-binding site490 •

Adenosine receptors modulate Ca 2+ currents in hippocampal neurones 42-44-14: The selective activation of adenosine receptor subtypes has different effects on Ca2+ channels from acutely isolated pyramidal neurones from the CA3 region of guinea-pig hippocampus. Activation of adenosine Al receptors primarily inhibited N-tvre Ca2+ current, while activation of A2b receptors produced potentiation of P-type, but not N-type, Ca2+ current. The potentiation was blocked by inhibition of protein kinase A491.

Adenosine counteracts cAMP-dependent stimulation of cardiac L-type channels 42-44-15: The effect of PI purinoceptor (adenosine Al receptor) stimulation on L-type channels in cardiomyocytes depends on the whether the preparation is in a 'basal' state or in a cAMP-mediated state of activation. The Al receptors are coupled to inhibitory Gi, so that adenosine depresses f3-adrenergic stimulation of adenyl cyclase activity, cAMP formation and PKA-mediated stimulation of L-type Ca2+ currents. The isoproterenolstimulated L-type Ca2+ currents in guinea-pig ventricular492, guinea-pig atrial493, rabbit sinoatrial node494 and frog ventricular495 myocytes is markedly diminished by adenosine acting via a PTx-sensitive G protein. The suggestion that adenosine acts by activation of a phosphatase496, via Gi, rather than by inhibition of adenylate cyclase, remains plausible.

Dopamine D 1 receptors activate L-type channels in chromaffin cells 42-44-15: Stimulation of the dopamine D I receptors in bovine chromaffin cells activates Ca2+ currents in the absence of pre-depolarizations or repetitive activity. This activation by D I agonists, mediated by cyclic AMP and protein kinase A, results from prolonged openings of an otherwise quiescent 27pS L-type channel298 .

Angiotensin II stimulates L-type currents in an adrenal cortical cell line 42-44-16: The vasoactive octapeptide angiotensin IT is the major stimulator of aldosterone secretion from adrenocortical glomerulosa cells, an effect that is dependent on Ca2+ influx through voltage-dependent Ca2+ channels. Studies with the murine adrenocortical cell line, Y1, showed that angiotensin IT (1 nM to 1 J,lM) stimulates a slowly inactivating L-type current, on average 1.7-fold. A rapidly inactivating T-type current was not affected by angiotensin IT. The stimulatory effect of angiotensin II on L-type currents was blocked by pertussis toxin (100ng/ml) acting to modify the a subunit of a Gi-like G protein461 . A similar stimulatory effect of angiotensin II (1 J,lM) on L-type currents was shown in freshly isolated porcine glomerulosa cells461 .

II

_L.-.---------------------e-n-try-4-2--1

Q-type Ca 2+ channels respond to neuromodulators 42-44-17: The Ca2+ currents required to stimulate glutamatergic t synaptic transmission between hippocampal CA3 and CAl neurones are carried by N-type and Q-type channels61 . Blockage of the N-type channels with w-conotoxin GVIA (1 J.1M) allows the effects of neuromodulators on Q-type channel activity to be monitored. Stimulation of metabotropic glutamate (IS,3R-ACPD, 200J.1M)), 1'-aminobutyric acid type B ((-)-baclofen, 5 J.1M), adenosine (2-chloroadenosine, 5 J.1M) or acetylcholine (carbachol, 10 J.1M) receptors significantly depressed synaptic transmission under these conditions. Prior application of the phorbol ester phorbol 12,13-dibutyrate (1 J.1M) to stimulate PKC activity, reduced or abolished the inhibitory effects of the neuromodulators on Q-type channel activity61. An endogenous brain peptide modulates L-type and T-type currents 42-44-18: Calcium channels can be modulated following application of a low molecular weight endogenous peptide purified from rat brain: in cardiac cells L-type Ca2 + currents are enhanced while in neuronal cells both L-type and T-type currents are inhibited497.

Galanin inhibits Ca 2+ channels via two species of G protein 42-44-19: Galanin, a neuropeptide of 29 amino acids that is widely distributed throughout the central t and peripheralt nervous systems, inhibits voltagegated Ca2 + channels by interaction with a G proteint -linked receptor in rat insulinoma RINm5F cells and in rat pituitary GH3 cell line498-5oo. Microinjection of antisense t oligonucleotides t designed to inhibit expression of genes encoding different G protein subunits shows that both these cell types couple the galanin receptor to the Ca2+ channel mainly via the Go protein consisting of o.Ol{32,2, with the o.Ol{33,4 species also being used, but less efficiently501. Cannabinoids inhibit N-type Ca 2+ channels via a pertussis toxin-sensitive G protein 42-44-20: The cannabinomimetic aminoalkylindole WIN 55,212-2 reversibly inhibits ICa in the neuroblastoma-glioma cell line NGI0S-15, with an ICso of 'less than 10nM'. The effect is stereospecifict , the enantiomert WIN 55,212-3 being without effect at 1 J.1M, and is blocked by pertussis toxin (500 ng/ml). The inhibitory effect of the cannabinoid was eliminated in the presence of w-conotoxin (1 mM), a specific blocker of N-type channels, which decreased ICa by 380/0, but was insensitive to dihydropyridine antagonism that reduced ICa in NG108-15 cells by 27%502.

Fatty acids modulate lea in smooth muscle 42-44-21: Free fatty acids such as arachidonic acid (AA) have potential roles in the physiological and/or pathological modulation of lca in smooth muscle. In smooth muscle cells from rabbit ileum 10-30 J.1M AA causes a gradual depression of lca 503. This inhibitory effect is not prevented by the cyclooxygenase inhibitor indomethacin (10 J.1M) or the lipoxygenase inhibitor nordihydroguaiaretic acid (10J.1M) (see also lLG K AA, entry 26). The PKC inhibitors H-7 and staurosporine do not mimic this action of AA. Certain

II

1L...-__ _ _ry_42

en t

_

other cis-unsaturated fatty acids (palmitoleic, linoleic and oleic acids) can also depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic and palmitic acids) show no inhibitory effects on ICa. Myristic acid consistently increases the amplitude of lca at negative membrane potentials503 .

Ca 2+ influx into hypothalamic neurones can inhibit voltage-gated Ca 2+ currents 42-44-22: In cultured rat hypothalamic neurones both NMDA and nonNMDA receptor-channels (see ELG series, entries 04-11) are permeable for Ca2+. Calcium influx through these channels activates a calmodulindependent mechanism, which can lead to high voltage activated Ca2+ current inhibition504.

Inositol phosphates modify gating of Ca 2+ channels 42-44-23: Intracellular InSP3 and InsP4 have been shown capable of eliciting Ca2+ entry into rat cerebellar neurones by modifying the gating characteristics of voltage-dependent calcium channels. InsPa (ECso 0.5 JlM) shifted the steady-state inactivation curve towards more positive values 505.

Modulation of voltage-gated Ca 2 + channel function by ethanol 42-44-23: Ethanol inhibits voltage-dependent Ca2+ flux in vitro in synaptosomes and pre-synaptic nerve terminals (threshold, 25 mM; ICso > 150 mM)506 and in cultured phaeochromocytoma (PC12) cells 94,507. The electrophysiological and pharmacological characteristics of the channel involved in PC12 cells identify it as an L-type channel507. Chronic ethanol treatment increases dihydropyridine-binding sites in membranes from whole brain and cerebral cortex of mice and rats, an effect that is blocked by Ca2+ channel antagonists. Increases in dihydropyridine binding are also found in heart tissue of ethanol-dependent rats and in adrenal chromaffin cells and PC 12 cells after growth in the presence of 200 mM ethanol for 6 days 94. Menthol, a major constituent of peppermint oil, acts as an antagonist of L-type Ca2+ channels at concentrations of 10-100 JlM508 . A figure illustrating the relative potentiating effects of ethanol on GABAA mediated CI- flux and the depression of NMDA-, kainate- and voltagegated Ca2+ flux in synaptosomes from rat cortex appears as Fig. 6 in Channel modulation under ELG Cl GABAA, 10-44.

N-type Ca 2 + currents are inhibited by adenosine A 1 receptor activation 42-44-24: In acutely isolated pyramidal neurones from the CA3 region of guinea-pig hippocampus, the Al adenosine receptor agonist 2-chloroadenosine (2-CA) (100 JlM) inhibits ICa by 14%. The N-type Ca2+ channel blocker w-conotoxin GVIA (w-CTx) (5 JlM) removed most of the inhibition by 2-CA, confirming that the Al receptor agonist primarily inhibits N-type channels. The effect of 2-CA is voltage dependent, the I-V relationship being shifted toward a more negative potential in the presence of the agonist491 .

II

_L...-

e_n_try_4_2_1

Inhibition of N-type Ca 2+ currents by luteinizing hormone-releasing hormone 42-44-25: The chicken type IT luteinizing hormone-releasing hormone (LHRH) inhibited the N-type Ca2+ and Ba2+ currents of neurones from bullfrog paravertebral sympathetic ganglia with a half-maximally effective concentration of 20 nM509. There was no detectable effect on the nimodipine-sensitive L-type currents in these preparations. The inhibition of N-type currents was sensitive to the activation state of the channels: the hormone had little effect when applied during a long depolarization (-20mV) that opened the channels, but was effective when applied in the resting state (-90mV) and currents were elicited by short (5ms) depolarizations (-20mV) every 200ms. Inhibition was relieved when channels were activated by short (3 s) pre-pulses to membrane potentials above -30 mV. A kinetic model to fit the data proposes that inhibition results from the binding of activated G proteins to N-type channels to stabilize the closed state, and that activation of the channel complex destabilizes the binding of the G protein. The preferred version of the model suggests the binding of four G protein subunits per Ca2+ channel, one for each domain of the channeI509.

G protein-linked receptors can activate Ca 2+ currents 42-44-26: Activation of receptors in secretory cells can enhance Ca2+ currents via pertussis toxin-sensitive G proteins (reviewed in ref. 510). Thyrotropinreleasing hormone stimulation of Ca2+ currents in rat pituitary GH3 cells requires activation of both PKC and Gi2511.

P-type Ca 2+ currents are potentiated by activation of adenosine A 2b receptors 42-44-27: In the presence of the Al receptor antagonist 8-cyclopentyl-l,3dimethylxanthine (CPT), exposure of guinea-pig CA3 hippocampal neurones to adenosine caused a 63.5% increase in lca: w-CTx had no effect on the potentiated ICa, suggesting that N-type Ca2+ channels are not involved in this augmentation. The potentiationt was strongly inhibited by w-agatoxin NA (0.1 J.1M), a specific P-type Ca2+ channel blocker, establishing the involvement of P-type channels in activation via adenosine receptors. The specific adenosine A2 receptor agonist N6-[2-(3,5-dimethyloxyphenyl)-2-(2-methylincreased lca by 33% above the phenyl)ethyl]adenosine (DPMA) (0.1 ~i), control value. The highly selective adenosine A2a receptor agonist CGS 21680 (1 J.1M), had no effect on ICa, suggesting the conclusion that the A2b adenosine receptor is probably involved in the potentiation of lca by adenosine491 . The protein kinase A inhibitor peptide WIPTIDE (10 JlM) prevented ICa potentiation by CPT and adenosine, establishing that the potentiation via A2b activation involves cAMP-dependent protein kinase activity491.

T-type channels are modulated by G protein activation 42-44-28: The T-type Ca2+ channel currents from cultured rat dorsal root ganglion neurones can be modulated by G protein activation. Photorelease 5'-O(3-thio) trisphosphate (GTP,S) from a of intracellular ~anosine photolabile 'caged't precursor had dose-dependent effects on the T-type

1'--_e_n_try_4_2

_

current. At 6 J.1M, GTP,S enhanced the current, but higher concentrations (up to 20 J.1M) were inhibitory. The inhibitory response, but not the stimulation, was sensitive to pertussis toxin, suggesting the involvement of more than one G protein in T-type Ca2+ channel modulation. Low concentrations of the GABAB agonist (-)-baclofen (2 J.1M), potentiated the T-type current, but 100 J.1M(-)-baclofen was inhibitory512. Bradykinin (0.1 J.1M), baclofen (2 J.1M) and internal GTP,S (100J.1M) inhibit T-type currents in the dorsal root ganglion-neuroblastoma cell line, ND7_23 513 .

Endothelin enhances T-type Ca 2+ currents 42-44-29: Endothelin-l (ET-1), a 21 amino acid vasoconstrictive t peptide, increases intracellular Ca2+ levels and has hypertrophic action on ventricular myocytes. In cultured neonatal rat ventricular myocytes, ET-1 (10nM) increased the maximum current density of Ica,T from -3.0J.1A/cm2 to -4.4J,1A/cm2 . This enhancement by ET-1 was dose dependent, with the maximal response at approximately 10nM and a half-maximal dose of 1.3 nM. The stimulation of ICa,T was antagonized by protein kinase C inhibitors staurosporine (0.2 J.1M) and 1-(S-isoquinolinesulphonyl)-2-methylpiperazine (B-7, 20 J.1M) in the pipette solution. Extracellular application of phorbol esters, activators of protein kinase C, also increased the maximal current density of ICa,T' an effect that was blocked by H-7 (20J.1M) in the pipette solution. The application of ET-1 had no significant effect on ICa,L in this systeml14 .

Stimulation of the j-t-opioid receptor inhibits P/Q-type and N-type Ca 2+ channels 42-44-30: During heterologousteo-expressiont of cDNAst encoding the J..topioid receptor and Ca2+ channel subunits in Xenopus oocytes, activation of the receptor with the synthetic enkephalin [n-Ala2 , N-Me-Phe4 , GlyolS]enkephalin (DAMGO; 1 JlM) produced rv20% inhibition of Ba2+ currents from P/Q-type channels (alA, a2, ,84) -and rvS5% inhibition of those from Ntype channels (alB, a2, ,84). The inhibitory effect of DAMGO was reversible on washout and prevented by the opioid receptor antagonist naloxone (10 J.1M), and the G protein antagonist pertussis toxin (2 J.1g/ml). The inhibition was not obtained when the channels contained the alC or alE subunits. The currents obtained following expression of alA cRNA, in the absence of a2 and {3 subunits, were also sensitive to opioid inhibition, but the effect was enhanced about threefold in the absence of the ,8 subunit. The absence of the a2 subunit was without effect on the degree of inhibition. On the basis of these results, it has been suggested that the activated G protein, probably via the released {3" subunits (see paragraph 42-44-11), interferes with Ca2+ channel {3 subunit binding to the alA I-IT linker, or affects {3 subunit interactions in other regions of the al subunit514.

Equilibrium dissociation constant Dissociation constants for binding of drugs to L-type channels 42-45-01: The dissociation constants for the binding of several drugs to L-type Ca2+ channels are shown in Table 11. Note that the drugs are likely to have

II

II Table 11. Equilibrium dissociation constants for binding of various drugs to L-type Ca 2+ channels (From 42-45-01) Class

Dissociation constants (nM)

Ligand

Reference

Skeletal muscle

Heart

Brain

(-)-Azidopine (- )-Iodopine (- )-Sadopine (+ )-Sadopine

0.29-0.7 0.2 0.35 0.4 0.51 0.4

0.051 0.052 0.030 n.d. n.d.

0.075 0.044 0.096 0.06 n.d.

(- )-Desmethoxyverapamil (Devapamil) N-methyl-LU 49888

1.5-2.2 2.0

1.4-2.5 n.d.

1.6 1.4

620,621

Benzothiazepines

(+ )-cis-Diltiazem

39-50

40-80

37-50

622-624

Diphenylbutylpiperidines

Fluspirilene

0.100

0.070

n.d.

441,445,625

Benzothiazinones

HOE-166

0.100

n.d.

n.d.

428

1/4-Dihydropyridines

Phenylalkylamines

(+) PN 200-110

611 612 183,613-615 616 617

618,619

I

~

='

~

~ ~

1__e_n_t_ry_4_2

_

higher affinity for a particular state of the channel. For example, the apparent Ko for nitrendipine block of ICa,L in canine ventricular myocytes is 0.73 J.1M at a holding potential of -80 mY, but 0.36nM at about -15 my411 , reflecting the preferential binding to the open and/or inactivated states, compared to the resting state of the channel.

w-Conotoxin inhibits Ca 2+ channels in an electric ray 42-45-02: w-Conotoxin (w-CTx) gives dose-dependent inhibition of increases in free calcium concentration in, and acetylcholine release from synaptosomes isolated from a Japanese electric ray, Narke japonica, following

depolarization with a high concentration of potassium ions. Half-maximal inhibitions (IC so )of the increase in intrasynaptosomal Ca2+ and acetylcholine release were obtained using 8 and 7 J.1M w-CTx, respectively. Assay using radioiodinated toxin revealed a specific binding site with a Ko of 2.8 J.1M and a density of 45 pmol/mg synaptosomal protein. Binding assay with synaptosomal plasma membrane showed a K o = 7 J.1M and a density of 200pmol/mg protein. The radiolabelled toxin was covalently cross-linkedt (by disuccinimidyl suberate) to a protein that has a molecular weight of 170000 as determined by SDS-PAGEt and autoradiography515.

The peptide toxin w-agatoxin IVA is a potent blocker of P-type channels 42-45-03: The 48 amino acid peptide toxin w-agatoxin IVA (w-Aga-IVA) from the venom of the funnel web spider, Agelenopsis aperta, blocks P-type Ca2+

channel currents in rat Purkinje neurones with an ICso of "",2 nM68 • Note that this value differs markedly from the 200nM w-Aga-IVA that gives 50% block of currents obtained after co-expressiont of cRNAs encoding rabbit 0IA, 02/6 and {31 subunits in Xenopus oocytes 13 .

Cone snail toxin w-CTx-MVIIC is a potent blocker of a lA -containing channels 42-45-04: The 26 amino acid peptide toxin, w-conotoxin MVIIC (w-CTxMVIIC), from the piscivorous marine snail Conusrnagus, is a blocker of both N-type and P-type Ca2+ channels, with an ICso of 1-10 J.1M for P-type channels516 . In Xenopus oocytes expressing cRNAs encoding 0IA, 02/6 and {31 subunits, w-CTx-MVIIC blocked I Ba with an IC so of <150nM, but 5J.1M toxin was without effect on currents elicited via the 0IC (/L-type') subunit13.

Ligands 42-47-01: Available radioligands include: L-type: [3H] Nitrendipine, [3H] PN200-110, [3H] azidopine, [3H] diazipine,

[3H] isradipine. Note that dihydropyridine derivatives such as azidopine, diazipine, isradipine, nitrendipine and nifedipine are intrinsically photoreactive, so that their 3H-derivatives can be photoactivated to generate covalently labelled DHP-receptor sites on L-type channels. N-type: [1 2S I] w-Conotoxin.

II

_""'"--

en_try_42_._1

The anticonvulsant drug gabapentin binds to the (};2/6 subunit 42-47-02: Gabapentin (l-(aminomethyl)cyclohexane acetic acid; neurontin),

an orally active antiepileptic drug, has been shown to bind to a single, high-affinity (Ko = 38 nM) binding site in rat brains17• Purification of the gabapentin-binding protein from pig brain shows it to be the a'l16 subunit of Ca2+ channelss18• High levels of [3H] gabapentin-binding sites were detected in membranes from rat brain, heart and skeletal muscle, as well as in COS-7 cells heterologously expressing 0:2/6 cDNAs18 • The functional consequences of gabapentin binding to the 0:2/6 subunit were not reported.

Openers See Receptor agonists, 42-50.

Receptor/transducer interactions Reviews 42-49-01: The modulation of voltage-gated Ca2+ channel activity via

receptor/transducer interactions has been extensively reviewed98,380,s19-s21.

Activated Go inhibits Ca 2+ currents in cultured neurones 42-49-02: In a number of neuronal and neurosecretory systems, activation of a pertussis toxin-sensitive G protein causes inhibition of Ca2+ currents, involving both a slower activation and a reduced current amplitude (reviewed in ref. 41). In some cases, the G protein has been identified as the Go subtype by experiments involving injection of purified G proteins into neuroblastoma x glioma hybrid cells479 or rat dorsal root ganglion neuronesS22, use of subtype-specific antisera in cultured dorsal root ganglion neuronesS23 and neuroblastoma x glioma hybrid (NGI08-15) cellss24 or the use of antisense t oligonucleotides to deplete specific G protein subunitsS2S,s26. There is evidence that, in cultured rat dorsal root ganglion neurones, there is competition or allosteric interaction between activated Go protein and the Ca2+ channel {3 subunit for binding to the calcium channel 0:1 subunitS27• 42-49-03: Examples of the stimulation of L-type Ca2+ channel activities in

cardiac and smooth muscle preparations are given in Table 12.

Different receptor subtypes in sympathetic neurones 42-49-04: In rat sympathetic neurones, pertussis toxin-sensitive suppression

of Ca2+ current is mediated primarily by via M.t muscarinic receptors460, as distinct from the BAPTA(bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetate)-sensitive, Ml-receptor subtype slow mediation of M current in the same cells.

Coupling via specific G protein 1 subunits 42-49-05: Receptor linkage to voltage-gated calcium channel effector molecules has been shown to be determined by specific , subunit subtypes of G proteins551 : The voltage-sensitive L-type calcium channel in rat

II

1__e_n_try_4_2

_

,4 ,3

pituitary GH3 cells has been shown to require the G protein subtype for coupling of the somatostatin receptor while the subtype was found to be required for coupling of the muscarinic receptor to these channels551 .

Channel modulation during heterologous co-expression of components 42-49-06: The inhibition of N-type Ca2+ channels by G proteins can be reproduced by co-expression of the cRNAs encoding the essential components, a G protein linked seven-transmembrane spanning receptor, G protein subunits and Ca2+ channel subunits, in Xenopus oocytes. In oocytes expressing cRNAs encoding the alB, 02 and {3l subunits of N-type Ca2+ channels, the somatostatin receptor SSTR4, and the G protein {3l and ,2 subunits, the voltage-dependent Ba2+ currents were inhibited by somatostatin (",250/0 inhibition at 1 ~)552. Heterologous expression of a Gao subunit did not influence the degree of inhibition, suggesting the participation of an endogenous Go subunit. The degree of inhibition of Ca2+ currents by somatostatin depends on the particular 01 subunit in the functional channel: where this was alA, the inhibition by 1 J.1M somatostatin was only 9%, and there was no detectable inhibition with olC-containing channels. Neither replacement of the I-II loop of alB by those of alA or alC nor the complete absence of exogenous {3l subunit altered the susceptibility of the expressed channel to inhibition by somatostatin, suggesting that the latter does not simply involve interference with the al/{3l subunit interaction. Replacement of both motif I and the intracellular C-terminal domain altered the modulatory behaviour to that of the donor of the two domains552 • G protein inhibition of N-type channels during heterologous expression 42-49-07: Co-expressiont of cDNAs encoding the rat alB, a2 and 13lh subunits and the M2 muscarinic receptor in HEK-293 cells produced N-type Ca2+ channels that were sensitive to inhibitory modulation by M2 agonists. Application of carbachol (50 J.1M) 'substantially attenuated' N-type current elicited by a test pulse depolarization to +20mV and slowed the kinetics of activation. The inhibitory effect was due to a 10-fold increase in the time required for channels to open following the depolarization. There were no changes in the pattern of gating once channels opened during the test pulse, or in unitary currents553 • It is suggested that a simple delay in the first opening of individual channels, mediated by G protein-dependent inhibition, might be sufficient to switch off N-type channels during the action potential, the duration of which is brief (1-3 ms) compared with the delay ('first latency') in initial channel opening553 •

ATP is an lendogenous inhibitor' of lea adrenal chromaffin cells 42-49-08: The release of catecholamines from adrenal chromaffin cells is governed by Ca2+ entry through voltage-gated Ca2+ channels, which is in turn activated by the depolarization induced by acetylcholine released from splanchnic nerve terminals. The secretory granules of chromaffin cells contain a number of neurotransmitters, one or more of which acts as an

II

II

Table 12.. Stimulation of L-type Ca 2+ channel activity by receptor agonists (From 42-49-03)

Agonist

Receptor

Angiotensin IT (angIT)

ATP

P2

Calcitonin gene-related peptide (GGRP)

Preparation

Observations

Neonatal rat cardiomyocytes Human atrial cells Guinea-pig portal vein

Rabbit saphenous artery

nM angIT enhanced ICa,L by rv50% 10nM angIT increased ICa,L by 100% Doubling of current and a 10mV negative shift in Vpeak by 0.1-10nM angIT. 100nM angIT increased IBa,L by 50%

Frog ventricular myocytes

1 JlM ATP increased Ica,L by rv60%

382,495

Bullfrog atrial myocytes

0.3 JlM CGRP induced 8-fold increase in L-type current 0.1 JlM (no effect in ventricular myocytes) 10nM CGRP increased IBa,L by 40%

531,532

Guinea-pig atrial myocytes Rat vas deferens smooth muscle cells Endothelin (see note 1)

Porcine coronary artery Porcine portal vein myocytes

Glucagon Histamine Neuropeptide Y

H2

10nM endothelin increased IBa,L by 200% 10nM maximally effective; doubled mean open time, slightly curtailed closed times

References 528 529 530

264

532-534 535 536 537

Rat ventricular myocyte

glucagon maximally increased Ica,L 3-fold

538

Guinea-pig ventricular myocytes

Maximal stimulation of 3- to 4-fold being reached at rv10 f.1M

539-541

PTx-insensitive stimulation of ICa,L by rv50% (see note 2)

542

Rat ventricular myocytes

JlM

('D

=' ~ ~

t--J

Noradrenaline

(3

Parathyroid hormone

Serotonin

Frog, rat and guinea-pig ventricular myocytes

Neonatal rat ventricular myocytes

5-HT4

Human atrial cells

Rabbit basilar artery Thrombin

Frog ventricular myocytes

1 JlM isoproterenol caused a slow, ",S-fold increase in frog ventricular myocyte ICa,L. The stimulation was completely blocked by inhibitors of cAMP-dependent phosphorylation. Qualitatively similar observations were made using ventricular myocytes from rat and guinea-pig

543

0.1-1 JlM bovine PTH-(1-34) increased IBa,L by ",70%. Rat PTH-(1-34) increased ICa,L with an ECso of 43 pM

544,545

1-10JlM serotonin (5-HT) maximally increased ICa,L by ",7-fold and shifted the peak of the I-V curve by -12mV (see note 3) 10-100nM 5-HT increased 8Popen by up to 200-fold

547

4 nM 'nearly doubled' ICa,L 10 nM thrombin doubled average L-type currents by reducing closed times and the fraction of blank runs. Pre-treatment with the PKC inhibitor H-7 blocked stimulation by thrombin

549

j ~

t-.J

546

548

550

Notes:

II

1. Endothelin has proved to be without effect on L-type currents in some preparations or cell types, while showing slight reduction of ICa,L in others (reviewed in ref. 98). 2. In guinea-pig ventricular myocytes and rat sensory neurones, neuropeptide Y is reported to inhibit L-type Ca2+ currents (reviewed in ref.98). 3. The stimulation of Ica,L by 5-HT was not seen in atrial myocytes from frog, guinea-pig, rabbit or ratS47.

_1.-.

e_n_try_4_2

1

'endogenous inhibitor' of ICa to control secretion. Tests with a number of candidate neurotransmitters showed that ATP, which is present at high concentrations in chromaffin cell secretory granules, produced a voltagedependent inhibition of ICa (ECso 0.5 IlM) that mimicked, but was not additive with, the endogenous inhibition. The ATP-dependent inhibition was sensitive to pertussis toxin, and the relative responses to a range of P2 agonists and antagonists suggested that it acted through the P2Y recepto~54. The ATP-mediated inhibition affected both the N-type and the P/Q-type Ca2+ channels of the bovine adrenal chromaffin cells554.

Receptor agonists (selective) Some dihydropyridines act as selective agonists of L-type channels 42-50-01: Some dihydropyridine molecules, such as Bay K 8644, CGP 28392563 and (+}-(s)-202-791, enhance L-type Ca2+ currents by increasing the probability of channel opening by inducing or stabilizing long-lasting openings. Bay K 8644 is ineffective unless the L-type channels are at least partially phosphorylated225,346. Channel activity is more sensitive to these agonists at low depolarizations, when the probability of opening is lowest in controls. For example, L-type Ca2+ currents in rabbit atrial cardiac myocytes are enhanced 14-fold by 1 IlM (+)-(s)-202-791 at -30m~ but only three-fold at OmV (see Bean, 1994 under Related sources and reviews, 4256). The stimulation of L-type currents by DHP agonists is due to a large increase in Popen and the induction of long openings (see Single-channel data, 42-41). Studies with pure stereoisomers have shown that it is the (-)Bay K 8644 that is the active agonist in the racemict mixture564,565.

Activating and blocking dihydropyridines can show co-operative interactions 42-50-02: Activating and blocking dihydropyridine analogues compete with each other for the same binding site on inactivated L-type channels422. In contrast, in intact polarized cardiac myocytes the activating dihydropyridine (s)-202-791 shows positive co-operative interactions with the blocker (s)isradipine. The affinity of isradipine for the L-type channel was increased by low concentrations of (s)-202-791, and the activating potential of (s)-202791 was strongly increased by low concentrations of the blocking enantiomer, (r}-202-791 422. These observations suggest that the blocking and ~ctivating dihydropyridines interact with differen~ sites that are allosterically linked, allowing co-operative binding of the two types of analogue422 .

Dihydropyridine agonists slow the deactivation of L-type tail currents 42-50-03: Dihydropyqdine agonists characteristically slow the deactivation of L-type tail currents t on repolarization. This phenomenon is useful in identifying components of L-type currents in neurones, where other Ca2+ currents are present. Under normal conditions, the deactivation of tail currents is similar for N-type and P-type channels, with that of L-type channels being slightly faster. In the presence of Bay K 8644 or (+}-(s}-202791 (1 IlM) the deactivation of the tail currents of L-type channels is slowed

II

l_e_n_t_ry_4_2

_

several-fold, allowing the identification of the contribution resulting from Ltype channels (see Bean, 1994 under Related sources and reviews, 42-56).

FPL 64176 is a powerful agonist of L-type Ca 2+ channels 42-50-04: The benzoylpyrrole compound FPL 64176 is a powerful agonist of Ltype channels566 . When cRNAs encoding rabbit ale, a216, and {31 subunits were co-expressedt in Xenopus oocytes, the peak current was enhanced about six-fold by 5 J!M FPL 64176. The tail currentst obtained after step repolarization were also enhanced and prolonged. The peak I-V relationship was displaced by 20-30 m V towards more negative potentials13 . In parallel experiments, FPL 64176 had no detectable effect on channels containing 13 alA subunits . In A7r5 smooth muscle cells, the stimulatory action of FPL 64176 (1 J!M) was allostericallyt inhibited by (-)-Bay K 8644 (100nM), suggesting that these two compounds have different but interacting binding sites567.

Peptide toxins can activate L-type channels 42-50-05: The peptide toxins gonioporotoxin (ICso = 5.3 J!M), from the toxic coral Goniopora 568 , and atrotoxin (Ko = 5 x 10-7 g/ml), from the rattlesnake, Crotalus atrox569,570, activate cardiac L-type Ca2 + channels and allostericallyt inhibit binding of dihydropyridines to the channel. Note that these toxins are lethal at high dosage: 0.4mg (rv 20nmol) of gonioporotoxin kills a mouse within an hour of injection568 .

Receptor antagonists (selective) Caveat on the targets of 'calcium antagonists' 42-51-01: It can not be assumed that voltage-gated calcium channels are the exclusive targets of calcium antagonist drugs. Non-channel targets that have been identified include Ca2+ ATPases, cell nuclei, contractile proteins, endoplasmic reticulum, intracellular Ca2 +-binding proteins, lysosomes, mitochondrial Ca2 + transport enzymes, mitochondrial respiratory enzymes, Na+ I Ca2+ exchangers, sarcoplasmic reticulum and transcription factors. Indirect modification of calcium channel properties can also occur by drug effects on receptor and second messenger components which may normally regulate native channels (see Receptor/transducer interactions, 42-49). Drugs which modulate calcium channel fluxes through components such as adenylate cyclase or G proteins should not be classified as 'calcium antagonists' (see ref.555).

Classification of drug-binding sites on L-type Ca 2+ channels 42-51-02: By convention555, class 1 drugs selectively interact with binding sites on L-type channels. Class 2 drugs interact with other voltagedependent Ca2+ channels. Class 3 drugs are classified as non-selective channel modulators (see below). These sites have been subclassified555 as shown in Table 13. 42-51-03: L-type: The 60 aa residue natural snake venom polypeptide calciseptine has been described as a specific blocker of L-type channels in

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_2_1

Table 13. Classification of drug receptor sites on calcium channel proteins555 (From 42-51-02) Class

Sub-class

Class I: L-channelselective agents IA

Act at the dihydropyridine (DHP) site including amoldipine, darodipine, elgodipine felodipine, flordipine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, nitrendipine, oxodipine and riodipine. Several dihydropyridines have very high affinity for the L-channel. Act at the benzothiazepine site - including clentiazem (TA-3090), diclofurime and diltiazem. There is a distinct binding site for benzothiazepines on the (}:1 subunit, and this is probably allosterically linkedt to the DHP site. Act at the phenylalkylamine site - including anipamil, devapamil, gallopamil, levemopamil, tipamil and verapamil Act at undefined sites on L-channels including SR33557, HOE 166, McN6186, MDLI2330A, MCI176, pinaverium. Not assigned at time of compilation.

IB

IC

IX

Class 2: Agents interacting with other voltagedependent Ca2+ channels (non-L-type)

There are no highly selective blockers of Tor N-type channels. Ni2+ has some selectivity for T channels in comparison with Cd2+. Cd2+ is more potent at L-type and N-type channels. Gd2+ may be a selective antagonist at N channels405 • w-Conotoxin GVIA has selectivity for N-type channels, while funnel web spider toxin is selective for P-type channels.

Class 3: Non-selective channel-modulating agents

Agents which do interact with L channels but with apparently low selectivity, e.g. bepridil, caroverinej' cinnarizine, fendiline, flunarizine, prenylamine.

Class 4: Agents acting at other Ca2+-selective channels

e.g. see ILC; Ca Ca RyR-Caf, entry 17, ILG CAT cGMP, entry 22, ELG series, entries 04 to 11.

Class 5: Agents acting at intracellular sites affecting Ca2+ sensitivity or mobilization

e.g. see IL(; Ca InsPg, entry 19.

l_e_n_try_4_2

_

smooth and cardiac muscle and in neuronal cells. L-type channels are also inhibited by a variety of pharmacological agents used in the treatment of cardiovascular disorders including the dihydropyridines (e.g. nitrendipine, PN200-110) and the phenylalkylamines. Both movement of gating charge for excitation-contraction coupling and muscular contraction are reduced by these agents; ~5% of DHP-binding sites in skeletal muscle fibres are active in ion conductances556,557 (see also footnotes to Table 2 in Subtype classifications, 42-06). Heparin has been shown to bind to L-type channels with high affinity558 see also ILG Ca InsPa, entry 19. OPC-13340, a dihydropyridine derivative, is a calcium channel blocker with potent antimitotic effects in rat vascular smooth muscle cells590. N-type: The peptide w-conotoxin MVII A (25 aa residues)559 is a selective channel antagonist. N-type and P-type: The peptide w-conotoxin MVII C (26 aa) is a blocker of wconotoxin GVIA-insensitive Ca2+ channels, including P-type Ca2+ channels in cerebellar Purkinje cells516. P-type: The 48 amino acid peptide w-Aga-IVA, isolated from the venom of the funnel web spider, Agelenopsis aperta, is a potent blocker of P-type Ca2+ channels. The toxin inhibits voltage-dependent Ca2+ entry into rat brain synaptosomes with an 1Cso of rv20nM66 . High-threshold P-type Ca2+ currents in rat Purkinje neurones are inhibited by w-Aga-IVA with an ICso in the range 2-10nM, with saturation blocking at 60-200nM toxin66 . T-type: There is no known high-affinity, selective toxin affecting T-type Ca2+ channels. These channels are characterized by insensitivity to dihydropyridines, w-CTx VIA, w-Aga-IVA and the polyamine toxin FTx, from the funnel web spider.

Dual action of nitrendipine as anticonvulsant 42-51-04: The dihydropyridine nitrendipine may exhibit anticonvulsant and neuroprotectant activity via the combined ability to modulate both NMDA-associated ion channels and L-type voltage-sensitive calcium channels560.

Imidazole antimycotics are antagonists of low selectivity 42-51-05: Imidazole antimycotics (e.g. econazole and miconazole) inhibit voltage-gated Ca2+ channels561, but are of low selectivity as they can also block Mn2+ entry currents observed in cells with empty intracellular Ca2+ stores562.

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 42-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction eiJ layout of entries, entry 02). Database locus names and accession numbers immediately follow the

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_ntry_42_1

colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources,

which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in Introduction eiJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouringr analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers), author/reference or nomenclature. Following direct accession, however, neighbouring t analysis is strongly recommended to identify newly reported and related sequences.

II

Nomenclature (see note 1)

Species DNA source

Original isolate Accession (sequence motifs)

Sequence/ discussion

al

subunit, DHP-sensitive 2 Ca + channel

Human

64 aa (partial)

pir: 177689

Powers, Genomics (1991) 10: 835-9.

al subunit, DHP-sensitive Ca2+ channel

Rabbit cardiac cloneHTDHP 2.0 (partial)

1331 aa (fragment)

pir: S05011

Slish, FEBS Lett (1989) 250: 50914.

al subunit homologue A

Rat brain (fragment, not verified)

233 aa

pir: A35901 Snutch, Proe Natl Aead Sci USA (1990) 87: 3391-5.

al subunit homologue B

Rat brain (fragment, not verified)

164 aa

pir: B35901

al subunit homologue C

Rat brain (fragment, not verified)

246 aa

pir: C35901 Snutch, Proe Natl Aead Sci USA (1990) 87: 3391-5.

al subunit homologue D

Rat brain (fragment, not verified)

188 aa

pir: D35901 Snutch, Proe Natl Aead Sci USA (1990) 87: 3391-5.

Snutch, Proe Natl Aead Sci USA (1990) 87: 3391-5.

al

subunit

Rat kidney

308 aa

gim: 207892 Yu, Proe Natl Aead Sci USA (1992) 89: 104948.

al

subunit

Rat kidney

283 aa

al

subunit

Rat kidney

95 aa

gim: 207890 Yu, Proe Natl Aead Sci USA (1992) 89: 104948. gim: 207888 Yu, Proe Natl Aead Sci USA (1992) 89: 104948.

1L..-_e_n_t _ry_42

_

Nomenclature (see note 1)

Species DNA source

Original isolate Accession (sequence motifs)

01 subunit, DHP-sensitive Ca2+ channel

Rat

147 aa

gim: 209517 Ma, TBiol Chern (1992) 267: 22728-32.

01 subunit, DHP-sensitive Ca2+ channel

Rat

1146 aa

gim:207729 not found

01 subunit, Ntype calcium channel

Human

478 aa

gim: 177456 Ellis, Science (1992) 257: 38995.

01.7

subunit, DHP- Rat aorta sensitive Ca2+ channel

96 aa

pir: A39227 Koch, TBiol Chern (1990) 265: 17786-91.

01.7 subunit, DHP- Rat aorta (fragment, not sensitive Ca2+ channel verified)

96 aa

pir: B39227 Koch, TBiol Chern (1990) 265: 17786-91.

01 subunit, DHP- Rat aorta sensitive Ca2+ channel 01 subunit, DHP- Rat sensitive Ca2+ channel

2169 aa

gim: 212166 Koch, FEBS Lett (1989) 250: 386-8.

01 subunit, DHP- Rabbit, cardiac sensitive Ca2+ muscle channel

Sequence/ discussion

2169 aa gb: M59786 Koch, TBiol N.Gly: 100, 183, sp: P22002 Chern (1990) 265: 17786-91. 358, 493, 928, 1246, 1417, 1468 Phos: 1574, 1626, 1699, 1847, 1927 2171 aa N.Gly: 100, 183, 358,493,859, 928, 1247, 1418, 1469

emb: Mikami, Nature (1989) 340: 230-3. X15539 prr: S05054 sp: 15381

2181 aa gim: 177450 Seino, Proc Natl 010 subunit, DHP- Human sensitive Ca2+ pancreatic islets N.Gly: 155,215, gb: M83566 Acad Sci USA 329 (1992)89: 584-8. channel Phos (PKA): 464, 465, 802, 869, 1510, 1679, 1720,1793, 1820, 1942, 1952 Phos (PKC): 1605 0lC

subunit rbC-ll Rat, neuroneal 2143 aa pir: JH0427 Snutch, Neuron differential splice Phos (Sel): 1545, (1991) 7: 45-57. variant 1597, 1670, 1818, 1898

0lC

subunit rbC-I Rat, neuroneal; 2140 aa pir: JH0426 Snutch, Neuron differential splice Phos (Sel): 1545, gb: M67516 (1991) 7: 45-57. variant 1597, 1670, 1818, 1898

II

-

entry 42

Nomenclature

(see note 1)

Species DNA source

Original isolate Accession (sequence motifs)

subunit A isoform

Rat cerebellum (not verified)

2212 aa

al

Sequence/ discussion

pir: A41098 Starr, Proc Natl

Acad Sci USA (1991) 88: 5621-5.

subunit, DHP- Rabbit, skeletal sensitive Ca2+ muscle channel

al

al

subunit

Rat

emb: Tanabe, Nature 1873 aa N-Gly: 79, 257 X05921 (1987) 32.8: 313Phos/PKA: 687 gb:M2319 18. pir: A30063 sp: P07293 1634 aa pir: JH0422 Hui, Neuron N-Gly gb:M57682 (1991) 7: 35-44. (carbohydrate, Asn): 154, 224, 328 Phos (Ser)/PKA: 464, 848, 1489, 1584 Ca2+ binding (EF hand): 1463-1491.

al

subunit, DHP- Carp (Cyprinus sensitive Ca2+ carpio) white channel skeletal muscle

1852 aa gb:M62554 Grabner, Proc Phos/PKA: 407, pir: A37860 Natl Acad Sci sp: P22316 USA (1991) 88: 1471, 1523, 727-31. 1738 (database comment - may not be phosphorylated)

al subunit, DHP- Mouse sensitive Ca2+ channel

2139 aa

gim: 197460 Ma, TBiol Chern (1992) 2.67: 22728-32.

Mouse erythro- 1864 aa leukaemia (MEL) cell line

Ma, TBiol Chern (1995) 2.70: 48393.

subunit, truncated form

al

al subunit, DHP- Rat sensitive Ca2+ channel

2212 aa

gim: 207622 not found

subunit, Ntype calcium channel

Rat

2336 aa

gim: 211091 Dubel, Proc Natl

subunit, Ntype calcium channel

Human

alB

alB

Acad Sci USA (1992) 89: 505862. 2237 aa

aID

subunit, Ca2+ Human, neuronal 2161 aa channel

em: M76558 Williams, Neuron (1992) 8: 71-84.

2261 aa subunit, P/Q- Human, (47 exons) cerebellum type calcium CACNL1A4 gene channel

em: X99897 Ophoff, Cell em: Z80114- (1996) 87: 543-52. Z80115

subunit, P/Q- Mouse, brain type calcium channel

gb: U76716

alA

alA

III

gim: 177442 Ellis, Science (1992) 2.57: 38995.

2164 aa

Fletcher, Cell (1996) 87: 607-17.

l_e_n_t_ry_42

Nomenclature (see note 1)

_

Species DNA source

Original isolate Accession (sequence motifs)

Sequence/ discussion

0:1

subunit (doe-I) Marine ray, Discopyge ommata, forebrain

2223 aa

gb:L12531

Home, Froc Natl Acad Sci USA (1993) 90: 378791.

0:1

subunit (doe-4) Marine ray, Discopyge ommata, forebrain

2326 aa

gb:L12532

Home, Froc Natl Acad Sci USA (1993) 90: 378791.

0:2a

subunit

Mouse, skeletal muscle

1096 aa

gb: U73483 Angelotti, FEBS Lett (1996) 397: 331-7.

0:2b

subunit

Mouse, brain

1084 aa

gb: U73484 Angelotti, FEBS Lett (1996) 397: 331-7.

0:2c

subunit

Mouse, heart

1079 aa

gb: U73485 Angelotti, FEBS Lett (1996) 397: 331-7.

0:2d

subunit

Mouse, heart

1072 aa

gb: U73486 Angelotti, FEBS Lett (1996) 397: 331-7.

0:2e

subunit

Mouse, smooth muscle

1077 aa

gb: U73487 Angelotti, FEBS Lett (1996) 397: 331-7.

0:2b subunit, DHP- Rat, brain sensitive Ca2+ channel

1091 aa

gim: 208474 Kim, Froc Natl gb:M86621 Acad Sci USA (1992) 89: 3251-5.

subunit, DHP- Rabbit, skeletal muscle sensitive Ca2+ channel precursor

1106 aa N.Gly: 94, 138, 186, 326, 350, 470, 477, 606, 615, 678, 784, 827, 891, 898, 988, 1001 Phos/PKA: 503, 848

gb: M21948 Ellis, Science pir: (1988) 2.41: 1661-

1091 aa Sig: 1-24 N.Gly (carbohydrate, Asn): 92, 136, 184, 324, 348, 468, 475, 585, 594, 663, 682, 769, 812, 876, 883,973,986

em: M76559 Williams, Neuron (1992) 8: 71-84.

0:2a

subunit, Ca2+ Human, channel neuronal

0:2B

CHRBA2

4.

sp: P13806

II

-

entry 42

Nomenclature (see note 1)

Species DNA source

Q2B subunit, Ca2+ Human,

Original isolate Accession (sequence motifs)

Sequencej discussion

PhosjPKA (Ser): 833 Phos/PKC (Ser): 91, 142, 250, 625, 817 PhosjPKC (Thr): 501; predicted PKC bindingsites 32, 268, 326, 539, 635, 1087

channel

neuronal

{3 subunit ({3lb)

Rat brain (not verified)

597 aa

{3 subunit, DHPsensitive Ca2+

Rabbit skeletal muscle

gb:M25817 Ruth, Science 524 aa N.Gly: 189,470, sp:P19517 (1989) 245: 111518. 499 524 aa

channel

Rabbit skeletal muscle (not verified)

{3 subunit ({32)

Rat brain

gb: M80545 Perez-Reyes, T 604 aa N.Gly: 148 BioI Chern (1992) 267: 1792-97. Phos/PKA (Thr): 164 Phos/PKA (Ser): 591 Phos/PKC (Thr): 23,489 Phos/PKC (Ser): 142, 159, 160, 190, 225, 235, 345,587

{3 subunit ({32)

Human hippocampus

478 aa

em: M76560 Williams, Neuron (1992) 8: 71-84.

{3 subunit CaB2a

Rabbit cardiac (not verified)

606 aa

pir: S21046 Hullin, EMBO T (1992) 11: 885-90.

{3 subunit CaB2b

Rabbit cardiac (not verified)

632 aa

pir: S21048

{3 subunit CaB2c

Rabbit (not verified)

229 aa

pir: S21047 Hullin, EMBO T (1992) 11: 885-90.

{3 subunit CaBa

Rabbit cardiac

477 aa

pir: S21049 Hullin, EMBO T (1992) 11: 885-90.

channel (3 subunit, DHPsensitive Ca2+

Human skeletal 478 aa voltage-dependent muscle and brain Ca2+ channel (two isoforms, single gene)

{31 subunit,

{31 subunit ({3la)

II

I

Human skeletal 523 aa muscle and brain (two isoforms, single gene)

pir: S18304 gb: X61394

Pragnell, FEBS Lett (1991) 291: 253-8.

pir: A41347 Ruth, Science (1989) 245: 111518.

Hullin, EMBO T (1992) 11: 885-90.

TBioI Chern (1992) 267: 22967-72.

gim: 177502 Powers,

gim.: 177500 Powers, TBioI Chern (1992) 267: 22967-72.

_

~e_n_try_4_2

Nomenclature (see note 1)

Species DNA source

(31 subunit ((31b)

Human skeletal 596 aa muscle and brain (two isoforms, single gene)

(32 subunit Ca2+

channel

Human, hippocampus

em: M76560 Williams, Neuron 478 aa N.Gly: (1992) 8: 71-84. (carbohydrate, Asn, covalent): 189,425 Phos/PKA: 205 Phos/PKC (Thr): 64,201 Phos/PKC (Ser): 32, 167, 209, 348, 374, 450, 464

(33 subunit

Rat brain

484 aa

gim: 207733 Castellano" BioI gb: M88751 Chern (1993) 268: 3450-55.

Rat brain

519 aa

gb: L02315

522 aa

gim: 184749 Collin, Cire Res

Original isolate Accession (sequence motifs)

(putative) (34 subunit

(3A,B or c? subunit, Human, cardiac L-type Ca2+

Sequence/ discussion

gim: 177504 Powers,' BioI Chern (1992) 267: 22967-72.

Castellano" BioI Chern (1993) 268: 12359-66. (1993) 72: 1337-

44.

channel, (3A,B or c? subunit, Human, cardiac

477 aa

gim: 184747 Collin, Cire Res

L-type Ca2+ channel,

(1993) 72: 1337-

44.

(3A,B or c? subunit, Human, cardiac L-type Ca2+

597 aa

gim: 184745 Collin, eire Res

(1993) 72: 1337-

44.

channel, Ca2+ channel

Rabbit lung

2166 aa

pir: 811339 Biel, FEBS Lett (1990) 269: 40912.

Ca2+ channel

Rabbit (PCRt origin, not verified)

347 aa

pir: 812955

Huang, FEBS Lett (1990) 274: 20713.

Ca2+ channel BI-1 Rabbit brain

2273 aa

prf: 1709354A

Mori, Nature (1991) 350: 398402.

Ca2+ channel BI-2 Rabbit brain

2424 aa

prf: 17093454B

Mori, Nature (1991)350:398402.

Ca2+ channel Bll-2 Rabbit brain

2259 aa

gim: 260373 Niidome, FEBS

Lett (1992) 308: 7-13. Ca2+ channel Bll-2 Rabbit brain

2178 aa

gim: 260375 Niidome, FEBS

Lett (1992) 308: 7-13.

II

-

entry 42

Nomenclature (see note 1)

Species DNA source

Original isolate Accession (sequence motifs)

Ca2+ channel protein 1

Human (not verified)

294 aa

pir: A23660 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

Ca2+ channel protein 2

Human (not verified)

294 aa

pir: B23660 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

Ca2+ channel protein 3

Human (not verified)

283 aa

pir: C23660 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

Ca2+ channel Bill Rabbit brain (type N)

2339

gim:402531 Fujita, Neuron (1993) 10: 585-98.

6 peptides Rabbit associated with the Ca2+ channel 02 subunit

13 aa

pir: A39518 Jay, J BioI Chern (1991) 266: 328793.

222 aa N.Gly: 43, 79

emb: X56031 gb:M32231 pir: A34857; S10728 sp: P19518

Rabbit, skeletal subunit precursor for Ca2+ muscle channel Human skeletal "y subunit muscle

222 aa

giro: 161310 Jay, Science (1990) 248: 490-2.

222 aa

em: X56031 Powers, J BioI Chern (1993) 268: 9275-9.

L-type Ca2+ channel

Human, fibroblasts

2220 aa

gim: 177510 Soldatov, Proc Natl Acad Sci USA (1992) 89: 4628-32.

L-type Ca2+ channel

Mesocricetus auratus

281 aa

gim: 195825 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

L-type Ca2+ channel

Mouse, skeletal muscle

300 aa

giro: 196814 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

L-type Ca2+ channel

Mouse

281 aa

giro: 196816 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

L-type Ca2+ channel

Mouse, cardiac

294 aa

gim: 196818 Perez-Reyes, J BioI Chern (1990) 265: 20430-6.

Partial, 121 aa

bbs: 121551 Oguro-Okano, Mayo Clin Proe (1992) 67: 1150-9.

"y

subunit (L-type) Rabbit, skeletal muscle cDNA

"y

Human, small N-type VGCC class D 01 subunit cell lung carcinoma cell line SCC-9 cDNA

II

I

Sequence/ discussion

Bosse, FEBS Lett (1990) 267: 153-6. Jay, Science (1990) 248: 490-2.

II.--_e_n_t _ry_4_2 Nomenclature (see note 1)

_

Species DNA source

Original isolate Accession (sequence motifs)

Sequence/ discussion

N-type VGCC Human, small class D 01 subunit cell lung carcinoma cell line SCC-9 cDNA

293 aa

bbs: 121550 Oguro-Okano, Mayo Clin PIoe (1992) 67: 1150-9.

Neuroendocrine L-type Ca2+ channel

Mouse

296aa

gim: 195823 Perez-Reyes, J Biol Chem (1990) 265: 20430-6.

Neuroendocrine L-type Ca2+ channel

Mouse

281 aa

gim: 196820 Perez-Reyes, J Biol Chem (1990) 265: 20430-6.

Synexin

Human voltagedependent Ca2+ channel (see Gene family, 42-

(51 kDa) gb: J04543 Unrelated to S4type family of VDCC.

Burns, PIoe Natl Aead Sci USA 86: 3798-802.

05)

Miscellaneous information Members of the annexin family can act as voltage-gated Ca 2+ channels 42-55-01: The Ca2+ -dependent phospholipid-binding protein annexin-V (CaBP33) can act as a voltage-gated Ca2+ channel through its membrane fusion activity571, as can synexin572 and other members of the annexin gene family573. These channels are structurally unrelated to the 'SI-S6 + H5' family of voltage-gated channels (see VLG key facts, entry 41).

[Ca 2+Ji rise due to intracellular Ca 2+ release 42-55-02: It has been shown that a major component of the glucose-activated intracellular calcium rise associated with insulin secretion in pancreatic beta cells represents voltage-dependent intracellular Ca2+ release (not Ca2+ influx through voltage-dependent calcium channels) (see Voltage sensitivity under JLG Ca JnsPa, 19-42).

Related sources and reviews 42-56-01: Major sources:33,41,98,555,574,575j channel-associated proteins576j excitation-contraction couplin~91j exocytosis and Ca2+ channels577,578j Ca2+ channel subunit structure_function34,35,191-193,420,579j genetic diseases affecting Ca2 + channels580j G protein coupling of Ca2+ channels456,521,581-585j direct G protein gating of cardiac L-type Ca2+ channels (critical discussion)586j selectivity and gatint87,588j facilitation of L-type Ca2+ currents300j Ca2+ channel antagonistsl99j heterologous expression of Ca2+ channels169j pharmacology of Ca2 + channels76.

Book references: Bean, B.P. and Mintz, I.M. (1994) In Handbook of Membrane Channels (ed. C. Perrachia), pp. 199-210, Academic Press, San Diego.

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Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA. Kostyuk et al. (1988) In The Calcium Channel: Structure, Function and Implications (eds Morad, M., Nayler, W., Kazda, S. and Schramm, M.), pp.442-64. Springer, Berlin.

Feedback Error-corrections, enhancement and extensions 42.-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource J, Search Criteria ei) CSN Development). For this entry, send email messages To: CSN-42.flle.ac.uk, indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 42-14-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback ei) CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

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NOTE ADDED IN PROOF

The molecular basis of T-type currents Reports describing the molecular cloning of cDNAs encoding (};lG subunits that form Ca2+ channels with the characteristics of T-type channels (PerezReyes et a1., Nature (1998) 391: 896-900) and of low-conductance IT-type' channel openings associated with other (};1 subunits (Meir et a1., Neuron (1998) 20: 341-351) have recently been reviewed (Bean, Neuron (1998) 20: 825-828).

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William

Voltage-gated chloride channels

J. Brammar

Entry 43

NOMENCLATURES

Abstract/general description 43-01-01: Included in the category of voltage-gated CI- channels are those channels in which open probability shows strong dependence on membrane voltage or for which changes in membrane potential represent the primary regulatory mechanism. The classification is crude and does not necessarily correlate with structural features. Also included are channels that have significant sequence homology with the family of voltage-gated channels, but which are themselves not strictly voltage dependent. 43-01-02: The physiological functions of the voltage-gated CI- channels vary in different tissues. The CIC-O and CIC-l channels stabilize the membrane potential in the electric organ of Torpedo and in skeletal muscle cells respectively. In the former case, the non-innervated surface of the cell is maintained close to the chloride equilibrium potential t while the innervated side is depolarized by the action of the nicotinic acetylcholine receptor in passing inward cation currents. In skeletal muscle, the activity of the CIC-l channel ensures electrical stability of the excitable membrane. The widely expressed CIC-2 channel is activated by unphysiologically strong hyperpolarization t and by extracellular hypotonicity t. The physiological role of the channel is to contribute to regulatory volume decrease in a wide range of cell and tissue types. The (:lC-Kl channel, largely expressed in the thin ascending limb of Henle's loop in the kidney, is probably involved in urinary concentration. Genes encoding proteins with homology to the CIC family of chloride channels have been identified in yeast (Saccharomyces cerevisiae) and in the gram-negative bacterium Escherichia coli, though the functions of the proteins in these microorganisms have not yet been defined. 43-01-03: The CIC-l mRNA is at very low levels in embryonic skeletal muscle, but increases continually over the first 30 days after birth, in parallel with the increase in muscle CI- conductance. The expression of the C1C-l gene is stimulated by muscular activity, and denervation t of the muscles leads to a reduction in the level of CIC-l mRNA. The promotert region of the C1C-l gene contains potential binding sites for transcription factors of the MyoDjmyogenin family, making it likely that CIC-l transcription is stimulated by myogenic t factors. The abundance of CIC-Kl mRNA in the kidney increases in response to deprivation of water and it is likely that the CIC-Kl channel activity may be important in urinary concentrating mechanisms. 43-01-04: Myotonia is a condition in which stiffness and impaired relaxation of skeletal muscle are caused by repetitive firing of action potentials. Several myotonic disorders are due to mutations in the CIC-l gene. The myotonic ADR (/~rrested gevelopment of righting response') mouse mutant, myotonia

1'--_e_n_t_ry_43

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in other mouse strains, and the recessive (Becker type) and dominant (Thomsen's disease) types of human myotonia are all caused by mutations in C1C-1 (see Phenotypic expression, 43-14-02 to 43-14-10). Dent's disease, an X-linked familial renal tubular disorder involving hypercalciuric nephrolithiasis ('kidney stones') and eventual renal failure, is caused by mutations in the C1CN5 gene (see 43-14-11). 43-01-04: Members of the CIC family have well-defined and overlapping tissue specificities. The mRNA for CIC-l, the 'skeletal muscle channel', is also found in kidney, liver and heart. The swelling-activated CIC-2 channel is 'ubiquitously' expressed, while CIC-3 mRNA is preferentially found in the brain, with lower levels in lung, kidney and adrenal gland. CIC-4

mRNA is prevalent in liver and brain, but also detectable in heart, spleen and kidney. Kidney is also a major site of expression of the gene encoding CIC-5, but the message is also found in brain, liver, lung and testis. The C1C-K genes appear largely kidney specific (see 43-13-01). 43-01-05: The structures of the genes encoding mammalian CIC channel proteins are complex and variable. While the human C1CNl gene has 23 exonst, the human C1CN5 gene has only 12, and there is no correspondence in the positions of the intronst in these two genes (see 43-20-05 and 43-2007). The sequences upstream of the gene encoding the 'skeletal muscle CIchannel', C1C-l, contain consensus binding sites for muscle-specific transcription factorst (see 43-20-01 and 43-20-02). 43-01-06: The transmembrane topology of the CIC proteins is ill-defined, being largely based on hydropathyt analyses. There are 13 discernable hydrophobic! sequences, Dl to D13 but an even number of transmembrane passes, since the N- and C-terminiT are intracellular. The favoured models suggest that neither D4 nor D13 are amongst the 10 or 12 transmembrane domains. The CIC-O and CIC-K2 channels have been shown to be glycosylated in the region between D8 and D9, placing this sequence on the outside of the membrane. 43-01-07: The CIC-O channel of Torpedo electroplax shows slow (seconds)

transitions from the closed to the open state, but more rapid (rv l0ms) transitions between open states with single-channel conductances of 10 and 20pS. The model to explain these observations involves twin ('doublebarrelled') 'two-state' channels as a single conducting unit. The slow transition from the closed to the open state always affects both twin channels simultaneously, so that the 'open' substates always appear and disappear together. The individual 'protomeric' channels can then undergo fast gating independently (see 43-38-01). The single-channel conductances of CIC-l and CIC-2 are rv 1pS and rv3-5 pS respectively, making it very difficult to study the kinetics of these channels directly at the singlechannel level. 43-01-08: CIC-2-mediated CI- currents are slowly and reversibly increased at physiological voltages when cells are subjected to hyposmotic t conditions. The regions of the CIC-2 channel involved in the activation by osmolarityt have been identified. The CIC-3 channels, which are open at all membrane voltages, are completely blocked by activation of protein kinase C.

II

Voltage-gated CI- channels

entry 43

43-01-09: The voltage-dependent opening of ClC-O and ClC-I channels is sensitive to the external CI- concentration. The permeant ion interacts with an externally available site, probably within the channel pore, to change the conformation of the channel protein and influence the voltagedependent gating process. Some organic anions, such as methanesulphonate, can apparently mimic the activity of Cl- in influencing gating without being permeant. 43-01-10: Members of the CIC family of voltage-gated chloride channels are blocked by the common Cl- channel blockers 4,4'-diisothiocyano-2,2'stilbenedisulphonate (DIDS), 9-anthracene-carboxylic acid (9-AC) and diphenylamine-2-carboxylate (DPC), though the sensitivities of individual channels to a particular blocker vary greatly. Inhibition of CIC-l by 9-AC can give rise to myotonia in tissue preparations or in vivo in treated animals. Inorganic anions (e.g. Cl04 -, Re0 4 -) and divalent cations (e.g. Cu2 +, Zn2 + and Cd2+) also block CIC channel activity (see Blockers, 43-43-01 to 43-43-07). 43-01-11: The activation of protein kinase C (PKC) has a profound inhibitory effect on CIC-l and CIC-3 activity. Activation of PKC in muscle fibres induces myotonict hyperexcitability similar to that of fibres from congenitally myotonic animals (see 43-,44-01). The activity of CIC-3 heterologously expressed in Xenopus oocytes is eliminated by treatment of the oocytes with phorbol esters to induce PKC activity (see 43-44-02).

Category (sortcode) 43-02-01: VLG Cl j Le. voltage-gated Cl- channels. The suggested electronic retrieval code (unique embedded identifier or DEI) for 'tagging' of new articles of relevance to the contents of this entry is DEI: VLGCL-NAT (for reports on native t channel properties) and DEI: VLGCL-HET (for reports or reviews on channel properties applicable to heterologouslyt expressed recombinantt subunits encoded by cDJNAst or genes t ). For a discussion of the advantages of UBIs and guidelines on their implementation, see the section on Resource Tunder Introduction etJ layout of entries, entry 02, and for further details, see Resource T- Search criteria.

Channel designation 43-03-01: ClC-O, CIC-I, etc. More rarely, some authors use Clc-I, etc.

Current designation 43-04-01:

IC1(CIC-O),

etc.

Gene family Scope of entry: the C1C gene famil), 43-05-01: Several distinct gene families are known that encode CI- channels, including those that are gated by the extracellular ligands ,-aminobutyric acid (ELG Cl GABAA , entry 10) and glycine (ELG Cl GLY, entry 11), the

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_

ATP-binding-cassette channels CFTR (JLG Cl ABC-CF, entry 23) and Pglycoprotein (JLG Cl ABC-MDR/PG, entry 24). This entry is concerned with the voltage-gated CI- channels encoded by the CIC gene family, defined by homologyt to the sequence encoding CIC-O, the voltage-gated CI- channel from the electric organ of Torpedo, the 'electric ray'. Without molecular information, it is not always possible to be sure of the identity of the ion channels giving rise to electrophysiologically or pharmacologically based data, and there is a danger that 'currents' or other consequences of ion channel function will be wrongly ascribed to members of the CIC family.

Genetic nomenclature 43-05-02: Note that the gene encoding the human CIC-5 chloride channel expressed in kidney was originally termed hCIC-K2, but has been renamed CICN5 on the advice of the Genome Database Nomenclature Committee1 . The rat CIC-K22 and CIC-5 3 sequences are quite distinct and are clearly the products of different genes.

Trivial names 43-07-01: CIC-O: double-barrelled chloride channel, Torpedo electroplax chloride channel. CIC-l: skeletal muscle sarcolemma chloride channel, the muscular chloride channel. CIC-2: the ubiquitous chloride channel. CICKl, CIC-K2, CIC-Ka, CIC-Kb: the kidney chloride channels.

EXPRESSION

Channel density C1C-O is abundant in Torpedo electroplax membranes 43-09-01: The Torpedo electroplax CIC-O protein has been estimated to constitute 200-1000 channels/llm2 of plasma membrane from conductance measurements on reconstituted planar lipid bilayers4,5. A method based on measurements of 36CI tracer-efflux from liposomes reconstituted at various protein concentrations yields a value of 630pmol channel/mg membrane protein, which corresponds to a channel density of 1"'J2000 channels/llm2 in the electroplax membrane6 . Note that this abundance is only 5- to 10-fold lower than the very highly concentrated packing of the acetylcholine receptor channels in the innervated face of the same cells 7, and represents 2-4% of the membrane protein in the Torpedo electrocyte8 .

Cloning resource cDNAs encoding C1C-O channels were isolated by expression cloning 43-10-01: DNAs encoding a member of the voltage-gated chloride channel family were first cloned from a cDNA library made from mRNA from the electric organ of the electric ray, Torpedo marmorata, by 'expression cloning't 9 . (Cells from the electric organ contain an unusually high density of voltage-gated CI- channels: see 43-09-01). The electric organ mRNAI

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was enriched for species encoding CI- channels by size-fractionationt , active mRNA fractions being detected by their ability to specify channels following injection into Xenopus oocytes. A cDNA libraryt was made from the enriched fraction, and cDNA clonest were screened by their ability to deplete CI- channel RNA from the enriched mRNA fraction. Candidate clones were characterized by their ability to hybridizet to an RNA species of appropriate size (9-10kb) on Northem t blots that was most abundant in electric organ. RNA transcribed from the full-length cDNA was shown to specify a voltage-dependent CI- channel when injected into Xenopus oocytes 9 . This CIC-O cDNA was subsequently used as a probet to screen cDNA libraries from rat skeletal muscle, leading to the isolation of CIC-l cDNAs 1o.

The human gene encoding G1G-4 was isolated by positional cloning 43-10-02: The human gene (C1CN4) encoding the CIC-4 channel protein was cloned because of its position in a region of interest on the human X chromosome (Xp22.3, see Chromosomal localization, 43-18-03). An evolutionarily conserved CpG islandt was recognized on a recombinant YACt clone, cloned from a cosmidt library and a restriction fragment containing the corresponding gene was used as a probet to identify a cDNA clone in an adult retina cDNA libraryt. When the cDNA isolate was sequenced it proved to encode a protein with homology to members of the CIC familyll.

Rat G1G-5 sequences cloned using .RT-PGR with degenerate primers 43-10-03: Rat CIC-5 cDNAs were initially cloned using RT-PCRt on rat brain poly(A)+ RNAt, with overlapping primers directed against the highly conserved amino acid sequence GKEGPLVH after domain D3 and allowing for all possible codonst. A 200 base-pair DNA fragment of novel sequence, closely related to CIC-3 and CIC-4, was used as a hybridization probet to screen a rat brain cDNA library3.

A GiG gene homologue on the E. coli chromosome 43-10-04: An open reading frame encoding a homologue of the Torpedo CIC-O chloride channel was revealed by sequencing an 83 kb region of the Escherichia coli chromosome corresponding to minutes 2.4 to 4.1 on the E. coli genetic map12.

Yeast G1G-l gene discovered during genome-sequencing 43-10-05: The gene encoding the yCIC-l chloride channel of the yeast, Saccharomyces cerevisiae, was recognized as an open reading frame t (ORF) during systematic sequencing of the yeast genome. Database searches revealed significant similarity of the putative gene product to members of the CIC family of voltage-gated CI- channels 13.

Developmental regulation G1G-l mRNA levels increase continually in neonatal rat muscle 43-11-01: The 4.5 kb mRNA of CIC-l is at very low levels in embryonic rat skeletal muscle, but increases continually over the first 30 days after birth,

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_

in parallel with the increase in muscle CI- conductance1o. The CIC-l mRNA is just detectable by Northern blotting t in neonatal muscle, but not in cultured myotubes t14 . The more sensitive RT-PCRt technique detects CIC1 mRNA in mouse myotube cultures, at levels estimated to be 100-fold lower than those of adult muscle. A combination of RT-PCR and hybridizationt to a CIC-l specific probet allowed detection of CIC-l mRNA in myoblasts and liver, at levels of 0.01 % or less than that of adult skeletal muscle15.

Expression of ClC-l is stimulated by muscular activity 43-11-02: Denervation t of the muscles of the mouse hindlimbs, by sciatic nerve transsectiont, leads to a reduction in the level of CIC-l mRNA in

the gastrocnemius and anterior tibial muscles to 20% of those of the control animal after two days. The mRNA levels in the muscles of the contralateral limb are very slightly reduced. Similar procedures carried out on adr and adt< myotonic mice (see Phenotypic expression, 43-14-02) produce no reduction in CIC-l mRNA levels. The concentrations of mRNAs encoding nAChR a subunit, myogenint and the muscle-specific transcription factor, MyoD, increased on denervation of wild-type muscle, but were not affected by denervation in adr animals. The promotert region of the C1C-1 gene contains potential binding sites for transcription factors of the MyoD/myogenin family16, making it likely that CIC-l transcription is stimulated by myogenic t factors. The spontaneous excitation that is characteristic of myotonic muscle may substitute for nerve activity in maintaining transcription of the C1C-l gene16.

Mouse ClC-5 mRNA increases in embryo 43-11-03: The rat CIC-5 mRNA is most abundant in the adult kidney, with a

broad intrarenal pattern of distribution. Within total mouse embryo RNA, the level of CIC-5 mRNA shows 'a marked increase' from day 7 to day 11 3 .

ClC-Kl expression is stimulated by dehydration 43-11-04: The abundance of CIC-Kl mRNA in rat kidney increased about four-fold as rats became dehydrated by deprivation of water for 5 days. The

predominant expression of CIC-Kl in the thin ascending limb of Henle's loop and the regulation by dehydration suggest that the CIC-Kl channel activity may be important in urinary concentrating mechanisms17.

mRNA distribution 43-13-01: The distribution of the mRNA encoding members of the CIC family

of chloride channels is shown in Table 1.

Phenotypic expression The ClC-O channel is involved in the generation of transcellular voltage 43-14-01: The CIC-O chloride channels in Torpedo species ('electric rays'),

located predominantly in the non-innervated membrane of the polarized

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_3_1

Table 1. The tissue distribution of mRNAs encoding members of the C1C family of chloride channels (From 43-13-01)

II

Channel

mRNA distribution

Refs

rCIC-I

43-13-02: The 4.S kb mRNA for the rat skeletal muscle CIC-I channel is also present in kidney, liver, heart, and the smooth muscle cell line AID. The level of CIC-I mRNA is 2-3 times lower in the slow-twitch soleus muscle of the rat, which predominantly contains type I fibres, than in the tibialis anterior and extensor digitorum longus, largely constituted of fast-twitch fibres.

10,16

rCIC-2

43-13-03: The rat CIC-2 mRNA is readily detectable by Northern blott in all tissues examined (skeletal muscle, heart, brain, lung, kidney, pancreas, stomach, intestine, liver) and in a wide range of c;elilines, including fibroblast (3T3), epithelial (T84, human colon cell line CFPAC-I, human pancreatic cystic fibrosis cell line), 'neuronal-like' cell-lines (Neuro-2a, PC12). I-fuman CIC-2 mRNA is detectable by Northern blotting most readily in pancreas and brain, less readily in heart, placenta, skeletal muscle and kidney and weakly in lung and liver18 . The CIC-2 mRNA was also present in human epithelial cell line, T84, and the cystic fibrosis bronchial epithelial cell line, rn3-1.

18,19

rCIC-3

43-13-04: CIC-3 mRNA is most abundant, as detected by Northern blotting, in rat cerebrum and cerebellum, with moderate levels in the lung, kidney and adrenal gland. ISH: strong in olfactory bulbi' cerebellum (abundant in Purkinje cell layer, moderate in granular cell layer) and hippocampus: moderate in cerebral cortex.

20

rCIC-4

43-13-05: CIC-4 mRNA is most prominent in rat liver and brain, but is also readily detected in heart, muscle, spleen and kidney. The mRNA was not detected in lung and testis by Northern blotting. ~rhe human CIC-4 mRNA is most abundant in skeletal muscle and readily detectable in brain and heart by Northernt blotting. The more sensitive RT-PCRt technique: detects CIC-4 mRNA in liver, adrenal gland, lymphoblast, adult retina and a teratocarcinoma cell line.

11,21

rCIC-S

43-13-06: Rat CIC-5 mRNA is most abundant in kidney, but is also detected in brain, liver, lung and testis. It is also detectable by Northern t blotting in mouse neuroblastoma (Neuro-2a), rat adrenal phaeochromocytoma (PC12), pig kidney (LLC-PKI), Syrian hamster pancreatic b-cell (HITTIS) and mouse fibroblast (3T3) cell lines. CIC-S message is readily detectable in kidney cell lines, being highest in the mTAL cell line derived from the thick ascending limb of the loop of Henle in mouse kidney. RT-PCRt on

1L....-__ _ _ry_43 en t

_

Table 1. Continued Channel

rCIC-Kl

mRNA distribution

Refs

microdissected rat kidney tubules showed CIC-S mRNA to be very low in glomeruli and S2 segments of the proximal tubule, but 'significant' in all other segments. Strong signals were obtained from cortical collecting tubules, the S3 segments of the proximal tubule and the medullary thick ascending limb.

3

43-13-07: The message for rat CIC-Kl was found

2,17

predominantly in kidney, especially in the inner medulla, and also at low levels in the bladder. RT-PCRt analysis on microdissected nephron segments showed the main site of expression in kidney to be the thin ascending limb of Henle's loop, which has the highest CI- permeability among the nephron segments and is thought to be involved in a counter-current system for urine concentration in the inner medulla. The CIC-Kl mRNA is also found in the S3 segment of the proximal tubule, the cortical thin ascending limb, the thin descending limb of Henle's loop, the distal convoluted tubule and the cortical collecting tubule. The sensitive RT-PCR technique also reveals CIC-Kl mRNA in rat brain. rCIC-K2

43-13-08: A Northernt blot with total rat CIC-K2L eDNA

as a probet under high stringencyt revealed the mRNA predominantly in the outer and inner medulla of the kidney. RT-PCRt analysis on RNAs from microdissected nephron segments showed the main site of expression in kidney is the thick ascending limb of Henle's loop and collecting ducts. The rat CIC-K2 mRNA is also detectable in glomerula, the S2 and S3 segments of the proximal tubule, the cortical thin ascending limb, the distal convoluted tubule and the cortical collecting tubule. 2,22

Note: The human CIC-K mRNAs are found 'exclusively in the kidney'. Analysis of mRNA from rabbit kidney shows expression in both the medulla and the cortex2 . (Note that because of the similarity between the C1C-Ka and C1C-Kb sequences, Northern blotting is unable to distinguish between them.)

cells of the electric organ, act to clamp the local membrane potential close to the resting potentialt. This function is essential for the generation of the large transcellular voltages ("J 100 mV) on strong depolarization of the innervated side by the action of the nicotinic acetylcholine receptors (see ELG CAT nAChR, 09-14-01). Hundreds of the flat cells are arranged in parallel stacks, and the transcellular potentials add to produce the large potential difference that is responsible for the stunning electrical discharge.

II

Voltage-gated CI- channels

entry 43

Inactivation of the mouse C1C-l gene causes myotonia 43-14-02: Mice homozygous t for the recessive t autosomalt mutation ADR ('~rrested ~evetopment of ~ghting response') lack the skeletal muscle chloride channel CtC-l and suffer from myotonia, stiffness and impaired relaxation of skeletal muscle caused by repetitive firing of action potentials. The classical mouse ADR mutation is caused by insertion of a member of the ETn family of mouse transposonst into the CIC-l gene, downstream of the exont encoding the putative D9 transmembrane donlain23 . Sequencing of ADR mouse muscle cDNAs shows a variety of truncated mRNAs resulting from splicingt to ETn-derived sequences and result events that fuse the D9 coding sequenct:~ in non-functional CI- channels23 . Other myotonic mutations of the mouse adr gene have been identified as changes of CGA (Arg) to TGA (Stop) at codon 47 in adrn to and I553T in the adt< allele24.

Mutations in the human C1C-l gene can cause congenital myotonia 43-14-03: The heritable muscle myopathyt myotonia congenita (Thomsen's disease) is caused by autosomal t dominant t mutations in the CIC-l gene25. Autosomalt dominantt myotonia congenita was first described by Thomsen26, who suffered from the disease. A mutation causing a P480L change in the short region connecting putative transmembrane segments D9 and DI0 cosegregatedt with the disease in the Thomsen family pedigree25 . On expression of a CIC-l cRNA sequence containing the P480L mutation in Xenopus oocytes, chloride currents were abolished in the physiological voltage range (-120 to +80mV), but slow activation of a CIcurrent could be detected after strong hyperpolarization (below -120mV). Co-expression of wild-type and P480L mutant cRNAs into Xenopus oocytes gives rise to hetero-oligomeric t channels in which the voltage dependence of gating is shifted to more positive voltages by +45 mY. Such channels would be closed at the resting potential of skeletal muscle and unable to contribute to the repolarization t of an action potential t27 (see Voltage sensitivity, 43-42-02). A G230E mutation found in Canadian myotonia congenita families 28 abolished CI- currents altogether in the heterologous Xenopus oocyte system25.

Mutant C1C-l subunits show dominant negative behaviour in Xenopus oocytes 43-14-04: Co-injection of P480L or G230E cRNAt with wild-type t CtC-l cRNA into Xenopus oocytes leads to specific suppression of the chloride currents in proportion to the concentration of the mutant cRNA. The degree of inhibition was much greater with the P480L cRNA. Assuming that the presence of a single mutant subunit in a channel multimert is sufficient to eliminate channel activity, the data obtained by titration of P480L versus wild-type cRNAs are consistent with a tetrameric arrangement of subunits for CCI-1 25 . The G230E protein has a weaker dominant negativet effect than the P480L variant, and the cRNA titration data suggest that more than one G230E mutant subunit would be necessary to inactivate the multimeric channel. Incorporation of only one G230E mutant subunit leads to functionally altered channels, with slower channel activation and less sensitivity to blockage by 1- at positive potentials25 .

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1'--_e_n_t_ry_4_3

_

Dominant negative C1C-l mutations act by shifting the voltage dependence of gating 43-14-05: The behaviour of a series of dominant negativet mutations in the human CIC-l gene (I290M, R317Q, P480L and Q552R, all isolated from patients with Thomsen's disease) has been examined by co-injection of wild-type and mutant cRNAst into Xenopus oocytes. In all cases, gating of the mutant/wild-type hetero-oligomeric l channels is strongly shifted to more positive voltages: shifts of +25 to +90mV are observed. The wildtype channel is rv 15% open at the resting potential of skeletal muscle (- 70 to -80mV), but the hetero-oligomeric channels are virtually closed at this voltage and therefore cannot contribute to the repolarizationt of a single action potential27.

Repetitive action potentials ('myotonic runs') can activate mutant chloride channels 43-14-06: In the muscles of patients with dominant myotonia congenita, the inability of the hetero-oligomerict chloride channels to repolarize action potentialst leads to repetitive action potentials ('myotonic runs') characteristic of myotonia. When such myotonic runs are mimicked with heterologouslyt expressed CIC-l channel subunits in Xenopus oocytes, by imposing a 66 Hz train of 5 ms pulses to +40 mV from a holding potential of -80mV, the Popen of the hetero-oligomeric channels increased from 050/0 to 18% over 700ms. This regime produces a Popen for the mutant channels that is similar to that of the wild-type channel under resting conditions and may therefore limit the length of the myotonic run27.

Becker's disease is caused by recessive mutations in the C1C-l gene 43-14-07: Autosomal t recessive t generalized myotonia t (RGM), 'Becker's disease', is usually caused by mutations in the CIC-l gene. An F413C mutation is commonly present in recessive myotonia29,,3o. Mutation of the last nucleotide of exonl 8 (G to A), probably giving rise to a splicingt defect, and a R496S replacement in exon 14 have also been identified in Becker families 3o. Individuals carrying one of these alleles are clinically normal, while affected members of the families have two mutant alleles30, as expected for an autosomal recessive disease. A 14 bp deletion in exon 13, in the region encoding the D9-DI0 loop and the beginning of DI0, has been shown to recur in a survey of unrelated Becker's disease patients in Germany31. The deletion causes a shift in the reading framet, resulting in a premature translation-terminationt codon 23 codons beyond the deletion breakpoint and a truncated CIC-1 protein of only 502 amino acids31 . A deletion of 4 bp, causing a frameshiftt mutation leading to translation terminationt in the region between D8 and D9, is the cause of recessive myotonia in an affected Turkish family32.

A mutation affecting voltage-dependent gating of C1C-l channels

43-14-08: A missense t mutation causing an amino acid replacement (D136G) within the first transmembrane segment of CIC-l causes an unusually severe recessive myotonia32. On heterologous expression in Xenopus oocytes or HEK-293 cells, the D136G channels show unusual gating properties, slowly

II

_L....-

e_n_t _ry_43_

activating over several seconds after stepping from a holding potential of -30mV to more negative potentials. (Wild-type channels deactivate on hyperpolarization.) The mutant channels also exhibit strongly increased inward rectification t , with a complete absence of steady-state outward currents at voltages positive to the CI- reversal potential (Ecd. This absence of outward current at voltages positive to ECI is explained by rapid, voltagedependent channel deactivation at these potentials. The activation of the D136G channel, in contrast to that of the wild-type CIC-l, is strongly coupled to the transmembrane CI- gradient, and Popen approaches 0 at voltages positive to ECI at physiological chloride distributions. These observations suggest that D136 is normally involved in determining the channel's response to changes in membrane potential and explain the severe phenotypet of the homozygoust D 136G mutant33 .

Myotonia can be induced by chemical block of Cl- channel activity 43-14-09: The chloride conductance of intercostal muscle fibres from myotonic t goats are reduced about eight-fold from those of fibres from normal control animals. Intravenous injection of anthracene-9-carboxylic acid (8mg/kg) produced, within 1-2min, a myotonic state resembling that of a severely affected congenitally myotonic goat. The dosage giving '50% goats, ",,4 mg/kg, was estimated response' in pentobarbitone-anaesthett~ed to correspond to ",,5 x 10-5 M in the blood and extracellular fluid 34.

PKC activation causes myotonia in vitro 43-14-10: Activators of protein kinase C (PKC), such as the phorbol ester 4-{3phorbol-12,13-dibutyrate (0.1-2.0)lM), cause significant (up to 76%) block of the chloride conductance (Gcd in fibres isolated from goat intercostal muscles, and thereby induce myotonic T hyperexcitability similar to that of fibres from congenitally myotonic goats35 (see Channel modulation, 43-44-01).

C1C-2 is involved in regulatory volume decrease 43-14-11: The ubiquitously expressed CIC-2 chloride channel is activated by non-physiological hyperpolarization and by hyposmotic conditions at physiological voltages ('swelling-induced CI- current'). The regions of the CIC-2 channel involved in the activation by osmolarityt have been identified and shown to function norrrlally when transposed in the CIC-2 channel protein36 (see Domain functions, 43-29-03 and 43-29-04). Activation of CIC-2 by cell swelling is part of the 'regulatory volume decrease', in which the cell loses salt and water and tends to return towards its original volume. Note that CIC-2 is not the only chloride channel involved in the volume-regulating phenomenon, since chloride channels differing from CIC-2 in voltage sensitivities, anion selectivities and pharmacological characteristics are induced by swelling in airway epithelia37, oocytes38 and other cell types39- 42•

Mutations in the human C1CNS gene cause Dent's disease 43-14-12: Dent's disease, an X-linked familial renal tubular disorder involving hypercalciuric nephrolithiasis ('kidney stones') and eventual renal failure, has

II

1

e_n_t_ry_4_3

---'_

been associated with a microdeletion at Xp11.2243 • The use of YACt clones covering the Xp11.22 region as hybridization probest to screen a human renal cDNA library led to the isolation of cDNAs corresponding to a member of the ClC family, originally designated hClC-K2 44 but more recently renamed hCICN5 1 . Disruption of the GlGNS gene has been demonstrated in several cases of Dent's disease 1 .

A member of the G1G family is involved in iron transport in yeast 43-14-13: The gene encoding the ClC family member yCIC-l in yeast complements t mutants of the GEFl gene ('glycerol/~thanol, Ee-requiring'j GEF1 mutants require high concentrations (5 mM) of iron for normal growth). Intact cells of the gefl- mutants show a 500/0 reduction in rates of 02 consumption compared to the wild-type, an effect that is confirmed with mitochondrial extracts. It has been suggested that the yeast GEF1 gene product 'is directly involved with the transport of iron across a cellular membrane', possibly the mitochondrial membrane45 .

Protein distribution G1G-l protein is in the sarcolemmal membrane of skeletal muscle 43-15-01: Antibodies raised against the last 15 amino acids (980-994) of the rat skeletal muscle chloride channel, CIC-l, detect a single immunoreactive protein of 130 kDa in skeletal muscle microsomes. Westernt blot analysis of membrane preparations from other tissues, including liver, kidney and heart, where CIC-1 mRNA can be detected, did not detect CIC-1 protein. In cryosectionst of skeletal muscle, immunoreactivity was detected exclusively in the cell periphery, suggesting sarcolemmal distribution of CIC-1 protein. Staining was evenly distributed along the sarcolemmal surface of all fibre types. Separation of sarcolemma membranes from transverse tubules and sarcoplasmic reticulum showed that the CIC-l protein remained exclusively with the sarcolemmal component. The CIC-1 protein was not detectable in skeletal muscle membranes from the ADR (~rrested gevelopment of righting response) myotonic mouse46 (See Phenotypic expression, 43-14-02). f".J

Subcellular locations G1G-O channels are located on the non-innervated face of the electroplax cell 43-16-01: The voltage-gated Cl- channels of the electric organ of the Californian ray, Torpedo californica, have been predominantly detected in the non-innervated membrane of these polarized cells by immunofluorescencet 47. Results consistent with this observation were obtained by comparing the yields of functional channels from membrane fractions enriched for either innervated or non-innervated face membranes 6 .

Transcript size 43-17-01: The sizes of the mRNA species encoding members of the CIC family of chloride channels are shown in Table 2.

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_3_1

Table 2. Sizes of mRNA species encoding members of the GlG family (From 43-17-01) Channel

Tissue

mRNA size

ctc-o CtC-l CtC-2

CtC-3

Torpedo marmorata electric organ Rat, skeletal muscle Rat, most tissues Rat, kidney and brain Human, wide range of tissues Rat brain

CtC-4

Human skeletal muscle, liver, brain

CIC-5

Human kidney, skeletal muscle Rat kidney Rat kidney Rat kidney

9-10kb 9 4.5kb1O 3.3kb19 3.3 kb + ",4.5 kb 19 3.3 kb + ",5 kb (minor)18 5.5 kb (major) and 3.0kb (minor)2o 7.5 kb (major) and ",4 kb (minor)ll 9.5kb44 9.5kb3 2.4kb 17 2.4 kb (major) and ",3.2 kb (minor)22

CtCK-l CtCK-2

SEQUENCE ANALYSIS

Chromosomal localization The C1C-l gene lies on mouse chromosome 6 and human chromosome 7 43-18-01: Using interspecies backcrossest, the CtC-l locus has been mapped to mouse chromosome 6, between the marker genes Tcrb and Hox1.1 23 . This region of mouse chromosome 6 shows strong syntenyt with a region of human chromosome 748 . The human CIC-l gene has been mapped to 7q32-7qter, tightly linked to the T cell receptor f3 (TGRB) locus at 7q35 29,49.

Human C1C-2 gene located on chromosome 3 43-18-02: The human C]CN2 gene has been localized to chromosome 3 using human/rodent somatic cell hybrid t panels, and mapped to 3q26-qter using a panel of human/hamster radiation hybrids t18 .

The human C1CN4 gene maps to Xp22.3 43-18-03: The human C]CN4 gene was isolated by positional cloningt during a detailed study of the Xp22.3 region. Southern blottingt of DNAs from cell lines with mapped deletions in the region of Xp22 with a CICN4 cDNA probet confirmed the position of the gene11 .

The human gene encoding C1C-5 maps to Xpll.22 43-18-04: The human C]CN5 gene, originally designated hCIC-K2, maps to Xpll.2244 . Mutation of this gene is responsible for Dent's disease, an Xlinkedt hereditary nephrolithiasis ('kidney stones,)l,44.

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1......._e_n_t_ry_43

----'_

Mapping of a yeast gene encoding a chloride channel 43-18-05: The gene encoding a voltage-gated CI- channel in the budding yeast, Saccharomyces cerevisiae, is located on chromosome X, between genes PET191 and SSC1 5o . (An earlier report has suggested that the yeast gene GEF1, encoding yCtC-l, is located on chromosome XIV45 .)

Location of a chloride channel gene on the E. coli chromosome 43-18-06: A gene encoding a protein with homologyt to CIC-O was located by sequencing the region of 83 kb corresponding to minutes 2.4 to 4.1 on the E. coli genetic map. The chloride channel gene lies between the known E. coli genes gsa and dgt, and is transcribed rightwards (clockwise) on the standard genetic map12. No function has been associated with the product of the E. coli gene.

I

Encoding 43-19-01: The predicted sizes and molecular weights of the different CIC proteins are shown in Table 3.

Gene organization Sequences upstream of the mouse C1C-l gene 43-20-01: The immediate 5' flanking region of the mouse C1C-l gene contains a TATAA box (-132 to -128, where A of initiating ATG is +1) and four closely spaced E boxes (CANNTG), consensus t binding sites for musclespecific transcription factors t of the MyoDjmyogenin family. A CCCCACCCC sequence at -228 to -219 is a motiit that is found in a subset of muscle-specific genes that are subject to late foetal or early postnatal development16. A palindromict sequence (AGGGAATTCCCT) at -271 to -260 is conserved in the 5'-untranslated region of the human C1C-1 gene16,30. Sequences upstream of the human C1C-l gene 43-20-02: The 5' upstream region of the human C1C-l gene has a TATAA box centred at -132 bp with respect to the first base of the initiator (ATG) codon. Consensus t 'E-box' sequences (CANNTG), putative binding sites for transcription factors t of the MyoDt family, are present at -146 to -lSI, -166 to -171 and -190 to -195. A CCAC box (CCCCCACCCCC, -219 to -208) and a palindromic t dodecameric sequence (AGGGAATfCCCT, -260 to -249) are features that are shared with the mouse C1C-1 gene30 (see Gene organization, 43-20-01). Sequences upstream of the human C1C-2 gene lack an obvious TATA box 43-20-03: The upstreamt flanking region of the human CIC-2 gene does not contain obvious TATA boxt or CCAAT boxt consensus sequences, but there are three consensus sequences for the transcription factor t SP-1 at -183 to -188, -194 to -199 and -215 to -220 (numbered from the first base of the translation initiationt (AUG) codon). Primer extensiont on the mRNA

II

Voltage-gated CI- channels

entry 43

I

Table 3. Predicted sizes of CiC proteins (From 43-19-01)

Channel protein

Size (aa)

Mr

CIC-O hCIC-l rCIC-l hCIC-2 rCIC-2 rCIC-3 hCIC-4 hCIC-5 rCIC-5 rCIC-KI rCIC-K2L rCIC-K2S hCIC-Ka yCIC-l

805 988 994 898 907 760 760 746 746 687 687 632 687 779

89000 110000 110000

Refs 9 30 10

18

99000 84000 84000 83000 83000 76000 75000 69000 75000 87000

19 20

11 1 3

2,17 22 22 2 45

The prefixes h, rand y refer to channel proteins of human, rat and yeast (S. cerevisiae) origins.

from the human epithelial T84 cell line reveals transcription start sites at -100 nt (major) and -123 nt (minor) from the translation initiation codon18.

Sequences upstream of the yeast G1G gene 43-20-04: The DNA upstream of the presumed ATG translation-start codon of

the S. cerevisiae GEFl (yCIC-I) gene contains potential TATA boxes t at -51 to -54 and -151 to -154, a match at five out of eight bases to the HAP2/3/4 transcription factort-binding motif (TNATTGGT) at -184 to -177 and a match at eight out of ten bases to the ROX1 consensus sequence (YYYATTGTTC) at -334 to -325 45 •

Structure of the human G1GN1 gene 43-20-05: The human C]CNl gene occupies at least 40 kb of chromosomal

DNA, organized in 23 coding exonst. The positions of the introns with respect to the coding sequence are shown in Fig. 1.

The human G1GN4 gene occupies >60kb of X chromosome DNA 43-20-06: The human C]CN4 gene has been shown by long-range t mapping in

the Xp22 region to occupy 60-80 kb of chromosomal DNA and to contain at least ten exons11 .

The human G1GN5 gene has 12 exons 43-20-07: The coding region of the human C]CN5 gene has been shown

to consist of 12 exons l and the exon-intron t boundaries have been determined by a PCRt -based strategy on cDNA and human genomic DNA templates 1 . None of the introns t of theCICN5 gene correspond in position with any of the 22 introns of the CICNI gene1 .

II

-----l_

II--_e_n_t _ry_43

~

~

221 ~

1

===~I ==~::~~~_-_I_~_81

2

3

,_302_..~,.D.JIOo iI

.......4_34...-.....;,.m&&.

333 ~

4

6

;_! ~_63 ~1m1I~ ...........

8

:~_~4_ _.,lC&

.....'...,;;,;::_ .........'_7...-:m __'......

,~- '980

__........

ATG

1 ~

~

234 ~

~

220 ~

9

10

,."S~

,' l}(: ..

)6., ..

. ~

188 ~

12

11

14

11~83

, 111 •. . .1.6677 ' _115_2_ _ 11402 11472

"/~DJf~~

CL

l'2

~

bp

rl73

18

19

16

I>lti

S

CL

~

383

17

~

13

93 bp

20

22

21

~r--2~_96

....... ,

==

STOP

2967

Figure 1. Structure of the human CLCNI gene. The complete protein-coding region of the gene is shown as three open boxes, with the first base of the initiation codon (ATG) as nucleotide 1 and the last base of the termination codon as nucleotide 2967. The grey areas represent regions of exons that encode putative transmembrane domains (D1-D12). D13 is a highly conserved cytoplasmic segment near the C-terminus of the protein. Arrowheads indicate the positions of introns (1-22) that interrupt the coding region: the sizes of some introns are indicated above the arrows. The exons are drawn to scale, with the position of the first bases of the exons indicated by the numbers to the right of the arrowheads. (Reproduced with permission from Lorenz (1994) Human Mol Genet 3: 941-6.) (From 43-20-05)

Homologous isoforms 43-21-01: The homologies between the various CIC proteins are shown in

Table 4.

The tkidney G1G' subfamily 43-21-02: Several cDNAst with sequence homologyt to the CIC family have been cloned from rat2,17 and human2 kidney RNAs. The rat CIC-Kl cRNA

gives rise to 'slightly outwardly rectifying time- and voltage-independent chloride currents I in Xenopus oocytes 171 but there are reports that the kidney-specific channel cRNAs (ClC-Kl 1 ClC-K21 ClC-Ka and CIC-Kb)1 either singly or in pairwise combinationsl do not give rise to detectable currents in the Xenopus oocyte system2 . l

A short variant of G1G-2K in rat kidney 43-21-03: A cDNA isolated from rat kidney mRNA1 CIC-K2S1 encodes a

632 amino acid protein in which 55 amino acids containing the putative

II

II

Table 4. Identities (per cent) between the various CiC isoforms (From 43-21-01)

CIC-O CIC-O CIC-l CIC-2 CIC-3 CIC-4 CIC-5 CIC-Kl CIC-K2 CIC-Ka

CIC-l

CIC-2

CIC-3

55

49 55

20.9 24 26.3

CIC-4 <30 <30 78

CIC-5

78 78

CIC-Kl

CIC-K2

39 41 43 24.7

40 43 45

82

CIC-Ka

CIC-Kb

24 80 91

9 M-

~ ~ (J.J

1'--_e_n_t_ry_4_3

---J_

second membrane-spanning domain of CIC-K2L are deleted. The CIC-K2L cRNA induced chloride currents that were very similar to those induced by the longer CIC-K2L cRNA, with anion selectivity sequence Br- > 1- > CI- »cyclamate22 .

The yeast CiC protein 43-21-04: The yCIC-l protein from Saccharomyces cereVlSlae has 460/0 identity with the CIC-O protein in the region between yCIC-l residues 169-244 (roughly D2-D4), and 25% identity over the region 169-611 45 •

Protein molecular weight (purified) The purified Torpedo CiC-O protein is a homodimer of a 90kDa monomer 43-22-01: The CIC-O channel protein extracted from Torpedo electroplaxt membranes, immunoaffinityt purified in an active state using purified polyclonalt antibodies raised against an 18 residue peptide from CIC-O, consists of a 90 kDa polypeptide. The purified preparation contains no associated subunits. Direct protein sequencing reveals an N-terminus t at Ser2 of the cDNAt -derived sequence. The protein is N-glycosylatedt on Asn365. Sucroset density gradient sedimentationt measurements under activity-preserving conditions suggest the CIC-O channel is approximately 200 kDa, consistent with its being a homodimert of the purified 90 kDa protein8 .

Protein molecular weight (calc.) 43-23-01: See Encoding, 43-19-01.

Sequence motifs Consensus sites for N-iinked giycosyiation 43-24-01: The consensus t sites for N-linkedt glycosylationt , based simply on analysis of the protein primaryt sequence predicted from the nucleotide sequence of the coding region, are shown in Table 5.

Identification of the N-giycosyiated residue in CiC-O 43-24-02: Digestion of the purified Torpedo californica CIC-O protein with endoglycosidasetHor N-glycanaset reduces the apparent molecular mass by rv2 kDa, consistent with only one N-linked glycosylationt with a high mannose-type oligosaccharidet. Digestion of the purified protein with V8 proteaset before and after removal of the N-linked oligosaccharidet allowed identification of the glycosylated residue as Asn36S 8 . This conclusion is consonant with data obtained following in vitro translation of cRNAs encoding rat CIC-Kl and CIC-K2 in the presence of dog pancreatic microsomest: in both cases glycosylation occurred exclusively in the region between D8 and D9 2 .

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Table 5. Consensus sites for N-linked glycosylation (Asn-X-Ser/Thr) in CiC proteins (From 43-24-01) Channel

Position (sequence)

Refs

CIC-O rCIC-1 hCIC-2 rCIC-2 CIC-3 hCIC-4 rCIC-KI rCIC-K2 hCIC-Ka hCIC-Kb hCIC-5 rCIC-5 yCIC-l

365 (NYT) and 806 (NSS) 430 (NNT), 802 (NMS), 895 (NTT) 127 (NTS), 232 (NES), 784 (NFS) 238 (NES), 409 (NRT), 711 (NAT) and 793 (NFS) 119 (NET), 393 (NTS), 421 (NAS), 616 (NMT) and 627 (NET) 119 (NET), 141 (NQS) and 421 (NMT) 364 (NNS), 373 (NSS), 554 (NLT) 364 (NNS), 373 (NSS), 552 (NCT) 679 (NLT) 364 (NHS), 373 (NSS), 552 (~HS), 364 (NHS), 373 (NSS), 552 (NHS), 679 (NLT) 38 (NKS), 408 (NTS) 38 (NKS), 408 (NTS) 115 (NKS), 295 (NSS), 328 (NVT), 417 (NTS), 515 (NLT), 599 (NET), 657 (NST), 669 (NKS), 689 (NES)

9 10 18

19 20 11

2,17 2

2 2

1 3 45

The rat C1C-K2 protein is glycosylated in the D8-D9 region

43-24-03: Consensus t sites for N-linked glycosylationt at N364 and N373 are common to the four members of the CIC-K subfamily. The rat CIC-K2 protein is glycosylated when produced by heterologous t expression of cRNA in Xenopus oocytes, and the rat CIC-K1, rat CIC-K2, human CIC-Ka and human CIC-Kb proteins are glycosylated during synthesis by in vitro translation t in the presence of dog pancreatic microsomes t. Removal of the consensus glycosylation sites at N364 and N373 by in vitro mutagenesis giving N to Q replacements eliminated in vitro glycosylation of rCIC-Kl and rCIC-K22 . This observation suggests that the D8-D9 region of the rCIC-K channel proteins is extracellula~.

Southerns Southern blots reveal the C1C-l mutation in adr myotonic mouse DNA 43-25-01: Southern blots obtained by digesting mouse genomic DNA with the EeoR! restriction endonuclease and probed with a rat CIC-l cDNA probe show two bands, at 4.1 kb and 6.6 kb. In DNA from adr homozygous mice, which carry an insertion in the CIC-1 gene, the 6.6 kb fragment is replaced by a 10.5 kb fragment 23 • Aberrant restriction fragments were also revealed by digestion of the two DNAs with the BamID, PstI and SspI endonucleases.

Southern blots reveal a human C1C··l mutation causing recessive myotonia 43-25-02: Southern transfers of EeoR! digests of human genomic DNA, probed with human CIC-l cDNA, show four hybridizing bands. A similar analysis of the DNAs of chromosome 7-specific, human-mouse somatic cell hybrids

II

l_e_n_try_4_3

---J_

localized the human C1C-l gene to the chromosomal region 7q32-qter9 . One C1C-l mutation causing recessive generalized myotonia t , a T to G transversiont resulting in an F413C substitution can be detected by a change in the pattern of hybridizing bands seen on a Southern blot after digestion with NSl1. (The mutation changes the sequence A.TTC.AT to A.TgC.AT, thereby generating a new NSl1 recognition site29.)

Southern blots show the strong conservation of the ClCN4 gene 43-25-03: Southern blots produced by digestion of human DNA with the HindTII and EcoRI restriction enzymes and hybridizing with a human CICN4 cDNA probe, are shown in ref. 11. A Southern blot containing digests of DNA from monkey, cow, sheep, mouse, rabbit and chicken shows the high degree of conservation of the C1CN4 gene11 .

Restriction mapping of the human ClCNS gene 43-25-04: The seven EcoRI sites in the region of the CICN5 gene on the human X-chromosome have been mapped, and their relationship to the intron-exon t structure of the gene determined1 .

STRUCTURE AND FUNCTIONS

Domain functions (predicted) 43-29-01: Note that chloride channels of the C1C family lack an obvious voltage-sensor motif analogous to the S4 helix of the voltage-gated Ca 2+, Na+ or K+ channels. In addition, functional domains such as the poreforming regions have not been identified in any of the C1C family members.

An N-terminal region of C1C-2 required for gating by hyperpolarization 43-29-02: The channels produced by expression of CIC-2 cRNA in Xenopus oocytes activate slowly on strong hyperpolarizationt and their CIconductance is stimulated by hypotonicityi 19. Deletion of the first 62 amino acids from the N-terminus i leads to constitutively open channels, suggesting the presence of a slowly acting, voltage-dependent 'gate t , in this region of CIC-236 . Deletion of the first 15 amino acids did not affect the channel activity, but small deletions in the region of residue 24, including the deletion of only E24 and Q25, led to the 'open' phenotype. Amino acid changes leading to open channels included Q25L/T26L, increasing local hydrophobicityi, and T26E/L27E, generating increased charge. The 'open' mutants showed no response to hyper- and hypotonicity36.

A region of C1C-2 is responsible for response to osmolarity 43-29-03: Deletions affecting the N-terminal t region of rat CIC-2, removing small numbers of contiguous amino acids anywhere in the region 36-76, change channel activation parameters and apparent responses to changes in osmolarityt. These mutants pass significant CI- currents at resting

II

Voltage-gated CI- channels

entry 43

membrane potentials, show rapid activation on hyperpolarization and are further activatable by hypotonic conditions. In contrast to wild-type channels, the mutant channels are closed by hypertonic media. These 'intermediate' mutants behave as though the set points for cell volume activation are shifted towards hypertonicity and are thus partially open at isotonicity36.

The gating region of C1C-2 interacts with a channel-specific treceptor' 43-29-04: The function of the 'gate t ' region of CIC-2 (see 43-29-02) is position independent. Translocation t of the sequence encoding Metl-Arg74 to a position between His695 and Gly696, in the intracellular C-terminal t domain of the CIC-2 deletion mutant lacking residues 16-61, resulted in a CIC-2 channel with wild-typet characteristics. Insertion of a shorter region (GluI7-Lys41) produced channels with an 'intermediate' phenotype, partially activated at resting potentials and responsive to both hypo- and hypertonicity. Inclusion of the Q25L/T26L changes (see above) in the translocated region destroyed its ability to rescue the voltage and volume sensitivity of the d(16-61) mutant channel. The replacement of the entire intracellular N-terminal region of CIC-l by the analogous region of CIC-2 did not significantly change the properties of the CIC-l channel, suggesting that the N-terminal gating domain needs to interact with a 'receptor' site that is channel specific36 . Note the similarity of these findings to the 'balland-chain' model of inactivation proposed for voltage-dependent sodium channels51 and experimentally verified for Shaker K+ channels52,53 (see VLC K Kvl-Shak, entry 48).

Predicted protein topography Evidence for intracellular N- and C-termini 43-30-01: Hydropathyt analysis of the predicted CIC-O protein shows 13

hydrophobict, potential transmembranet regions and a hydrophilict Nterminus t lacking an obvious signal t sequence9. Deletion and mutagenesis experiments have located N-terminal domains of the related CIC-2 channel that are necessary for activation by both hyperpolarizationt and volume decrease36 . These regulatory domains remain fully functional after transplantation to a position C-terminal t to hydrophobic t domain D13, 'effectively excluding D13 as a translTlembrane domain', and suggesting that the C-terminus is intracellula~6.

The DB to D9 loop is extracellular

43-30-02: A consensust site for N-linked glycosylation t is present in the

region between hydrophobic regions DB and D9 (N365 in CIC-O)9 and is highly conserved among members of the CIC family. (A second such consensus site in the CIC-O protein is located at N801, very close to the intracellular C-terminust, and is therefore not utilized in vivo.) During in vitro translation in the presence of pancreatic microsomest the CIC-O, CIC-l, CIC-2 and CIC-K channel proteins are glycosylated2. Mutation of the consensus site between D8 and D9 in the CIC-K channels eliminates glycosylation21 and glycosylation of the equivalent segment (N365) of the

II

(1)

t:S

f"1"

~

~

o 136G has a strong effect on voltage sensitivity of CIC-1

G230E

D136G

(DMC) I 1 I

(RMC) 1 1 1

R300Q

Insertion

(DMC)

(ADR)

P480L (DMC)

1 1 1 1

'Y

I I I

I

1 1

I I I I 1290M (DMC)

NH

1

---I I

Q552R (DMC) 1 I I I I

I

Deletion of 4 base-pairs generating a stop codon (RMC)

2

(aal )

I Human skeletal muscle CI channel CIC-1 I

eOOH (aa988)

Channel symbol

NOTE:

II

All relative positions of motifs, domain shapes and sizes are diagrammatic and are subject to re-Interpretation

Il!llilmI~Ii 'il i

Figure 2. Monomeric protein domain topology model (PDTM) for the human skeletal muscle C1C-1 chloride channel. The positions of mutations causing Becker's disease (recessive myotonia congenita, 'RMC') and Thomsen's disease (dominant myotonia congenita, 'DMC') are shown. The topology in the M9-M12 region is not defined, but there is an even number of transmembrane passes. The position of an insertion in D9 in the C1C-1 channel of the ADR mouse23 is also shown. (Based on Heine (1994) Hum Mol Genet 3: 1123-8.) (From 43-30-02).

Voltage-gated CI- channels

entry 43

;

CIC-O protein has been shown for the native channel from Torpedo electric organ8 . These observations place the D8-D9 loop on the outside of the cell and imply that one of the hydrophobic domains N-terminal t to this region does not cross the membrane. Because the hydrophobicityt of D4 is relatively weak and not well conserved throughout the family, it is proposed that this region does not cross the membrane. A tentative topography model is suggested in which there are intracellular N- and Ctermini, 12 membrane-spanning domains, extracellular D4 and intracellular D13 regions21 (Fig. 2).

Protein interactions No evidence for heterologous interactions 43-31-01: Since the CIC-l chloride channel is multimeric t , it seems possible that some functional chloride channels might be heteromultimerict. Attempts have been made to obtain evidence for heteromultimers by coexpression of cRNAst encoding the closely related CIC-3, CIC-4 and CIC-5

Table 6. Consensus phosphorylation sites (From 43-32-01) Channel protein

Position (sequence)

Refs

PKA(RRXS) rCIC-1 hCIC-2 rCIC-3 hCIC-4 hCIC-5 rCIC-5 rCIC-Kl rCIC-K2 yCIC-l

682 (RKL~J 655 (RRAI) (not present in rat CIC-2) 362 (RRK.~) 56 (KKE~), 50 (RKII), 362 (RRK:I) and 363 (RKTI) 380 (TRM~), 350 (RKTI) 349 (KRKI), 350 (RKTI) none none 771 (KRFI)

10

PKC (S/TXK/R) rCIC-l h,rCIC-2

717 (~RK), 756 (~HK,I, 787 (~TK), 892 (~FR); 898 (~IR); 980 (~LR) 54 (~SR), 76 (~VR), 181 (ILK), 392 (~QK), 491 (IYR), 596 (IFR), 738 (~EK); 831 (ILK); 856

18 20 11

1 3

17 22 45

10 18

(~FR)

rCIC-3 hCIC-4 hCIC-5 rCIC-5 rCIC-Kl rCIC-K2 yCIC-1

II

51 (~KK) and 362 (~l'K) 50 (ISK), 104 (ILR), 362 (ITR), 558 (ISK), 591 (IHR), 642 (~ER), 730 (~GR), 738 (IKK) 37 (INK), 291 (ILR), 397 (~SK), 628 (~QR), 676 (ILK), 724 (IKK) 37 (INK), 291 (ILR), 397 (~SK), 628 (~QR), 676 (ILK), 724 (IKK) 659 (ISR) 11 (~PR); 659 (ISR) 312 (TLK), 592 (SSK), 658 (STK), 694 (SVK)

20 11 1 3

17 22 45

._e_n_t_ry_43

_

proteins in Xenopus oocytes. The currents seen were identical to those obtained from CIC-5 mRNA alone and no evidence was obtained in favour of heterologous interactions3 .

Protein phosphorylation Consensus target sequences for PKA and PKC 43-32-01: The consensust sites for phosphorylation by protein kinase A (PKA) and protein kinase C (PKC) in the proteins of the CIC family are shown in Table 6.

Activation of PKC blocks C1C-3 channels 43-32-02: The chloride currents expressed in Xenopus oocytes injected with CIC-3 cRNAt were completely blocked by activation of protein kinase C by the phorbol estert 12-0-tetradecanoylphorbol 13-acetate. Activators of PKA were without significant effect20 .

ELECTROPHYSIOLOGY Methodological note: In a number of instances, cRNAs t derived by in vitro transcription t of cloned cDNAs t failed to elicit detectable currents after injection into Xenopus oocytes. In some of these cases it has been possible to overcome this problem by placing the specific coding sequence, including the ATC start-codon, immediately downstream t of the 5'untranslated region (83 bpJ from the Torpedo G1G-O cDNA 10,17,19,20,22. The G1G-O mRNA 5'-untranslated region is known to allow efficient translation initiation in Xenopus oocytes 9 • The alternative strategy of placing the G1G coding sequence immediately downstream t of the Xenopus f3-globin 5'untranslated region, in the expression vectort pTLN, has also been used successfully3.

Activation The C1C-O channel is activated by hyperpolarization 43-33-01: The ClC-O channel expressed from cRNAt in Xenopus oocytes is activated by 25 s steps of hyperpolarizationt to voltages more negative than -40mV from a holding potential of -lOmV. The activation is slow, taking several seconds, and inactivation after stepping back to -lOmV is even slower. Once activated, current flow increases with depolarization, leading to an apparent outward rectification t9. Channels produced by expression of total electric organ mRNA in Xenopus oocytes and by the T. californica voltage-gated chloride channel reconstituted in lipid bilayers show similar behaviour9.

C1C-l channels rapidly deactivate at hyperpolarizing potentials 43-33-02: The rat ClC-l channel heterologously expressed in Sf-9t insect cells gives CI- currents that rapidly deactivate at hyperpolarizing potentials and

II

_~

e_n_t_ry_43------1

saturate at depolarizing potentials54, similar to the macroscopic currents seen in native preparations55 •

C1C-2 channels are activated by hyperpolarization and hypotonicity 43-33-03: The expression of CtC-2 cRNAt in Xenopus oocytes gives a chloride conductance that is slowly activated (within rvlO-20 s) at voltages <-lOOmV. The voltage dependence of activation is not saturated at -180mV19. The CIC-2-mediated CI- currents are increased at physiological voltages when the oocytes are subjected to hyposmotict conditions, a reversible effect that takes about 10min to appear36 • The regions of the CIC-2 channel involved in the activation by osmolarityt have been identified36 (see Domain functions, 43-29-03 and 43-29-04). Note that hypotonicity induces a chloride current in uninjected Xenopus oocytes, but this is distinguishable from CIC-2 currents by its different voltage and time dependence and a reversed halide-selectivity sequence56 •

C1C-3 channels are open at all menlbrane voltages 43-33-04: Currents produced by the expression of rat CtC-3 cRNAt in Xenopus oocytes are not voltage dependent: the channels are open at all membrane voltages2o. Note that some laboratories have been unable to obtain functional expression of CIC-3 and CIC-4 cRNAs in Xenopus oocytes, despite obtaining measurable chloride currents with other CIC cRNAs (see ref.3, for example).

Current-voltage relation C1C-O shows very weak outward rectification 43-35-01: The injection of Torpedo marmorata electric organ mRNA or ctc-o cRNA into Xenopus oocytes generates very weakly outwardly rectifying t CI- currents over the range -100-0 m V 9,57. After heterologous expression of CIC-O cRNA in oocytes, the 'instantaneous I-V' relationship is linear in the range -160 to +100mV58 . A mutant form of CIC-O carrying the K5l9E replacement shows a strong outward rectification in the same voltage range. These mutant channels fail to inactivate completely at negative voltages, having a minimum Pop en of rvO.35 58 .

C1C-l is inwardly rectifying 43-35-02: The expression of CtC-l cRNAt in Xenopus oocytes generates CIchannels that are open under resting conditions. The I-V relationship is nearly linear between -80 and -20mV, but the conductance t decreases at more positive voltages. Hyperpolarizingt voltage steps elicit large inward currents which decay rapidly, the rate of deactivation increasing with hyperpolarization. At hyperpolarizing voltages}' steady-state currents decrease with hyperpolarization after passing through a maximum at rvlOOmV10. These observations are in close agreement with the behaviour of native macroscopic skeletal muscle CI- channels59 • A mutant form of hCIC-l, K585E, heterologously expressed in Xenopus oocytes, produces a channel

II

----I_

1~_e_n_t_ry_43

Table 7. 1-V relationships of members of the C1C family of chloride channels (From 43-35-04) Channel

I-Y relationship

Refs

CIC-O CIC-l CIC-2 CIC-3 CIC-5 CIC-Kl CIC-K2

Linear Inwardly rectifying Slight inward rectification Slight outward rectification Strong outward rectification Slight outward rectification Slight outward rectification

58

10 19,36

20 3 17 22

with a linear instantaneous 1-V relationship in 104 mM CI-, in contrast to the inwardly rectifying t behaviour of the wild-type channel60.

Hyperpolarization-activated C1C-2 channels show slight inward rectification 43-35-03: Expression of rat CIC-2 cRNA in Xenopus oocytes gave CIcurrents that were not detectable under resting conditions, but increased slowly (tens of seconds) after hyperpolarization. Instantaneous 1-V curves of activated channels were approximately linear, with slight inward rectification, over the range -180 to +50my19. The inward rectification was accentuated when the currents were measured at the end of a 12 s test pulse36 . 43-35-04: The I-V relationships for members of the CIC family are summarized in Table 7.

Inactivation C1C-l channels rapidly inactivate at hyperpolarizing potentials 43-37-01: The human CIC-l channel produced by transient t expression following transfection t of human embryonic kidney (HEK-293) cells gives voltage-activated CI- currents that deactivate within ",,50 ms when the voltage is stepped to values more negative than -100my61 . This deactivation is considerably faster than that seen for CIC-l channels produced in Xenopus oocytes 10, for reasons that are not apparent. Native chloride channels in rat psoas muscle fibre segments show exponentially decreasing current amplitudes at a fixed test potential (-75 mY) with increasing duration of a pre-pulse at +50mY. The time constants for this inactivation process were estimated to vary between 8 and 35 s, suggesting the process may be too slow to be important during a single action potential55 .

C1C-l inactivation is sensitive to external Cl- concentration 43-37-02: The rat CtC-l chloride channel expressed from recombinant viral vectors in Sf-9t insect cells gave rapidly inactivating CI- currents at hyperpolarizingt voltages. The time constants for the inactivation process,

II

Voltage-gated CI- channels

entry 43

and 72} show some voltage dependence} with the 'fase component} 71} tending to become faster at less hyperpolarizing potentials (71 = 7.1 ms at -140mV} but 3.3ms at -60mV} and the 'slow} component} 72} becoming several-fold slower over the same voltage range (72 = 22ms at -140mV and 78 ms at -60 mV). Deactivation is faster at reduced concentrations of external CI-} due to a change in 7260. The human CIC-l produced in Xenopus oocytes behaves similarly} though with a slightly flatter apparent Popen vs. V curve (V1/ 2 of -62mV} apparent gating charge of 0.89 at 103.6mM [CI-]o}60. The K585E mutant form of hCIC-l} equivalent to the K5l9E mutation of CIC-O (see 43-40-03)} shows slower gating than the wild-type channel and its inactivation at hyperpolarizing potentials can be described by a single exponential} with a time constant threefold larger than the slow time constant (72) of the wild-type channel at -140mV. With the mutant channel} closure of the fast gate at hyperpolarizing potentials is incomplete} and the apparent Popen remains at !"'V0.18 even at -160mV6o . 71

Inactivation of C1C-l is sensitive to external pH 43-37-03: Currents through the rat CIC,·I channel expressed in Sf-9t insect cells are reduced by lowering the external pH6o. Peak currents are reduced by about 20-300/0 on changing the pHo from 7.5 to 5.5} with a decrease in deactivation and an increase in steady-state inward currents. The effects appear to titrate with a pKa of !"'V6. At pHo 5.5} the sensitivity of channel gating to the external CI- concentration (see 43-37-02) is lost 60 . Increased protonation of an externally accessible site on the channel protein could increase the affinity of the binding site for CI- and thereby remove the apparent sensitivity to the external concentration of the permeant ion6o. Reduced internal pH slows inactivation kinetics of C1C-l 43-37-04: The deactivation kinetics of rCle-l} expressed in Sf-9t insect cells} are slowed by reducing the internal pH: a drop in pH from 7.2 to 6.2 increases both of the time constants for the inactivation process (see 43-37-02) by !"'V threefold. Raising internal pH to 8.0 accelerates deactivation of rCIC-l channels. The Popen vs. V curve is also shifted in the hyperpolarizing direction. Intracellular addition of benzoate (19.2mM free benzoate) reverses the effects of low internal pH on the inactivation kinetics and open probability curve60.

Kinetic model The C1C-O channel behaves as two linked protomeric channels 43-38-01: The CIC-O channel of Torpedo electroplax} studied at the singlechannel level in reconstituted lipid bilayers or after heterologoust expression of cRNAt in Xenopus oocytes} shows one closed and three open states} D} M and U. Single-channel conductances in the 'open} states are OpS (D}} lOpS (M) and 20pS (U). Transitions between the closed and open states are slow (seconds}} while transitions between the three open states are much faster (!"'V 10 ms). The channel can enter the closed state from either the M or the U state} but transitions between U and D states always

1__e_n_t_ry_43

_

go via the M state. These relationships between states can be summarized as follows: closed

/ D

+---+

I M

+---+

U

The model to explain these observations involves twin 'two-state' channels as a single conducting unit. The slow transition from the closed state to the open state always affects both twin channels simultaneously, so that the three 'open' substates always appear and disappear together. The individual 'protomeric' channels can then undergo fast gating independently62.

Models for gating of C1C-l channels 43-38-02: The voltage-dependent gating of human CIC-! channel heterologouslyt expressed in human embryonic kidney (HEK-293) cells is mediated by two qualitatively distinct kinetic processes, one on a microsecond timescale and the other taking milliseconds. It has been suggested that the first, rapid process involves two identical voltage sensors, each consisting of a single, titratablet residue, probably a carboxylic acidt side-chaint. The slower gating has been proposed as being due to the interaction of a cytoplasmic gate with the ion pore of the channel63 . This model has been challenged and an alternative proposed which suggests that the voltage sensitivity arises indirectly from the voltage dependence of channel activation by external CI- ions 6o (See 43-42-02 to 43-42-05).

Selectivity C1C-O is highly selective for Cl-

43-40-01: The native t CIC-O channel of Torpedo cali/ornica and the channel obtained by heterologous t expression of cRNA in Xenopus oocytes are highly selective for CI-. Br- ions are also carried, but 1- blocks the channel, an effect that increases with depolarization4,57. Measurements of inhibition of uptake of 36CI- into reconstituted liposomes give a selectivity series CI- > Br- > F- > S042-6.

C1C-O behaves as a channel with a multi-ion pore

43-40-02: The CIC-O channel produced by heterologous t expression in Xenopus oocytes shows typical 'anomalous mole fraction behaviour t, when mixtures of CI- and NO.3 are used to provide the permeant anion. Both gating, as measured by V 1/ 2, the voltage giving half-maximal PopertJ and conductance of the channel are minimal at a NO.3 mole fraction of 1"'..10.2, increasing steeply above or below this value. Similar effects are found with Br- /N0.3 mixtures, but not with CI- /Br-, CI- /1- or Br- /1- mixtures. Such anomalous behaviour is an indication that the CIC-O channel has a multi-ion pore58 .

An amino acid substitution affects selectivity and gating of C1C-O 43-40-03: The CIC-O channel carrying the K519E substitution close to the intracellular end of D12 shows altered anion selectivity. The wild-type

II

Voltage-gated CI- channels

entry 43

Table 8. Selectivity sequences for C1C chloride channels (From 43-40-04) Channel CIC-O

CIC-l

CIC-2 CIC-3 CIC-S CIC-Kl

CIC-K2

Selectivity

Refs

CI- > Br- > NO.3 > 1- (heterologous expression in Xenopus oocytes); CI- > Br- > F- > SO~- (inhibition of uptake of 36CI- into reconstituted liposomes) CI- > Br- > 1- (Xenopus oocytes and rat psoas muscle fibre segments, measured by the 'vaseline-gapt ' technique) (see note 1) CI- >Br- >11- > CI- = Br- > acetate> gluconate (Xenopus oocytes) NO.3 > CI- > Br- > 1-. Glutamate inhibited CI- currents but was not itself permeant (Xenopus ocytes) Br- > CI- > 1- (heterologous expression in Xenopus oocytes and in vitro perfusion studies with thin ascending limb preparations) Br- > 1- > CI- »cyclamate (both CIC-K2L and CIC-K2S in Xenopus oocytes)

4,57,6

25,55

19

64 3

17,65

22

Note: 1. A mutant CIC-l channel containing the P480L replacement found in Thomsen's disease (see Phenotypic expression, 43-14-03 to 43-14-06) shows 1- > CI- selectivity after strong hyperpolarization25 . channel shows ion selectivity in the sequence CI- >Br- > NO.3 >1-, but the KS19E mutant channel passes CI- = Br--, and the NO.3 and 1- conductances are increased. This changed selectivity of conductance is reflected by the effects of the different anions on gating, suggesting that only permeant ions directly affect the gating process58 . 43-40-04: The selectivity sequences for the various CIC chloride channels are

summarized in Table 8.

Single-channel data Single-channel conductances of G1C channels are small 43-41-01: The single-channel conductances of the CIC channels that have

been heterologouslyt expressed are characteristically small, as shown in Table 9. Those of CIC-l and CIC-2 are too small to be well resolved by single-channel recording and have therefore been determined by nonstationary noise analysis

r.

Unusual gating behaviour of G1G-O channels 43-41-02: The CIC-O channel, studied after insertion into planar lipid bilayers1

shows unusual gatingt behaviour at negative voltages, involving a bursting l process in which the channel displays two distinct conducting states ('U' and 'M' states) and a short-lived, non-conducting 'D' state (average dwelltime r-..; 10 ms), as well as a longer-lasting inactivated 'I' state (average dwelltime r-..;SOOms)62,66. The U and M states have single channel conductances

II

l'--e_n_t_ry_43

_

Table 9. Single-channel conductances of C1C chloride channels (From 43-41-01) Channel isoform

Single-channel conductance (pS)

Method of measurement

Refs

CIC-O CIC-1 CIC-2

10,20a

",1 3-5

Single-channel recording Non-stationary noise analysis t Non-stationary noise analysis t

67 61 21

The CIC-O channel shows an unusual gating behaviour, involving 'bursting' and two conducting substates with approximate conductances of 10 and 20 pS. It is suggested that these substates result from independent opening and closing of two identical 'protochannels' that are intimately associated in a channel complex62?66. a

of approximately 20 and lOpS respectively67. All three active states (U, M and D) can be reached from the inactivated state but within a burst, transitions between the U and D states must go via the M state. At highly positive voltages (+90mV), only a single open state is seen, with a conductance of ",20pS. Addition of the CI- channel blocker, 4,4'-diisothiocyano-2,2'stilbenedisulphonate (DIDS; 10 J.lM) to the cis side of the bilayer inhibits channel activity after about 5 s delay. After this time, the three-state

u

/n I,~

o Figure 3. Single-channel bursts, obtained with single chloride channels from Torpedo electroplax inserted into a planar lipid bilayer. Recordings were

made at a holding potential of -90mV The four states of the single channel labelled on the recording are I (inactivated state), U (two tprotochannels' open, conductance ",20mV), M (a single protochannel' open, conductance ",lOpS) and D (active state, but both tprotochannels' closed, conductance 0 pS). (Reproduced with permission from Miller, Proc Natl Acad Sci USA (1984) 81: 2772-5). (From 43-41-02)

II

Voltage-gated CI- channels

entry 43

behaviour of the channel is lost, to be replaced by conventional opening and closing of a channel with a single conductance state of rvl0pS. Further exposure to the inhibitor results in complete disappearance of all channel fluctuations after about 25 s. Similar single-channel behaviour is seen after expression of the ClC-O cRNA in Xenopus oocytes, with chordt conductances of 9 and 18 pS for the two conducting states 57. These observations have been interpreted to support a 'double-barrelled' model for ClC-O, with two interacting 'protochannels' that open and close independently but enter and leave the inactivated state in concert67. Reconstitution of immunoaffinitypurified ClC-O protein into planar lipid bilayers gives channels with the characteristic 'binomial' single-channel behaviour8 .

Conformations of C1C-O are coupled to the Cl- electrochemical gradient 43-41-03: Analyses of single-channel recordings of the Torpedo CIC-O channel reconstituted into a planar bilayer show a highly asymmetric distribution of the transitions into and out of the inactivated (I) state: the channel tended to enter the I state from M, the state with one open protochannel, but leave I directly into V, the state with two open protochannels. This asymmetry increases with the transmembrane electrochemical gradient t for the Clion, and the conformational changes among the I, V and M states are coupled to the movement of the Cl- through the channe168 .

A mutant C1C-O with altered single-channel conductance 43-41-04: A CIC-O channel variant with a K519E replacement at the intracellular end of the D12 region shows lTlarked outward rectificationt, with a single-channel conductance of between rvO.8 (at -140mV) and rv3pS (at +60 mV). The wild-type ClC-O channel has a single-channel conductance of rv8 pS measured under the same conditions 58 .

Voltage sensitivity Voltage-dependent gating of C1C-O is facilitated by the permeant anion 43-42-01: When the Torpedo electric organ Cl- channel CIC-O is produced by heterologoust expression of cRNA in Xenopus oocytes, the steady-state open probability, PopellJ increases with transmembrane voltage over the range -180 to +120m~ in accordance with a Boltzmann t distribution Popen == II (1 + exp(zneO(V1/2 - V)lkT)), (where eo is the elementary charge, V 1/2 is the voltage of half-maximal activation, k is the Boltzmann constant and T is the temperature) with a nominal gating charget rv 158 . At 100 mM [Cl- ]0, the V1/'f is rv -110mV. (The channels obtained by reconstitution of immunoaffinityT purified CIC-O protein into planar lipid bilayerst show essentially similar behaviour8 ). The Popen/V curve is shifted to more positive voltages by reduction in the extracellular CI- concentration, without significant change in slope. Changes in intracellular CI- concentration have little effect58 . Only permeant ions affect voltage-dependent gating, and the ion selectivity of conduction is reflected in the influence of the ions on the gating process 58 . These observations suggest that the conformationt of the channel is influenced by

II

l_e_n_t_ry_43

_

binding of a permeant anion, and that the voltage-dependent gating of CIC-O is conferred by the permeating ion itself58 .

Voltage dependence of gating of C1C-l channels 43-42-02: The human CIC-l channel produced by transient t expression in human embryonic kidney (HEK-293) cells shows a Popen that relates to voltage by a Boltzmannt function with an effective gating valencet of 0.86 and half-maximal activation at -100 my 61 . Essentially similar behaviour is found on heterologous expression t of CIC-1 cRNAt in Xenopus oocytes1 though here the half-maximal open probability is -20 my27, and in Sf-91 insect cells, where the V 1/ 2 is -90my60. The voltage dependence of steady-state activation of native CIC-1 has been determined in studies with rat psoas muscle fibre segments. With 5 s pre-pulses at various potentials between -145 and +45 mY, followed by a test pulse at -5 mY, the instantaneous current amplitude vs. the pre-pulse potential gives an activation curve that can be fitted with a single Boltzmannt equation, with an inflexion point at -39mY and a slope of 1/17.2my55 . r-..J

r-..J

Voltage dependence of C1C-l gating is sensitive to external Clconcentration 43-42-03: The apparent Popen of rat CIC-l channels expressed in Sf-9t insect cells is voltage dependent, increasing with membrane potential according to a Boltzmann t distribution with a V 1/ 2 of -88.2mY and a slope factor of -21.3 at 174mM [CI-]o. The Popen vs. voltage curves are shifted to more depolarizing potentials as external CI- concentrations are reduced (cf. similar behaviour of CIC-O channels, 43-42-01). The curves are not shifted by changes in the internal CI- concentration, showing that Popen is not linked to the transmembrane CI- gradient 6o . (Note the similarity to the behaviour of inwardly rectifying potassium channels, where currents are dependent on the external but independent of the internal K+ concentration: see entry 29.) It is suggested that permeation through the CIC-1 channel requires binding of a permeant ion to a site within the conducting pore that is accessible only from outside60 . Some organic anions, such as methanesulphonate, can act as CIC-1 channel openers without being permeant ions 6o .

Voltage dependence of C1C-l is affected by internal pH 43-42-04: Reduction of the internal pH during whole-cell recording of currents induced by expression of rCIC-l in insect cells slows the inactivation kinetics (see 43-37-04) and shifts the Popen vs. V curve towards hyperpolarizing voltages (V 1/ 2 is shifted 50mY more negative by a change in internal pH from 7.2 to 5.6)60.

External pH affects channel kinetics in native C1C-l channels 43-42-05: Hyperpolarizationt of frog skeletal muscle fibres at low external pH (pHS.O) induces a slow increase in CI- conductance. The conductance/ voltage relationship can be fitted to a Boltzmannt distribution, with a slope factor of 0.05 mY and a V 1/ 2 of 4S mY more negative than the resting potentialt 69 . Similar behaviour can be seen with heterologouslyt expressed CIC-l channels in Sf-9t insect cells 60 (see 43-37-03).

II

Voltage-gated CI- channels

entry 43

PHARMACOLOGY

Blockers C1C-O is blocked by DIDS 43-43-01: The CIC-O channel from Torpedo electroplax is sensitive to the Clchannel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), which irreversibly blocks when applied to the cis t side of the lipid bilayer, but not from the trans t side. With CIC-O channels in planar phospholipid bilayers, at a holding potential of -50 mV, DIDS (20 JlM) inhibition of macroscopic Cl- currents is rapid and complete (tl/2 == ",,4 s). The inhibition by DillS can also be studied at the single-channel level, where 10 JlM DillS eliminates the highest conductance (20pS) substate after a few seconds delay, before inhibiting currents completely after about 25 S67.

SCN- produces voltage-dependent block of C1C-O 43-43-02: The 'pseudohalide' SCN- blocks the Torpedo CIC-O channel in reconstituted planar lipid bilayers in a reversible and voltage-dependent manner with a Ki at -30 mV of 3.3 mM (200 mM KC1)4,70. In reconstituted liposomes, internal SCN- blocked Cl- uptake half-maximally at 2-3 mM 6. In similar experiments, Cl- uptake was also blocked (degree not specified) by 'millimolar' amounts of internal NC)3 6 .

9-Anthracene-carboxylate blocks C:1C-l

43-43-03: The injection of cRNA t transcribed from rat CIC-l cDNA into Xenopus oocytes gives rise to an inwardly rectifying Cl- current that is sensitive to 9-anthracene-carboxylic acid (9-AC). The CIC-1 conductance was >800/0 inhibited by 0.1 mM 9-AC. The inhibition by 9-AC takes several minutes to reach its maximum and is nlore rapid at higher concentrations10. Very similar behaviour is seen from the native channels in segments of rat psoas muscle fibres, where Cl- currents are completely blocked by 0.1 mM 9_AC 55 . In preparations from excised rat diaphragm, 9-AC (20 JlM) reduced the chloride conductance by ""600/0 71 . The application of 9-AC at concentrations sufficient to block >60-700/0 of the skeletal muscle Clconductance results in myotonic t symptoms72. Injections of 9-AC (8mgjkg) into the external jugular vein of goats resulted in a myotonia 'resembling that of a severely affected congenitally myotonic goat'34 (see Phenotypic expression, 43-14-09).

Summary of C1C responses to COmlTIOn blockers 43-43-04: The responses of the CIC family members to common Cl- channel blockers are shown in Table 10.

Internal OH- ions and benzoate block C1C-l 43-43-05: The inactivation kinetics and the Popen vs. V curve for rCIC-l expressed in insect cells are sensitive to internal pH. Increases in the internal pH from 5.6 to 7.2 shift the Popen curve towards depolarizing potentials by 50mV. ~enzoate (free concentration, 19.2mM) added internally was able to mimic the effects of increasing the internal pH to

1L..-_e_n_ _ry_4_3 t

_

Table 10. Responses of C1C channels to common chloride channel blockers (From 43-43-04) Channel DillS CIC-O CIC-1 CIC-2 CIC-3 CIC-K1 CIC-K2

9-AC

DPC

67

~10JiM

71

~20JiM

1 mM (900/0 inhibition) 20 JiM (50/0 inhibition)

Refs

~lmM

~lmM

Insensitive 0.7mM

Insensitive

100 JiM

19

64 17

1 mM (150/0 inhibition)

22

8.0. It has been suggested that intracellular OH- ions are able to block the CIC-1 channel, either allostericallyt or directly, and that benzoate ions can mimic this intracellular block60 .

Inorganic anions can block C1C-l 43-43-06: Inorganic anions such as SCN- 1 CIOi and ReOi can block the CIC-1 chloride channels of amphibian and mammalian skeletal muscle 73 . The most effective such blocker is Re04" which, at 5 mM, blocks 80% of the chloride conductance of mammalian skeletal muscle 73 .

Some inorganic cations block C1C-l 43-43-07: Several divalent cations, including UOi+, Cu2+, Zn2+ and Cd2+, block CI- conductances in amphibian and mammalian skeletal muscle (reviewed in ref. 73). Zn2+ (0.5 mM) reduced chloride conductance in the isolated rat diaphragm by 770/0 and induced myotoniat. Cd2+ is more t potent than Zn2+ as an inducer of myotonia, but other transition metal ions such as Ni2 +, C0 2 + and Mn2 + are ineffective 73.

Sulphonamide derivatives block kidney Cl- channels 43-43-08: The sulphonamide-type diuretic torasemide inhibits CI- channel activity in isolated preparations of the thick ascending limb of the loop of Henle with an ICso of 30 JiM 74?96. (This agent is not specific to kidney chloride channels, since it inhibits the cation/CI- co-transporter in the same preparations with an ICso of 0.3 JiM 75 .)

Arylaminoalkyl benzoates block kidney chloride channels 43-43-09: Arylaminoalkyl benzoates, analogues of diphenylamine-2carboxylate, are effective inhibitors of chloride transport and act as blockers of the CI- channels of mammalian kidney. Diphenylamine-2-carboxylate (DPC) inhibits the CI- channels in preparations of the thick ascending limb (TAL) of the loop of Henle with an ICso of 30 JiM. Screening of structural analogues for their effect on CI- channels in in vitro perfused TAL preparations identified 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) as the most potent blocker (ICso == 8 nM)76. The effects of modifications of the structure of NPPB are discussed in ref. 76. NPPB is not specific for kidney

III

Voltage-gated CI- channels

entry 43

chloride channels, since at micromolar concentrations it inhibits the CFTR CI- currents in respiratory epithelia37 and pancreatic duct cells 77, and blocks cation/CI- exchangerst and non-selective cation channels (reviewed in ref. 78).

Channel modulation Activation of PKC blocks Cl- conductance in skeletal muscle 43-44-01: The protein kinase C (PKC) activator 4-{1-phorbol-12,13-dibutyrate (0.1-2.0 IlM), blocks up to 760/0 of the chloride conductance (Gcd in fibres from goat intercostal muscles. In so doing, the phorbol estert induces myotonict hyperexcitability similar to that of fibres from congenitally myotonic goats. The block of GCI was alleviated by pre-treatment with the PKC inhibitor staurosporine (10 IlM). 'Inactive' 4-a-phorbol-12, 13didecanoate (50 IlM) had no effect, whereas the 'active' 4-{1 isomer at 11lM blocked 41 % of GCI ' The very low GCI of congenitally myotonic goat fibres was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Compounds which may increase cAMP levels, forskolin and cholera toxin, and the R(+) clofibric acid enantiomers and taurine, which increase GCI in normal fibres, were ineffective in restoring GCI in myotonic goat fibres 35 . Similar effects of phorbol esters can be seen using rat extensor digitorum longus muscle fibres: 500/0 block of the membrane GCI required 230M 4-{1-phorbol-12,13dibutyrate, and the effect was prevented by pre-incubation of the preparations with the PKC inhibitors staurosporine (1-5 IlM) and tetrahydropapaverolone (50-100 flM)79.

C1C-3 channels are closed by PKC activation 43-44-02: Heterologouslyt expressed rat CIC-3 channels in Xenopus oocytes are completely inactivated by treatment of the cells with the phorbol ester t 12-0-tetradecanoylphorbol 13-acetate (TPA, IIlM), a potent activator of PKCt. Phorbol esters that are known not to activate PKC are without effect on these CI- currents. The inhibitory effect of TPA was abolished by co-treatment with 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7; 11lM) or staurosporine (5IlM), inhibitors of protein kinase activity64. It is suggested that the blockade of CI- currents by PKC could induce after-depolarization t of neuronal cells and thereby lead to excitation of the synaptic membrane64 .

Openers Some impermeant anions can act as channel openers 43-48-01: For both CIC-O and CIC-l, permeant anions act as channel openers, and the Popen vs. voltage curves are shifted to more depolarizing potentials as external CI- concentrations are reduced (see 43-42-01 and 43-42-03). This opening effect is largely restricted to permeant ions, but some organic anions, including methanesulphonate, can act as channel openers without being significantly permeant themselves 60 .

II••-

entry 43

I-

_

- - - - -

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 43-53-01: The relevant database is indicated by the lower case prefix (e.g.

gb:),which should not be typed (see Introduction and layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in the Introduction and layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouringt analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, author/reference or nomenclature. Following direct accession, however, neighbouring t analysis is strongly recommended to identify newly reported and related sequences. Nomenclature

Species, DNA source

Original isolate

Accession

Sequence/ discussion

CIC-O

Electric ray (Torpedo marmorata) electric organ cDNA Electric ray (T californica) cDNA

805 aa

gb: X56758

810 aa

gb: X60433

Jentsch, Nature (1990) 348: 510-14. O'Neill, Biochim Biophys Acta (1991) 1129: 131-34.

991 aa

em: X62894

CIC-l

Rat skeletal muscle cDNA library

988 aa

Human cDNA plus genomic libraries

5'-untrans- lated region exon 1 exon 2 Human genomic exons 8-22 DNA library exon 23

CIC-2

Rat brain cDNA 907 aa library 898 aa Human colonocyte cell line cDNA library

Steinmeyer, Nature (1991) em: Z25884 354: 301-4. gb: M97820 Koch, Science (1992) 257: 797-800. gb: Z31372 Steinmeyer, EMBO T(1994) gb: Z25768 13: 737-43. gb: Z25872 Lorenz, Hum gb: Z25753- Mol Genet (1994) 3: 941-6. Z25767 gb: Z25752 em: X64139

Thiemann, Nature (1990) 356; 5760. Cid, Hum Mol Genet (1995) 4: 407-13.

II

Voltage-gated

CI~

entry 43

channels

Nomenclature

Species, DNA source

Original isolate

Accession

Sequence/ discussion

CIC-3

Rat kidney eDNA library

760 aa

gb: D17521

Kawasaki, Neuron (1994) 12: 597-604.

CIC-4

Human retinal 760 aa cDNA library Rat brain cDNA

em: X77197

van Slegtenhorst, Hum Mol Genet (1994) 3: 547-52. Jentsch, T Physiol 482P: 19S-25S.

CIC-5

Human renal cDNA library

em: X81836 em: (see Fisher) gb: 256277

Fisher, Genomics (1995) 29: 598606. Steinmeyer, T BioI Chem (1995) 270: 31172-77.

746 aa

Rat brain cDNA 746 aa

CIC-Kl

Rat kidney eDNA

686 aaa

gb: D 13927

Uchida, TBioI Chem (1993) 268: 3821-4. a Kieferle, Proc Natl Acad Sci USA (1994) 91: 6943-7.

CIC-K2

Rat kidney cDNA

687 aa (CIC-K2L) 632 aa (CIC-K2S)

gb: D26111

Adachi, J Bioi Chem (1994) 269: 17677-83. Kieferle, Proc Natl Acad Sci USA (1994) 91: 6943-7.

gb: 230663

CIC-Ka

Human kidney cDNA

687 aa

gb: 230643

Kieferle, Proc N atl Acad Sci

USA (1994) 91: 6943-7.

II

gb: 230644

Kieferle, Proc Natl Acad Sci USA (1994) 91: 6943-7.

Escherichia coli 436 aa (ORF 102) genomic DNA

gb: D26562

Fujita, Nucleic Acids Res (1994) 22: 1637-9.

Yeast 779 aa (Saccharomyces cerevisiae) genomic DNA

em: L29347

Greene, Mol Gen Genet (1993) 241: 542-53. Huang, TMol BioI (1994) 242: 595-8.

CIC-Kb

Human kidney eDNA

ecCIC

yCIC-l

687 aa

--I_

1'--_e_n_t_ry_43

Nomenclature

Species, DNA source

Original isolate

Accession

ESTs

Picked out by homology with CICN4

",1000/0 homology gb: M79019 ",100% homology gb: Z19419 ",730/0 homology gb: Z21258

Sequence/ discussion van Slegtenhorst, Hum Mol

Genet (1994) 3: 547-52. a It

is suggested that this cDNA sequence lacks three cytosines at positions 1311, 1344 and 1403 (initiator A= I), leading to three frameshifts in the DI0-coding region and loss of one net amino acid from the translated protein2 .

Miscellaneous information Chloride currents induced by phospholemman 43-55-01: Phospholemman, a small (72 amino acids, 8 kDa) ubiquitous transmembrane protein, induces hyperpolarization-activated, CI- selective currents when produced by heterologous t expression of cRNA in Xenopus oocytes. Although superficially similar to CIC-2-dependent currents, the phospholemman-induced currents are distinguishable by their more rapid activation, their biphasic deactivation at positive voltages80 and their 1- > Br- > CI- selectivity sequence81 . The phospholemman-induced currents are also distinguishable from the endogenous r, hyperpolarization-activated CI- currents in some batches of Xenopus oocytes, but these two currents are sufficiently similar as to suggest the possibility that phospholemman activates the endogenous CI- channels and in so doing slightly alters their voltage dependence and pharmacological profile80 .

Chloride currents induced by heterologous expression of 1sK

43-55-02: The protein known as IsK (or minK), a 14.5 kDa glycoprotein t with a single transmembrane segment, induces slowly activating, voltagedependent K+ currents when produced by heterologous t expression of cRNA t in Xenopus oocytes 82. At increased concentrations, IsK cRNA also induces a hyperpolarization-activated, inwardly rectifying CI- current in Xenopus oocytes, 'very similar' to that induced by phospholemman83 (see 43-55-01). The suggestion that IsK can activate endogenous K+ and CIchannels in Xenopus oocytes 83 is supported by the finding that the K+ currents are elicited following the injection of a C-terminal 27-mer peptide from IsK, and the CI- currents are induced by the extracellular application of an N-terminal 13-residue peptide (amino acids 31-43; 50 JlM)84. The K+ channel subunit with which IsK interacts has been identified as the Xenopus homologue of K v LQT1: the Kv LQTl and IsK proteins associate to constitute the channel responsible for I Ks , the slowly activating, delayed rectifier K+ current involved in the repolarization of cardiac action potentials85?86. The identity of the endogenous Xenopus CI- channel activated by IsK remains unknown (see VLC K minK, entry 54).

A protein that regulates activation of swelling-induced Cl- currents 43-55-03: A sequence encoding a 235 amino acid protein, plein, cloned as cDNA from the Maclin Darby canine kidney (MDCK) epithelial cell line, gave rise to an

II

Voltage-gated CI- channels

entry 43

outwardly rectifying CI- selective current when expressed in Xenopus oocytes. The ICIn current was sensitive to the Cl- channel blockers 4,4'-diisothiocyanatostilbene-2-2'-disulphonic acid (DillSj ICso = 20 IlM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB j ICso = 1.5 IlM), and to externally added nucleotides (cAMP and cGM~ ICso ,-v 100 JlM)87. Currents with the same characteristics are induced by hypotonic media in uninjected Xenopus oocytes87,88. Western blots t showed that PleIn is an abundant, cytosolic protein in a wide variety of cells, where it associates with actin and other cytosolic proteins. Monoclonal t anti-plCIn antibody injected into Xenopus oocytes completely suppressed the activation of swelling-induced CI- currents within 24 hours. It is proposed that plCIn is a regulator of swelling-induced CIcurrents, linking actin-bound cytoskeletal proteins to a volume-sensitive CIchannel88. The characteristics of the swelling-activated CI- current, ICIswe1l56, distinguish it from the candidates CIC-236 and the P-glycoprotein89. '

A voltage-dependent Cl- current li.n.ked to the cell cycle in ascidian embryos 43-55-04: A voltage-dependent, inwardly rectifying CI- channel in early

embryos of the ascidian r Boltenia villosa, is transiently activated during each cell division. The CI- current begins to increase dramatically about 100 min after fertilization, increasing by a factor of 10-30 as first cleavage approaches, then rapidly decreasing in both daughter cells after cleavage. Oscillations in the current density can be triggered in the unfertilized egg by the Ca2 + ionophore t A23187, continuing with a periodicity of about 60 min for up to 6 hours, in-phase with oscillations in capacitance which monitor surface area 90. The molecular basis of this CI- eurrent and the mechanisms that link the activity of the channel to the cell cycle have not been determined.

An (intermediate' Cl- channel in cultured skeletal muscle 43-55-05: Whole-cell CI- currents obtained from cultured myoballs t derived from skeletal muscle were strongly increased by the presence of 0.1 mM GTP,S. This effect was insensitive to protein kinase inhibitors PKI (25 JlM) and H-8 (10 IlM), to an antagonist of Ca2+ /calmodulin (trifluoperazine, 50 JlM) and to quinacrine (10 JlM), an inhibitor of phospholipase A2 . Pre-incubation of the preparations with pertussis toxin or cholera toxin did not prevent the activating effect of GTP,S. The effects of GTP,S on channel gating could be seen in singlechannel recordings with inside-out patches as a change from short-openings to long-lasting bursts: Popen increased more than 10-fold at holding potentials more negative than -SOmV. These observations have been interpreted to suggest a direct gatingt of skeletal muscle chloride channels by activated G protein t subunits91 . The molecular identity of this 'intermediate CI- channel' is uncertain. It is unlikely to be CIC-l, since its conductance is too high (31 pS at the reversal potential) and the voltage dependence is different92.

Related sources and reviews 43-56-01: Properties of voltage-gated. chloride channels21,93, 94 j

muscle chloride channels 73j pharmacology of anion channels and transporters in mammalian cells 78 j ion channel defects in myotonia 95 .

II

43 _ I' entry -----------

Feedback Error-corrections, enhancement and extensions 43-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource T, Search Criteria etJ CSN Development). For this entry, send email messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 44-14-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback etJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

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Voltage-gated CI- channels

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entry 43

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I

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81 82 83 84 85 86

87 88 89 90

91

92 93

94 95 96

----I_ Kowdley, TGen Physiol (1994) 103: 217-30. Takumi, Science (1988) 242: 1042-5. Attali, Nature (1993) 365: 850-2. Ben-Efraim, TBiol Chern (1996) 271: 8768-71. Barhanin, Nature (1996) 384: 78-80. Sanguinetti, Nature (1996) 384: 80-3. Paulmichl, Nature (1992) 356: 238-41. Krapivinsky, Cell (1994) 76: 439-48. Valverde, Nature (1992) 355: 830-3. Block, Science (1990) 247: 1090-2. Fahlke, Pfluger's Arch (1992) 421: 566-71. Fahlke, Pfluger's Arch (1992) 421: 108-16. Jentsch, Curr Opin Neurobiol (1993) 3: 316-21. Pusch, Physiol Rev (1994) 74: 813-27. Cannon, Trends Neurosci (1996) 19: 3-10. Wittner, Pfluger's Arch (1987) 408: 54-62.

II

Rapidly inactivating, transient outward I A-type' K+ currents in native cell types of vertebrates Edward C. Conley

Entry 44

Notes on coverage: Independent of the molecular cloning of genes encoding Kvalpha, Kv-beta, eag/elk/erg and other potassium channel subunits (entries 46 to 52 inclusive), voltage-gated K+ currents in native cells have been 'classified' in terms of their responses to step depolarizations t. Thus native K+ currents were broadly divided into a 'transient, inactivating' class (this entry) and a 'delayed rectifying, non-inactivating' class (VLC K DR [native), entry 45). Several schemes to further 'subtype' currents using biophysical criteria have appeared (e.g. using rates, voltage dependence and kinetics of activation and inactivation). For the most part, these schemes for current classification have not proven rigorous, partly because of the extraordinary wide range of behaviours seen in native cells. Furthermore, with some notable exceptions, direct 'matching' of native cell current characteristics to 'cloned' channel properties has proven difficult (see discussions in Cene family, this entry, 44-05 and Channel designation under VLC K Kvl-Shak, 48-03). As a complement to the 'cloned' channel entries, however, this entry provides some cross-references for 'A-type' (rapidly inactivating, transient outward) potassium currents that have been described in native vertebrate cell types. The difficulty of specifying channel subunit composition in native cells has meant extensive descriptions of individual current properties have been excluded (these, however, have been extensively reviewed - see references in Related sources and reviews, 44-56). A similar organization has been used to accommodate properties of native delayed rectifier (the companion entry 45) and native inward rectifier K+ currents (Volume III, entries 29 to 32 inclusive). The coverage in this entry is limited to include references describing specific physiological functions, cell-type distribution patterns, receptor/transducer interactions and channel modulation. It is clear that the use of the terms 'A-type.:' (this entry) and 'delayed rectifier' (entry 45) will assume less significance in channel classification (see Subtype classifications, 44-06). Overlapping distributions of specific channel-forming and accessory subunits 'mapped' to identified cell types within defined developmental lineages may form a more stable framework for indexing native electrophysiological and pharmacological behaviours (e.g. see senselab.med.yale.edu/neurondb).

NOMENCLATURES

Abstract/general description 44-01-01: In the early 1970s, Connor and Stevens1,2 introduced the term lAcurrent' to distinguish a Itransient' K+ current from others present in the same cells. In these original descriptions, the' A-channels (underlying Acurrent) characteristically activated at 'very negative' membrane potentials (i.e. below the threshold for spike generation). Since the original descriptions of A-current, many other transient K+ currents with a wide range of rates and voltage dependence of activation and inactivation have been recorded (see Current designation, 44-04). As used today, the terms 'A-current', 'Achannels', 'A-type channels' or 'transient K+ channels' also describe K+

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currents and channels that inactivate during depolarizations but operate well outside the 'subthreshold range' of membrane potentials. 44-01-02: 'A-type' currents are ubiquitous in electrically excitable tissues (see Cell-type expression index, 44-14). Various schemes 'classifying' kinetic

properties of A-type currents recorded in native cells have appeared. These schemes have been confounded, and have had limited application due to the wide variability of properties observed in native cells. For example (and as indicated above) transient K+ currents have been described that start activating at membrane potentials across a wide range (between -80 and -10mV); midpoints of steady-state inactivation are also variable over a wide range (typically from -75 to about -15mV). Furthermore, inactivation rates of native A-currents may vary by at least 1DO-fold. Some of the factors accounting for this 'variability' in biophysical properties have now been established for the underlying channels (e.g. different 'intrinsic' inactivation mechanisms, redox or phosphomodulation status and/or accessory subunit composition (e.g. Kv;3) (see Cene family, 44-05 and VLC K Kv-beta, entry 47). 44-01-03: In general terms, however, A-type potassium channels (KA channels) are activated by depolarization but show rapid inactivation to zero conductance under continued depolarization (typically inactivating in rv< 100 ms). Expression of diverse A-type channels assists different types of neurones in the specific temporal integration of synaptic inputs and firing with distinct spike frequencies: KA channels can influence firing thresholds of neurones which have negative resting potentials. They are often associated with encoding membranes of sensory terminals or nerve cell bodies which are known to fire action potentials at a rate proportional to the intensity of stimulus. This general property of 'retarding' impulse frequencies (to a range of rvl-lOOHz) appears to be important for preventing 'overloading' or 'depletion' of responses in gland, muscle or axons. Furthermore, phosphoregulation of A-type channels linked to neurotransmitter receptor/second messenger systems (see Receptor/transducer interactions, 44-49 and Protein phosphorylation under the VLC K Kv series, entries 47 to 52) have great significance in neural mechanisms related to behaviour and learning. A-type channel activities have also been described influencing action potential shapes and rates of neurotransmitter release (for further examples, see Phenotypic expression, 44-14). 44-01-04: A number of observations have been reported detailing changes in A-current expression density in specified membranes during the course of organismal development, both in the presence/absence of various developmental cues and/or during the progression of disease (see Developmental regulation, 44-11). Availability of molecular probes for analysing co-localization and subcellular distribution of different Kva and Kv;3 subunits known to contribute to A-type currents in native cells is improving understanding of their specific biological roles at different stages in development. For example, there is direct evidence for subcellular 'targeting' of protein subunits conducting A-type currents in neurones (for details, see Kvl.4 under Subcellular locations in VLC K Kv1-Shak, 48-16 and Kv4.2 under Subcellular locations in VLC K Kv4-Shal, 51-16).

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44-01-05: Native lA-channel' pharmacology is poorly developed. Most studies have used compounds with relatively low pharmacological selectivity and potency (or those that have indirect mechanisms of action). Prior to the cloning of multiple cDNAs encoding Kv channel gene family members (entries 48 to 52), differences in sensitivity to the 'classical' blocker 4-aminopyridine (4-AP, over the 'high micromolar to millimolar' range) and variable inactivation properties suggested a subtype heterogeneity to KA channels in native cells. Properties of 'classical' IA blockers are in Table 4 under Blockers, 44-43. A large number of studies have described various 'modulatory' effects of external and internal inorganic ions (Ca2+, Mg2+, Na+, La3 +, Li+, Zn+) as modulators of native IA (see Channel modulation, 44-44). 44-01-06: Examples of receptor-coupled modulation of native A-currents/ channels by neurotransmitters are described under Receptor/transducer interactions, 44-49. Protein kinase/phosphatase-dependent phosphorylation/ dephosphorylation of KA channels coupled to receptor-second messenger systems constitute a fundamental mechanism for modulation of cellular excitation (ibid., see also Phenotypic expression, 44-14).

Category (sortcode) 44-02-01: VLG K A-T [native], Le. voltage-gated K+ currents in native t cells generally referred to as 'A-type' (for background, see Current designation, 44-04). Note: UEls (unique embedded identifiers) for this entry will be derived from specified gene family designations only in updates (see this field under the VIC K Kv series, entries 47 to 51). For advantages of UBIs and guidelines on their implementation, see Resource T- Search criteria.

Channel designation 44-03-01: Channel designations in the literature have included KA (Le. A-type K channels); 'T K+ channels' (T for 'transient', as opposed to 'DR K+ channels', see VIC K DR [native], entry 45 and 'IR K+ channels', see the INR K-series, entries 29 to 33). Most 'A-type' current designations in use are non-systematic and therefore do not make reference to specified gene family nomenclatures (see paragraph 44-04-02). Exceptions include those K+ channels that display 'intrinsic' fast inactivation, and'A-type' current is used in connection with some Kv subunit channels (e.g. Kvl.4 underlying axon/terminal A-type currents3 ,4 (which is often described as 'resembling' cardiac ITO, but see Cene family, 44-05), Kv4.2 underlying soma/dendritic A-type currents and for other subunits in the Kv4 subfamilyS-7 (see VIC K Kv4-Shal, entry 51).

Current designation 44-04-01: I A ; IK(A); I K .A ; ITO; Ito (Le. the 'to' subscript representing !ransient Qutward). Designations of ITO,fast and ITO,slow have been made in rat atrial myocytes; further source references and comparative biophysical data on

II

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ITO appears in the review by Boyett et al. (1996)8. In some studies (cited in ref. 9 ), A-type, voltage-sensitive transient outward, Ca2 + -independent currents have been designated 'Ito l' in order to distinguish them from Ca2 +dependent transient outward currents designated 'It02 '- (see also paragraph 44-07-02 and ILC K Ca, entry 27). Serodio et al. (1994)7 (see paragraph 4404-02) define the term 'subthreshold A-current' (ISA ) to distinguish Achannels that operate close to the subthreshold range of membrane potentials from other transient K+ channels (ibid.). Note: I SA values are of particular interest because of their role in governing the subthreshold behaviour of neurones (see Phenotypic expression, 44-14 and compare VLC K M-i, entry 53).

Perspective on the use of the term tA-current' (after ref. 7) 44-04-02: K+ currents mediated by channels that activate transiently in the

Isubthreshold' range of membrane potentials and involved in the control of repetitive firing were first described in molluscan neurones 1,2,10,,11. Connor and Stevens 1,,2 introduced the term'A-current' to distinguish the 'transient' K+ current from others present in the same cells. In these original descriptions, the A-channels (underlying A-current) characteristically activated at 'very negative' membrane potentials (i.e. below the threshold for spike generation, and additionally inactivate during depolarizing pulses of durations in which the other K+ currents present in the same cells do not inactivate). Since the original descriptions of A-current, other transient K+ currents with a wide range of rates and voltage-dependence of activation and inactivation have been recorded (see Related sources and reviews, 44-56). Thus, as used today, the terms 'A-currents' or 'A-channels', 'A-type channels' or 'transient K+ channels' include K+ currents and channels that inactivate during depolarizations but operate well outside the subthreshold range of membrane potentials (and thus may not govern firing properties in a fashion similar to the origina1' A-currents). Numerous attempts have been made at classifying transient K+ currents utilizing rates, or voltage dependence of activation or inactivation (or their role in excitation), but these have been of limited application. t

Gene family Defining tcontributions' of gene products to native A-currents is generally difficult 44-05-01: 'A-type' currents can be 'reproduced' in heterologous expression systems in several ways, including (i) expression of K+ channel subunits

with lintrinsic' rapid-inactivation properties (e.g. due to an N-type inactivationt mechanism - for subunit-specific listings, see Current type (field 34) and Inactivation (field 37) under entries 48, 50 and 51). (ii) coexpression of certain KVQ subunits with KvJ3 subunits conferring fast-inactivation properties (for full discussion, see VLC K Kv-beta, entry 47). Significantly, inactivation kinetics may critically depend on various protein modulatory factors (including redoxt modulation - for detailed examples see Channel modulation under VLC K Kv-beta, 47-44) and phosphomodulation (for example, loss of N-type inactivation from Kv3.4 following

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treatment of oocytes with activators of protein kinase C - see Protein phosphorylation under VLC K Kv3-Shaw, 50-32}.

Bases for tinterconversion' of native current types 44-05-02: Single cells are able to express multiple independent K+ channel

subtypes, but the contribution of Q subunit heteromultimers to native K+ channel diversity is generally limited to co-expression of members within a given K+ channel subfamily12,13 (see for example, Protein interactions under VLC K Kv1-Shak, 48-31). In keeping with this, many studies on native cell preparations specifically report 'co-expression' of 'A-type' with 'delayed rectifiers'. Strictly, however, established protein modulatory or subunit association properties (paragraph 44-05-01) constitute a mechanism for 'interconversion' of these channel types (sic). These mechanisms/ modulatory factors may therefore confound the 'matching' of native channel currents with those recorded in heterologous cells (see also Notes on coverage at the head of this entry and the related discussion in Channel designation under VLC K Kv1-Shak, 48-03).

Subtype classifications Various schemes classifying kinetic properties of A-type currents recorded in native cells have appeared (for references, see Related sources and reviews, 44-56). Because of difficulties in defining modulation status and subunit composition in native cells (see Cene family, 44-05) this entry only makes reference to the: most general properties of these currents.

44-06-01:

Trivial names tA-current'is a general term (see also Current designation, 44-03) 44-07-01: The A-current; neuronal fast-transient voltage-dependent K+

current; voltage-sensitive, rapidly inactivating (A-type) current; transient outward current (Ito). The term 'A-current' was originally used1 for native currents in molluscan and mammalian neurones which activated and inactivated at 'very negative' potentials. These channels activated close to, or sometimes more negative than, the IIlembrane resting potential (i.e. the 'subthreshold' range - see also VLC K l\t1-i [native], entry 53). The general 'A-current' term now tends to apply for all 'fast-inactivating' voltage-gated K+ currents (see refs. 14,15). Other terms in use include the '4-aminopyridine-sensitive, Ca2+ -insensitive A-type current' (see Blockers, 44-43) or the '4-aminopyridine-sensitive transient outward current (Ito )'.

Other ttransient outward' current names not referring to IA 44-07-02: The term 'spontaneous transient outward current' (STOC) is

frequently used to describe IKca components opening in response to periodic intracellular [Ca2+] oscillations (for details, see Protein interactions under ILC K Ca, 27-31). 'Transient outward' K+ currents are associated with activation of KNa channels (for illustration see Fig. 1 under ILC K Na, entry 28).

l"-e_n_t_ry_4_4

_

EXPRESSION For a background to the subunit diversity and expression patterns supporting 'A-type' currents, see the VLC K Kv series (entries 47 to 51).

Cell-type expression index tA-type' currents are ubiquitous in electrically excitable tissues 44-08-01: 'Fast-transient' or 'A-type' currents are of wide distribution in excitable cells (for established functions, see Phenotypic expression, 44-14; for full distributions, see refs 14,16). Table 1 lists selected references to tissue slice, enzymatically dispersed and cell culture preparations in which an Acurrent has been characterized as a substantial part of the work. Although not exhaustive, the source references may provide a link to extensive descriptions of current properties in a specific cell/preparation of interest.

Channel density Apparent IA texpression gradients' in cochlea 44-09-01: I A expression density in chick cochlear hair cells has been described as 'position dependent'24 as the magnitude of IA appears to depend upon cross-cochlear position. In this study, cells were isolated from 200-Jlm-Iong segments of the apical half of the cochlea. Cells that were isolated from the most apical tip of the cochlea expressed no lA, however in areas 'more basal than 200 Jlm' from the apex, the magnitude of I A correlates with cell morphology, as follows. (i) In each area, the tallest hair cells (i.e. cells with the smallest ratio of apical surface diameter to length) and occupying 'the first 400/0 of the distance from the neural side of the basilar papilla', express no lA. (ii) In cells displaying lA, the shorter cells (i.e. with larger ratio of apical surface diameter to length) express relatively more I A (i.e. cells nearer to the abneural edge express more IA)24. Note: In separate studies 63 it has been noted that (with respect to kinetic and voltage-dependent properties), the rapidly-activating and slowly-activating potassium currents of apical versus basal regions in the chick embryonic cochlea are similar (respectively) to the rapidly-inactivating A-current of mature short hair cells and to the delayed rectifier of mature tall hair cells.

Lower ITO density following streptozotocin-induced diabetes mellitus 44-09-02: ITO density in myocytes from rats with diabetes mellitus experimentally-induced by streptozotocin has been measured as rv300/0 less than control (at +60 m Vi P < 300) under basal recording conditions in the presence of 18 mM external glucose, while the density of IKI (entry 32) appeared without difference between groups. These and other data 64 have led to the suggestion that depressed glucose metabolism in the diabetic heart may be a key factor underlying changes in ITO channel function. Notably, agents that increase glucose utilization (e.g. insulin, 0.1 M, dichloroacetate,

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Table 1. Sample cell-type preparations and source references where an 'A-current' has been characterized as a substantial part of the work (From 44-08-01) Adrenal gland

Zona fasciculata (AZF) cells, enzymatically dissociated, bovine 17

Astrocytes

Spinal cord (2-13 days in culture)18 Chick24 (see Channel density, 44-09)

Cochlear hair cells Endothelium

Aortic endothelial cells, bovine, cultured (certain subpopulations only)25; corneal endothelium, rabbit, freshly dissociated26

Epidermis, footpad, Merkel cells

Enzymatically dissociated from 10- to 20-day-old rats 27 (non-excitablet)

Epithelium, retinal

Retinal pigment epithelium (RPE cells), toad, isolated28 (non-excitablet Note: lA-like currents are generally rare in epithelial cells)

Melanoma, cell line

(IGR 1)29 (non-exc.itablet)

Muscle, cardiac

Myocytes in primary culture, neonatal mouse19; ventricular muscle, canine2o; rat21,22; crista terminalis of rabbit heart, Purkinje fibres; atrial cells, rabbit23; quiescent atrio-ventricular node Vesicle preparations, frog Circular layer, from proximal colon of guinea-pig30; rabbit portal vein31; guinea pig portal vein32; tracheal, guinea-pig33; pulmonary artery, cultured cells33; aorta, rabbit33

Muscle, skeletal Muscle, smooth

Neurones

Bullfrog sympathetic; cerebellar granule cells34; cochlear ganglion of chick embryo; dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos; dorsal root ganglion (DRG) and afferent neurones innervating the urinary bladder of the adult rat35 (two types, note 3); globus pallidus, external segment, rat36; Hermissenda crassicornis type B photoreceptor neurones (I classical' preparation); hypothalamus, histaminergic tuberomammillary neurones, rat; intracardiac, rat37; isolated Anisodoris ganglion cells (I classical' preparation); islands of Calleja, granule neurones, rat38; medial nucleustractus-solitarius (mNTS) in adult guinea-pig39; major pelvic ganglion (MPG), acutely dissociated, rat40; mollusc (initial descriptions); neocortex, layer I neurones and layer IT/m pyramidal cells41 ; olfactory bulb (DB) output (Initral/tufted), rat42; periglomerular cells, olfactory

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Table 1. Continued Neurones

bulb, frog, in vitro43; primary sensory, dissociated (multiple cell types, including group 1 cells (capacitance 10-30 pF) and group 2 cells (capacitance 55-85 pF)44; hippocampus, guinea-pig, adult45; rat hippocampal, in dissociated cultures prepared at £17£19 46; spinal cord, sympathetic preganglionic, neonatal rat47; sympathetic, coeliac-superior mesenteric ganglia (C-SMG), adult rat, acutelydispersed48; stomatogastric ganglion, cultured49; suprachiasmatic nucleus 50 (see Channel modulation, 44-44); retinal ganglion cells (solitary retinal ganglion cells)51

Neurone-like, NGI08-15

Mouse neuroblastoma x rat glioma hybrid cells, differentiated with prostaglandin £1 52 (see also VLG K M-i [native), entry 53)

Neurosecretory cells

Supraoptic, adult rat Mouse53 (non-excitable t ) (see Phenotypic expression, 44-14) Acutely dissociated from adult rats 54

Pancreatic acinar cells Pineal cells Pituitary

Taste receptor

Melanotrophs, cultured, adult rat55; GH3 cellline56; intermediate lobe cells, rat (thin slice preparations)57; pars intermedia, melanotrophs, acutely dissociated58 (see Cd2+ and Zn 2+ under Channel modulation, 4544); posterior, neurohypophysial nerve terminals, rat, acutely dissociated and identified; melanotrophs, frog 59; luteinizing hormone-releasing hormonecontaining (LHRH), embryonic (identified by Lucifer yellow injection and LHRH immunocytochemistry)6o Cells, posterior, rat61; olfactory receptor neurones (ORN's), zebrafish62 (see also JLG CATcAM~ entry 21)

Notes: 1. The purpose of this table is to exemplify studies of native A-current; citations contain further reference lists for studies in the same or similar preparations. 2. The term 'cell-type expression' is normally used to describe distribution patterns of specified genes or gene products (as opposed to ionic currents of uncertain origin). 3. In this study, the majority (700/0) of bladder neurones expressed highthreshold tetrodotoxin (TTX)-resistant Na+ channels and slow-inactivating KA , which were'available at the resting membrane potential'. The remaining (300/0) neurones were larger DRG neurones with low-threshold TTX-sensitive Na+ channels and fast-inactivating KA channels; the latter were 'almost completely inactivated' at the resting membrane potential35 .

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1.5 mM, and L-carnitine, 10 mM) 'normalize' ITO density within 4 to 6 hours 64 .

See also intracellular protons under Channel modulation, 44-44.

Elevated [K+10 suppresses

ITO

in heart

44-09-03: Chronic membrane depolarization (induced by elevating [K+]o to 20 mM in the growth medium for 72 h) has been reported to 'significantly reduce' the density of ITO in cultured rat cardiac ventricular myocytes without affecting the channel kinetics and voltage-dependence 65 .

Developmental regulation Caveat for interpreting developmental electrophysiology data 44-11-01: A number of observations have been reported detailing changes in A-current densities in specified membranes during the course of organismal development, in the presence/absence of various developmental cues and/or during the progression of disease. While the majority of such studies use molecular probes to map specific developmental changes (see this field under entries 47-53), Table 2 lists a number of developmental studies involving I A employing electrophysiological methods alone, despite the inherent ambiguities of this approach (see the special note in this field under VLC K DR [native], 45-11).

Phenotypic expression Summary of general functions associated with A-type channels (see also Table 3) 44-14-01: Fast-transient channels operating in the subthreshold range t of action potentials are widespread in the central nervous system (see also Current designation, 44-04 and Ce1.l-type expression index, 44-08). Expression of diverse A-type channels assists different types of neurones in the specific temporal integration of synaptic inputs and firing with distinct spike frequencies 2,14. A-type channels have been implicated in multiple neuronal processes including: (i) control of action potential frequency and threshold of firing 1,73 {see Table 3}; (ii) determination of action potential shape 74,7S and neurotransmitter release7'S-77. Phosphoregulation of A-type channels linked to neurotransmitter receptor/second messenger systems (see Receptor/transducer interactions, 44-49 and Protein phosphorylation under the VLC K Kv series, entries 47 to 52) may be involved in neural mechanisms related to behaviour and lea:rning 78,79.

Subcellular locations 44-16-01: There is direct evidence for subcellular 'targeting' of protein subunits conducting A-type currents in neurones (for details, see Kvl.4 under Subcellular locations in VLC K Kv1-Shak, 48-16 and Kv4.2 under Subcellular locations in VLC K Kv4-Shal 51-16). J•

'---e_n_t_ry_44

_

Table 2. Developmental regulation studies involving A-current (From 44-11-01)

Feature/property

Descriptions and cross-references

Astrocyte-dependent development of IA

44-11-02: Patterns of IA expression have been compared in cultured hippocampal neurones growing on or touching astroglia expressing glial fibrillary acidic protein [==' on-glia neurones' or 'touching-glia neurones' respectively] with those in similar neurones growing directly on a coated glass substrate [== 'off-glia neurones']66. After 5-7 days in culture, A-current amplitude in 'off-glia' neurones is approximately 19% of that of neurones growing in the 'on-glia' configuration. A-currents are the major component of transient potassium current in 'on- and touching-glia' neurones, while delayed rectifier-type currents were more dominant in 'off-glia' neurones. These and other results 66 indicated that astrocytes were able to induce development of A-current; increased A-current amplitude was associated with an increase in membrane area, suggesting that glia promote insertion of an 'A-current-rich' membrane 66 . 44-11-03: A detailed account of properties and development of voltage-gated potassium currents (IKJ IA) in acutely-isolated rat hippocampal pyramidal cells (areas CAl and CA3) during postnatal development (P6-8, 9-14, and 26-29) has appeared67.

Cell-cell interactions regulating expression of IA in developing ciliary ganglion neurones

44-11-04: Expression of I A in chick ciliary ganglion neurones developing in situ has been shown to depend on target tissue interactions and preganglionic innervation68 . Both A-type and Kca channels (see Developmental regulation under ILG K Ca, 27-11) show altered functional properties in chick ciliary ganglion t neurones developing in vitro (Le. in the absence of normal cell-cell interactions, although other voltage-activated currents appear to develop normally under these conditions). Activation and inactivation kinetics of IA is two- to threefold faster in the absence of target tissues or in the absence of pre-ganglionic innervation, although amplitudes of IA are unaffected.

Development of spontaneous action potentials

44-11-05: Changes in outward current densities (including IA) and development of spontaneous action potentials in stomatogastric ganglion (STG) neurones (as a function of time) have been described following transfer to primary cell culture49.

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Table 2. Continued

Feature/property

Descriptions and cross-references

Epithelial ,de-differentiation'

44-11-06: Changes in IA and other membrane currents have been observed in human retinal pigment epithelial cells undergoing 'de-differentiation' in culture (see Developmental

regulation under VLC K DR [native], 45-11). Neonatal versus adult 44-11-07: In adult rabbit cardiac ventricular preparations, action potential duration is kinetics significantly increased when stimulation frequency (see also VLC K is increased from 0.1 to 1.0Hz. In neonatal Kv-beta, entry 47) preparations, a similar change in stimulation frequency produces 'no significant increase' in action potential duration. This difference in behaviour between adult and neonatal tissues is associated with marked differences in the rate of recovery from inactivation in Itol components, which is significantly faster in neonatal cells (time constant 113 ms) than in adult cells (1356 ms)69. Note: For definition of Itol, see Current designation, 44-03. Regulatory factors of postnatal development in ITO in cultured neonatal rat cardiac ventricular myocytes have been described and compared to current development in situ 7o .

I to1 inactivation

IA modulation in disease

44-11-08: Altered electrophysiology of A-type channels (underlying the transient outward current) in cardiac ischaemia have been discussed 71 . 44-11-09: Prolongation of cardiac action potential associated with ventricular hypertrophy is accompanied by alterations of the ITO (e.g. in reductions in density; increased time constants for recovery from inactivation) and may thus form part of a cardiac adaptation mechanism to pressure overload 72.

ELECTROPHYSIOLOGY For a background to the protein subunit diversity supporting lA-type' currents, see the VLC K Kv series (entries 47 to 51). As described in lnotes on coverage' at the beginning of this entry, these sections are limited to general properties of native currents (for detailed comparisons, see the references under Related sources and reviews, 44-56). Transient K+ currents have been described that start activating at membrane potentials across a wide range (between -80 and -10mV - for reference lists, see

l_e_n_t_ry_44

-----J_

Table 3. General physiological functions proposed for IA components (From 44-14-01) Feature/property

Descriptions and cross-references

General properties/ 44-14-02: For general roles of IA in the control of functions in the CNS action potential number, timing, duration and frequency in the CNS (see paragraph 44-14-01). Note: In computational models of neurones, IA (and IAHP, see ILG K Ca, entry 27) controls the rate of impulse production (e.g. in the 'silicon neurone' analogue integrated circuit model which is able to emulate the complex electrical behaviour of biological systems80 ). Fast transient (spike) 44-14-03: I A influences voltage trajectories following hyperpolarizing current pulses, and can therefore repolarization contribute to spike repolarization. I A (like 1M , see VLG K M-i [native], entry 53) generally activates at more negative potentials than do Ie, IAHP (entry 27) and I K (entry 45) (for comparative review see ref. 81). Whereas the 'low-voltage activating' channels (i.e. activating from -70 to -60mV) regulate firing patterns, A-type currents that activate at rv-40mV or rv-lOmV ('high-voltage activating') could play similar roles in cells where a depolarizing current (e.g. a Ca2 + current) is also activated at similar potentials 14 . 44-14-04: lA's influence on electrical firing in the

suprachiasmatic nucleus is strongly influenced by Zn2 + ion modulation (see Zn 2+ ions under Channel modulation, 44-44). Roles in cardiac pacemaking and action potential modulation

44-14-05: The physiological functions of I A in cardiac pacemaker (sinoatrial node) has been reviewed82,83. For listings of cardiac current components and their predicted functions, see also reviews listed under field 56 (this entry). In general, cardiac transient outward K+ current contributes to the early repolarization phase of the action potential as well as to the plateau height and total duration: ITO creates a 'notch' which 'resets' the early course of plateau, and also limits the duration at long cycles. Relatively slow recovery from inactivation of ITO (compared to that of the Ca2 + current) may contribute to premature excitation (reviewed in ref. 84). 44-14-06: General background: In cardiac ventricle,

each of the three major repolarizing potassium currents (ITO - see above, I KI , I K ) play different roles in modulating the action potential duration (APD; for descriptions of gene mutations influencing APD,

_'--

.

en_t_ry_4_4_

Table 3. Continued Feature/property

Descriptions and cross-references

see Chromosomal location under VLC K eag/elk/erg, IK1 (described under INR K [native], entry 32, see note below) contributes to maintenance of plateau

46-18):

and controls the course repolarisation during phase 3 of the action potential. IK (described under VLC K DR, entry 45, see note below) plays a major role in controlling action potential duration within a wide range of cycle lengths in Purkinje fibres, and when present, also in ventricular muscle fibres. Note: All 'current' components are heterogenous with respect to underlying channel protein contributions, see

respective entries for further description. A comparison of differences between ITO and Iso (slowly inactivating sustained outward current) in human cardiac atrial and subepicardial ventricular myocytes, attempting to account for differences in action potential shape has appeared85 . Roles of I A in excitation-secretion coupling

44-14-07: A voltage-sensitive transient potassium

current has been described in mouse pancreatic acinar cells 43 . This current is activated by depolarization and has many of the eharacteristics of the neuronal IA. Although acinar cells are non-excitable (i.e. do not carry action potentials) the membrane potential appears to be the primary regulator of the current in acinar cells 53 . 44-14-08: Rapidly inactivating A-type K+ current was

observed in each of 'more than 150 cells' from bovine adrenal gland zona fasciculata (AZF)17. Despite this abundance, the function of the current in steroid hormone secretion by endocrine cells (which do not generate action potentials) is unclear. I A in taste reception

A-type K+ currents are prominent in taste receptor cells where they help shape action potentials generated following stimulation with tastants. These action potentials may be important for communication within the taste bud as well as to afferent nerves in the eNS.

Potential roles of IA Taking into account its voltage dependence, I A has in circadian rhythms been hypothesized to play a role in determining the excitability of cultured neurones from the suprachiasmatic nucleus (i.e. through its probable influence on the action potential threshold and interspike interval). IA was determined to initiate

l_e_n_t_ry_44

_

Table 3. Continued Feature/property

Descriptions and cross-references

Potential roles of lA membrane repolarization following an action in circadian rhythms potential and possibly make a 'slight contribution' to resting membrane potential86 • Together with the lK cont. and lKCa component, lA magnitude affects spontaneous firing frequency, and hence the circadian rhythm in firing frequencies that have been described in suprachiasmatic hypothalamic neurones.

ref. 7}; midpoints of steady-state inactivation are also variable over a wide range, typically from -75 to about -15 m V (ibid.). Finally, inactivation rates of native A-currents vary by at least 100-fold (ibid.). These 'variable' properties have confounded attempts to 'classify' A-currents by biophysical properties alone; some of the factors underlying this variability have now been established, however (see Cene family, 44-05).

Activation Enhancement and suppression of fA components 44-33-01: In neurones with resting potentials between -60 and -50 mY, lA is completely inactivated and activation usually requires a brief hyperpolarization of the membrane. Experimentally, a hyperpolarizing ('conditioning') pre-pulse maximizes A-type current responses. In native cells, this hyperpolarization depends on opening of other K+ channels to 'deinactivate' KA. Conversely, lA components are frequently removed from superimposed records by incorporating an inactivating pre-pulse step (e.g. -lOOmV -----t -50mV) prior to the test depolarizing step to +20mV (i.e. current flowing during test pulses is limited to slowly rising lK). The voltage of the pre-pulse determines the amount of the A-current that is inactivated in the steady state t (complete inactivation generally occurs at about -50 mV, see above). Since some KA channels are also activated from negative potentials close to the resting potential, only relatively small depolarizations are required for their activation. Comparative note: Activation time courses of 'transient' voltage- and time-dependent K+ currents resemble those of native voltage-gated Na+ currents (see VLC Na, entry 55).

Current type 'Cloned' channels displaying 'intrinsic' fast-inactivation properties (cross-references) 44-34-01: Relatively few K+ channel cDNAs form 'fast-inactivating, lA-type' channels when expressed alone in heterologous cells; those that do mostly display a form of N-type inactivation t (for clarification, see the glossary; see also references listed under Kvl.4 in VLC K Kvl-Shak, entry 48; Kv3.4 under VLC K Kv3-Shaw, entry 50 and Kv4.1, Kv4.2 and Kv4.3 under VLC K

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_4_

Kv4-Shal, entry 51). Notably, Xenopus oocytes injected with rat brain mRNA express an A-type current activating transiently near the threshold for Na+ action potential generation seen in somatic recordings from neurones (I SA1 see Current designation, 44-04). In one study employing hybrid arrest' techniques7, an oligonucleotide t complementary to a sequence common to all (known) mammalian Shal- related mRNAs (Kv4.1, Kv4.2 and Kv4.3) blocked oocyte expression of ISA. An oligonucleotide complementary only to Kv4.2 mRNA (the most abundant Shal-related transcript in rat brain RNA, see entry 51) was also 'quite efficient' in arresting the expression of I SA from brain mRNA pools7. Notably, 'significant differences' between this IS A and currents from heterologously expressed Kv4.1 or Kv4.2 cRNA could be eliminated if Kv4.1 (or Kv4.2) cRNA was co-injected with (i) brain poly(A)+ RNA treated with antisense oligonucleotides which arrest the expression of the I sA , or (ii) a 'short size fraction' (2-4 kb) rat brain poly(A)+ RNA fraction which did not express detectable K+ currents under the same recording conditions 7 (but accelerates the rate of inactivation) (for likely significance, see paragraph 44-34-02).

Significance of Kv(3 'accessory'subunits 44-34-02: As described in detail under VLG K Kv-beta (entry 47), association of some Kv,8 (cytoplasmic laccessory') subunit variants (generally encoded by 'short' mRNA fractions between 2 and 4kb, compare paragraph 44-34-01) with certain membrane-bound, pore-forming (Kva) subunits can markedly influence inactivation properties and 'convert' a 'delayed rectifier-type' channel to a fast-inactivating 'A-type' channel. This factor, and the sensitivity of 'current type' to protein modulation (and other factors described under Gene family, 44-05) have determined that inactivation properties in isolation are insufficient for channel classification in vivo (see the NOMENCLATURES section).

Current-voltage relation Rectification induced by high concentrations of intracellular Mg 2+ or Na+ ions (example) 44-35-01: Unitaryt I-V relations of KA channels in cultured rat locus coeruleus neurones are non-linear, with a negative slope at very positive voltages in 3mM [K+]o87. When 140mM KCI solutions (containing no divalent cations or Na+) are applied to the intracellular face of excised, inside-out patches, unitary I-V relations become linear (slope conductance 17.8±1.8pS, n== 16, when [K+]o==3mM). Addition of Mg2 + (2-10mM) or Na+ (10-20mM) to the internal surface reduces outward currents and induces rectificationt similar to that seen during on-cell recording87 (see also INR key facts, entry 29, INR K native, entry 32, Equilibrium dissociation constant, 44-45 and Kinetic model, 44-38).

Inactivation Special note: Inactivation properties of native K+ channel complexes may depend on protein subunit composition (including the presence of K+ channel ,8 subunits and inactivation domains (see N-type inactivation t in

e_n_t_ry_44

1_ _

_

glossary and paragraph 44-34-02). For the range of observed characteristics in mammalian voltage-gated K+ channels, see also cross-references under Current type, 44-34, Domain functions, field 29 and Inactivation, field 37 of the VLC K Kv series (entries 47 to 51 inclusive).

General features and properties 44-37-01: Native A-currents display rapid inactivation with inactivating time constants t typically described as
Kinetic model 44-38-01: A-current in various preparations has been subject to extensive kinetic modelling (for references, see reviews under Related sources and

reviews, 44-56).

Selectivity 44-40-01: In general, fA components are 'highly selective' for potassium ions (i.e. K+» Na+). The 'classical' transient channel in Helix aspersa neurones shows an ionic permeability (PX/PK ) ratio series of TI+ : K+ : Rb+ : NHt : Cs+ : Li+ : Na+ : CH3NHt rv 2.04 : 1 : 0.73 : 0.18 : 0.14 : 0.07 : 0.09 : 0.06

(from ref. 92).

Single-channel data 44-41-01: In general, fA components in native cells have been reported with unitary conductances in the range 15-20pS (150mM symmetrical K+).

PHARMACOLOGY Most studies ofnative A -channel pharmacology (to date) have used compounds with relatively poor pharmacological selectivity, low potency or else have

II

_

entry 44

'-----------

Table 4. Basic properties of IA blockers in native cells (From 44-43-01) Blocker

Descriptions and cross-references

'Classical' blockers (non-specific dendrotoxins and aminopyridine compounds reduce other native K+ conductances in addition to IA )

44-43-02: a-Dendrotoxin (a-DTx - for details see VLC K Kv-beta, entry 47) blocks some fast-activating neuronal K+ channels in native cells with high potency (IC so < 50 nM, for IA in somata of hippocampal pyramidal neurones). Under experimental conditions, application of a-DTx can reduce the size 0.£ the short depolarizing current pulse required to bring neurones to firing threshold. Notes: Toxin I (from black mamba venom and related to a-DTx) has been reported to inhibit I A with an ICso of <5nM). 44-43-03: 4-Aminopyridine over a 'high micromolar to millimolar' concentration range blocks native IA dose-dependently in many preparations, by decreasing mean open time and conductance. Relatively greater 4-AP sensitivity has sometimes been adopted in distinguishing native IA from IK components (see Trivial names, 44-07). IA is generally less sensitive than IK to tetraethylammonium ions (TEA; 1Cso in the order of 10 mM). Notably, 4-AP sensitivity of A-type channels expressed from size-fractionated brain mRNA in oocytes increases when the channels are coexpressed with a 2-4 kb RNA fraction that expresses no K+ channel activity alone 93 (for significance, see paragraph 44-34-02 and VLC K Kv-beta, entry 47).

Extracellular lithium 44-43-04: Extracellular Li+ (140mM) suppresses both ions (see also channel IK and IA in rat anterior pituitary cells to 71 % and 69 % of control, respectively, in a reversible manner94 . modulation, 44-44) Supplementary note: Extracellular Li+ facilitates basal secretion of growth hormone from anterior pituitary cells (and of catecholamine from chromaffin cells); in both cases the intracellular accumulation of Li+ and the presence of extracellular Ca2+ is required for the effect. Internal block by other inorganic ions

44-43-05: Native A-currents are modulated (inhibited) by internal Mg2+ and Na+ ions (e.g. ref. s7, but generally require the ion to be in the 'millimolar range' (cf. fast intracellular ionic block of 'classical' KIR channels occurring in the 'micromolar range', see INR K [native], entry 32.) Functionally, ionic block of A-current may limit the contribution of A-current to repolarization during the 'overshoot' of the action potential. Pathologically, 'excitotoxict' build-up of intraeellular sodium ions may contribute

Il..-.-e_n_try_4_4

_

Table 4. Continued Blocker

Descriptions and cross-references to 'feed-forward excitation' by reducing the effectiveness of the A-current87. For the role of intracellular ion blockade in generation of rectification properties of unitary A-type currents, see also Current-voltage relation, 44-35.

Dihydropyridines (various, both Ca2+ antagonists and agonists)

44-43-06: The dihydropyridine (DHP) Ca2+ antagonist nicardipine (see Blockers under VLC Ca, 42-43) has been reported to block intracellular [Ca2+ hindependent transient outward K+ current (Ito) in myocytes from rabbit cardiac atrium 95 • The inhibitory effect occurs at similar doses to those that block lea, but by a distinct mechanism. Ito is also inhibited by other DHP compounds (with rank order for potency being nicardipine > benidipine > nisoldipine > BAY K 8644 > nitrendipine > nifedipine 95 ). 44-43-07: In independent studies of bovine adrenal zona fasciculata cells 96, both DHP Ca2 + channel antagonists (nimodipine and (+)-Bay K 8644) and agonists ((-)-Bay K 8644 and RS 30026) were shown to reversibly reduce A-type K+ current amplitude and 'markedly accelerate' the apparent rate of IA inactivation (at concentrations of 1-100 JlM). Effects of DHP agonists and antagonists on I A were qualitatively indistinguishable. 44-43-08: Comparative note: The calcium antagonist

D600 (a phenylalkylamine) has been reported to inhibit calcium-independent transient outward current in isolated rat ventricular myocytes 97. Other descriptions of relatively nonselective or weak blockers (with specific citation of I A block)

44-43-09: (i) Non-selective (but dose-dependent) block of rat cardiac ventricular Ito by positive inotropic, negative chronotropic agent thaliporphine (1-30 JlM) has been reported98 . (ii) Non-selective, weak block of rat cardiac IA by salicylaldoxime (0.1-2.0mM) and 2,3-butanedione monoxime has been described99• (iii) The bradycardic t and vasodilating agent E4080 {(E)-N-[3-((N' -(2-(3,S-dimethoxyphenyl) ethyl)-N'methyl)amino)propyl]-4-(4-( 1H-imidazol-1-yl)phenyl)3-butenamide dihydrochloride dihydrate} nonselectively inhibits IA (1 JlM), Ih (0.1 JlM, see entry 34) and lea (1.0-10JlM, voltage-dependent) in smooth muscle cells dispersed from rabbit portal vein3l . (iv) A comparative discussion of tedisamil inhibition of IA (as potential positive inotropic compounds for

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_4_----J1 4

Table 4. Continued Blocker

Descriptions and cross-references treatment of heart failure) has appeared10o. (v) Relatively weak block of an lA-type current in cultured rat cerebellar granule cells by two novel toxins purified from the venom of the Mexican scorpion Centruroides limpidus limpidus has been described (approx. 50% of the peak currents are reduced by application of a 1.5 JlM solution of C. 1. limpidus toxins 1 and 2)101. (vi) Men peptide (22 aa residues, isolated. from bee venom) blocks some native and cloned voltage-gated K+ channels (see VLG K Kv1 to VLG K Kv4, entries 48 to 51) and has potent convulsant activity.

indirect mechanisms of action. These factors make it difficult to relate pharmacological properties to specific ion channel proteins in cells. In consequence, properties described in this section may not have a clear 'counterpart' in studies of 'heterologously expressed' Kv channels. Studies have been included only where direct reference to 'lA' or 'Ito' components has been made.

Blockers Early indications of protein subunit heterogeneity underlying native IA 44-43-01: Prior to the cloning of multiple cDNAs encoding Kv channel gene

family members (entries 48-52), differences in sensitivity to the 'classical' blocker 4-aminopyridine (4-AP, over the 'high micromolar to millimolar' range) and variable inactivation properties suggested a subtype heterogeneity to KA channels in native cells. As described elsewhere (entry 47 and Table 4, this field), discovery of various Kv,B subunits and their selective functional properties in association with pore-forming (Kva) subunits has profoundly altered views of 'KA channel' composition (see Gene family, 44-05).

Channel modulation 44-44-01: Studies citing 'modulatory' effects of arachidonic acid, multiple ionic and chemical species on I A components in native cells are listed in Table 5. For scope, see general note at the head of this section.

Receptor/transducer interactions Kinases and phosphatase modulation of KA as effectors of G proteinlinked receptor signalling 44-49-01: Protein kinase/phosphatase-dependent phosphorylation/dephosphorylation of KA channels constitute a fundamental mechanism for

l'----e_n_t_ry_44

-----I_

Table 5. Examples of studies citing modulatory effects on native IA components (From 44-44-01) Modulator

Descriptions and cross-references

Suppression of native 44-44-02: In dissociated bullfrog neurones under whole-cell voltage-clamp conditions, arachidonic acid (AA) reduces IA in a dose-dependent and reversible neurones by arachidonic acid (AA) manner without a shift in the pre-pulse inactivation Non-specific K+ voltage-current relation102. In this preparation, current modulation 1.75 JlM non-metabolized AA inhibits IA by 50%; by long-chain higher concentrations induce 'a total suppression'. See also/compare: (i) AA suppression of Kv4 subfamily polyunsaturated fatty acid channels heterologously expressed in Xenopus oocytes5 , this field under VLG K Kv4-Shal, 51-44}; (ii) modulation in pineal gland cells AA-enhancement of native M-current; (iii) ILG K AA [native], entry 26.

I A in bullfrog

Potential role of AA modulation in cardiac action potential duration

44-44-03: Application of arachidonic acid to individual rat cardiac ventricular myocytes results in inhibition of the transient outward K+ current103. Functional note: Liberation of arachidonate by phospholipases in response to cell surface receptor activation (see ILG Ca AA-LTC4, entry 15 and ILG K AA, entry 26) has clear significance in inotropic t control of cardiac muscle. The effect of arachidonate on cardiac IA (above) is additive to effects of endothelin, which also inhibits distinct delayed rectifier-type currents (for details, see Receptor/transducer interactions under VLG K DR, 45-49). However, effects of both arachidonate and endothelin can be blocked by staurosporine (a relatively non-specific (protein kinase C (PKC) inhibitor). PKC-dependent inhibition of these distinct K+ channel types would result in prolonged action potential duration and increased Ca2+ influx across the sarcolemma.

Docosahexaenoic acid (22:6n3)

This has been reported to act at an extracellular site to produce a voltage- and time-dependent 'block' of the IK or IA in pineal gland cells in a manner 'similar to that classically described for intracellularly-applied quaternary ammonium compounds,104: Analyses of voltage- and timedependent block by supports a hypothesis that 22:6n3 blocks the K+ channel open state. Note: 22:6n3 fails to block either I K or I A in the presence of Zn2+ or Cd2+, although extracellular Ca2+ does not affect the response 104.

_ 1.....---

entry 44 _

Table 5. Continued Modulator

Descriptions and cross-references

Extracellular Ca2+ ions inducing shifts in voltage dependence of inactivation

44-44-04: Switching from salines containing 4 mM Mg2+ to salines containing 4 mM Ca2+ induces a large positive shift in the voltage dependence of steadystate inactivation of A-currents in chick autonomic neurones. Inactivation shifts appear to act following ion binding to extracellular sites l05 . [Ca2 +]0 (Note: Intracellular modulation of an IA component with divalents Ca 2+transients may interacting with a site on the external side of the cell trigger STOCs which membrane has also been characterized in rat are distinct from IA - cerebellar granule cells l06 . see Trivial names, 44-44-05: Elevations of [Ca2+]0 shift the steady-state 44-07) activation and inactivation parameters of the transient K+ current in rat cardiac ventricular myocytes, such that a greater proportion of ITO are activated at the less negative holding potentials22 • 44-44-06: Extracellular Ca2+ (at physiological concentrations) has been reported to 'mask' the activity of an A-type IA in guinea-pig cardiac cells l07. Removal of extracellular Ca2+ induces an I A at membrane potentials more positive than 0 m V, reaching a peak within a few milliseconds of stimulation (then decreasing exponentially). Note: External Cd2+ (0.1 mM) mimicks the inhibitory effect of Ca2+. 44-44-07: Modulatory effects of various divalent cations on the A-current in cultured rat dorsal root ganglion cells (DRGs) include a depolarizing shift of both the activation and inactivation gates (causing either depression or augmentation of lA, depending on the species and concentration of the divalent cation, and also on the pre- pulse potential used to de-inactivate IA (for details see ref. 10B).

La3+ ions enhancing A-current in CG neurones by effects on gating

44-44-08: Lanthanum ions (at low micromolar concentrations) induce 'a pronounced enhancement' in depolarization-sensitive outward current in rat isolated cerebellar granule neurones under whole-cell voltage-clamp l09. (i) The steady-state inactivation curve for the ITO (evoked by depolarizing steps from -140mV) is shifted by approximately 40mV in the depolarizing direction by 10 JlM La3 +, accompanied by a slight increase in the slope factor. (ii) Kinetics of activation and inactivation of ITO are slowed in the presence of La3+ 109.

II.-_e_n_t_ry_4_4

_

Table 5. Continued Modulator

Descriptions and cross-references Modulatory effects in the 'micromolar range' have been described for ITO in rat hippocampal neurones by extracellular Pb2+, La3 +, and Gd3 + ions, with relatively low activity for Cd2+ or none for Fe3 +, Cu2+, Ni2+ (in the same concentration range).

Extracellular Zn2+ ions inducing Acurrent potentiation - evidence for synaptic (vesicular) release of zinc ions

44-44-09: In neurones of the suprachiasmatic nucleus, micromolar concentrations of Zn2+ ions 'markedly potentiate' IA activated from a holding potential of -60 m V, which is the resting potential of these neurones 50. Zn2+ potentiation occurs at 2 JlM Zn2+ concentration or higher, and arises from a shift in the steady-state inactivation of I A to more positive voltages. At 30 JlM, Zn2+ shifts the halfinactivation voltage by +20mV (from -BOmV to -60 mV), while 200 JlM Zn2+ induces a +45 mV shift (from -BOmV to -35mV)50. In the same study, histochemical localization of Zn2+ ions showed intense staining in the (ventrolateral) region of the nucleus that receives major fibre inputs. These and other findings 50 suggest that Zn2+ ions (stored in pre-synaptic vesicles and released during synaptic activity) may modulate electrical firing of suprachiasmatic nucleus neurones through effects on IA - compare effects of Zn 2+ ions on

NMDA receptor-channel modulation (entry ELG CAT NMDA, field 08-44) and GABAA receptor-channel block (entry ELG Cl GABAA, field 10-43). In the presence of micromolar concentrations of

external (but not internal) Zn2+ ions, the voltage dependence of activation and inactivation of ITO in rat hippocampal neurones is shifted to more positive membrane potentials. Acutely-dissociated melanotrophs of the rat pituitary pars intermedia express a transient outward potassium current (IK£)58. Micromolar concentrations of external Zn2+ (or Cd2+) induce 'parallel and nearly equal rightward shifts' along the voltage axis of the activation and steady-state inactivation curves for Ikf. These and other observations58 have led t~ the suggestion that Zn2+ (or Cd2+) ions can interact with a binding site on (or close to) the channel and modify the electric field 'detected by' the voltage sensors.

_'--

e_n_t_ry_4_4_

Table 5. Continued Modulator

Descriptions and cross-references

Inhibition of ITO by intracellular H+ following streptozotocininduced diabetes mellitus

Intracellular protons (i.e. low pH conditions) inhibit ITO channels in cardiac ventricular myocytes from both streptozotocin-induced diabetic and control rats; for a given acid load t , however, inhibition appears 'markedly greater' in diabetics 11o. This difference has been explained by a 'diabetes-induced decrease in Na+/H+ exchange' that is expected to limit proton extrusion during intracellular acidosis. Note: This study also showed acidosis to suppressed native I K1 (entry 32) but with a different pH profile to the inhibition of ITO 110. See also Channel density, 44-09.

8-oxoberberine

8-oxoberberine (JKLI073A) has been described111 as exerting a positive inotropic effect in rat and human atrial tissues principally by inhibition of Ito (binding to open state channels or shifting of the steady-state inactivation curve).

Chemical dephosphorylating agents

Extracellular application of the chemical phosphatase 2,3-butanedione monoxime (BDM, IC so 14 mM) reversibly inhibits Ito in rat cardiac ventricular myocytes within 20 s of application in a concentration-dependent mannerl12. Application of 20 mM BDM shifts the Ito steady-state inactivation curve by 9 ± 2 m V in the negative direction. Application of isoproterenol (5 M), 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-AMP, 10mM) but not guanosine 3':5'-cyclic monophosphate (cGMP, l-lOmM) partially reverses the effect of BDM (supporting a role for protein kinase A in reversal of the dephosphorylating effects). The ventricular I K1 (entry 32) is relatively insensitive (e.g. at 20 mM BDM, Ito is reduced to a5.5 4.3% of control while I K1 is only marginally reduced to 92.9 4.00/0 of control112.

Cytochrome P450 inhibitors

Selective modulatory effects on lA, IKv and IKATP of a number of cytochrome P450 inhibitors (proadifen, clotrimazole and 17-octadecynoic acid) in rat portal vein have been describedl13 .

Redox status affecting losmosensitivity' and lmechanosensitivity' of ITO

Inactivation of ITO in NGI08-15 cells is markedly accelerated in hyperosmotic media (+30 mosmoll-l) while voltage-dependent Na+ and Ca2+ currents are unaffected. 'Osmosensitive' (and mechanosensitive) responses were interpreted as differential sensitivity of inactivation mechanism to the membrane electric field, a factor which appeared to be dependent on

----J_

l_e_n_t_ry_44

Table 5. Continued Modulator

Descriptions and cross-references cellular redox state l14 - for further background to inactivation kinetics depending on redox status, see Channel modulation under VLG K Kv-beta, 47-44.

Taurine

Taurine modulation (enhancement) of the transient outward K+ current and (suppression) of the delayed rectifier in embryonic chick cardiomyocytes has been describedl15 .

General anaesthetics, 44-44-10: Stereospecific effects of inhalational general optical isomer anaesthetics on classical neuronal currents (including selectivities IA) versus non-specific ('lipid membrane disruptive') effects have been compared and discussedl16 . Openers Note: The pharmacology of potassium channel openers has been reviewedl17, for more details on openers, see entries covering native channels such as SKca (ILG K Ca, entry 27), BKca (ibid. ) and KATP (INR K ATP-i [native], entry 30)

modulation of cellular excitation. Multiple studies have identified specific sites on K+ channels that are substrates for serine/threonine kinases and that contribute to short-term and long-term regulation of current amplitude and kinetics (reviewed in ref. 118, see also Protein phosphorylation, field 32 under entries covering 'cloned' channels). Receptor-coupled modulatory effects are not confined to neurones, and are likely to influence physiological functions such as cardiac action potential duration 103 or secretory cell activity (see also Channel modulation, 44-44). Some examples referring to receptor-coupled modulation of native A-currents/channels by neurotransmitters are listed in Table 6.

INFORMATION RETRIEVAL

Related sources and reviews 44-56-01: Early references/A-type current definitions 1,2,11,133; 1986 review of

voltage-dependent currents of vertebrate neurones (including IA) and their role in regulating membrane excitability 81; A-current distribution in native cell types (minireview)16; coupling to receptors for somatostatin and acetylcholine130; ion channel modulators as potential positive inotropic compounds for treatment of heart failure (including tedisamil block of ITO)100; phosphoregulation of K+ channels, including those contributing to IA 118; role of IA in cardiac pacemaker (sinoatrial node) cells82,83; see also general cardiac ion channel review (1993 }134; listings of vertebrate cardiac ionic currents - nomenclature, properties, function and cloned equivalents

_L..-

e_n_t_ry_4_4_

Table 6. Examples of neurotransmitter modulation of native A-type currents (From 44-49-01) Neurotransmitter/ receptor class

Descriptions and cross-references

General notes and effects

44-49-02: IA can be modulated by different neurotransmitter actionsl19-122. Generally, depression of IA leads to enhanced excitabilityt; conversely, increasing the amount of A-current that is activated lengthens the interval between one action potential and another and thus leads to reduced excitability.

Acetylcholine (ACh) (IA suppression in hippocampal neurones)

44-49-03: In hippocampal neurones, muscarinic suppression of A-current causes a reduction in latency (the time taken for an action potential to be initiated) as well as increasing the number of action potentials initiated by a constant depolarizing pulse (see also

Abstract/general introduction, 44-01). (IA enhancement in 44-49-04: Current-clamp studies in rat neostriatal neurones indicate that muscarinic receptors serve to striatal neurones, though effects are decrease their responsiveness to excitatory inputs, an membrane potential effect due in part to muscarinic modulation of the A-current123. Muscarinic agonists (i) shift the voltage dependent) dependence of A-current activation; (ii) shift inactivation towards more negative membrane potentials; and (iii) increase peak conductance. At relatively hyperpolarized resting potentials, ACh transiently alters the functional role of the A-current, allowing it to suppress excitatory inputs and further slow the discharge rate. At relatively depolarized resting potentials, ACh increases excitability by removing the A-current through inactivation123 .

Adenosine (IA enhancement in locus coeruelus)

44-49-05: Adenosine (300 JlM) enhances IA in rat locus coeruleus neurones by shifting the steady-state inactivation curve in the depolarizing direction (the mean shift of the. curve at 80% inactivation was 4.6 m ~ which increases the amount of I A available for activation at the threshold potential by 2.5-fold. These and other :results suggest that adenosine reduces action potential duration of locus coeruleus neurones through enhancement of I A 124). 44-49-06: Frog melanotrophs possess an A-type current that appears to play an important role in excitability: The melanotroph I A is modulated by adenosine Al receptors coupled through a Gi/Go protein, partly explaining the inhibitory effect of adenosine on eleetrical activity59.

1__e_n_try_4_4

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Table 6. Continued N eurotransmitter/ receptor class

Descriptions and cross-references

Angiotensin II (IA suppression in neurosecretory cells)

44-49-07: The excitatory action of angiotensin II on

Neuropeptide Y (NPY) (IA enhancement in cardiac ventricular myocytes, 'opposing' adrenergic modulation)

Noradrenaline (IA suppression in hippocampal pyramidal and dorsal raphe neurones)

Somatostatin (IA enhancement in somatotrophs)

rat supraoptic neurosecretory cells has been proposed to be 'in part' through suppression of I A (some neurones were 'insensitive', but in 'sensitive' cells, suppression appeared selective for I A over I K )136. 44-49-08: In rat cardiac ventricular myocytes NPY

activates ITO and attenuates the increase in the contractile response induced by isoprenaline125. These and other results were taken to indicate that in the rat the'antiadrenergic, negative contractile' effect of NPY results from its action on the ITO, and block of this current by 4-aminopyridine 'unmasks' a 'positive contractile' effect of NPY that is related to activation of the lsi (slow inward Ca2+ current) 125. 44-49-09: Noradrenaline (NA; norepinephrine)

suppresses IA in hippocampal pyramidal neurones. Noradrenaline also modulates cardiac Purkinje cell transient outward current by increasing fast inactivation; this modulation of I A kinetics can be mimicked by increasing intracellular cAMP concentration126. Noradrenaline also acts at Ql-adrenoreceptors resulting in suppression of I A in dorsal raphe neurones 122 . Comparative note: Acurrent in Aplysia bag neurones can also be modified in a cAMP-dependent manner127,128. 44-49-10: In primary cultured, identified rat

somatotrophs, somatostatin (100M) reversibly increases I A by 45 % (and I K by 75 %) 'without obvious effects' on steady-state voltage dependency of activation or inactivation129. This effect has been hypothesized to contribute (in part) to the inhibitory effect of somatostatin on growth hormone release129. Note: Modulation of ion channels (including A-type channels) by coupling to receptors for somatostatin and acetylcholine has been reviewed130 (see also INR K G/ACh [native], entry 31).

_ entry 44 - - - - - - - -

Table 6. Continued Neurotransmitter/ receptor class

Descriptions and cross-references

Other mechanisms Nonselective inhibition of ITO and positive inotropic/ chronotropic actions of capsaicin MCDP (comparative note only)

44-49-11: In rat atrial myocytes, capsaicin (10 JlM) induces a significant prolongation of action potential, accompanied by 'an initial rise and a sustained increase' in contractile force 131 . Over the range 1-100 JlM, capsaicin reversibly reduces ITO amplitude and late outward (IL) K+ currents in concentrationand voltage-dependent manners 131 . Note: Capsaicin also suppresses (ECso approx. 50 JlM) the amplitude of acetylcholine- ot' adenosine-induced K+ current (IKAch,IAdo, see I.NR K G/ACh [native], entry 31). Mast cell degranulating peptide (MCDP) has been shown to bind to a voltage-dependent A-type potassium channel with high- affinity «1 nM; cited in ref. 132 ). At >500nM MCDP induces long-term potentiation (LTP, see ELG CAT GLU NMDA, entry 08) in the CAl region of the hippocampus. Notably, the concentration of MCDP required for LTP induction is 'greatly reduced' by preactivation of G proteins 132 which are intrinsic to a K+ channel inhibition phenotype associated with LTP.

(1996 review)8; ion channel function in disease (1995 review), including discussion of transient outward current functions 135.

Book references: Halliwell, J. (1990) In Potassium Channels: Structure, Classification, Function and Therapeutic Potential (ed. N.S. Cook), pp.348-81. Ellis Horwood, Chichester. Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA.

Feedback Error-corrections, enhancements and extensions 44-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource f, Search Criteria ei) CSN Development). For this entry, send email messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 44-14-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback ei) CSN

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Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csnf).

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VLG K DR [native] Edward C. Conley

IDelayed rectifier' type K+ currents in native cell types of vertebrates Entry 45

Notes on coverage: Like the preceding companion entry covering Irapidly inactivating A-type' currents in native cells, a substantial literature exists on voltage-gated K+ currents which display 'slow-inactivating' or 'non-inactivating' responses following applied depolarizing voltage steps. The channels that lacked inactivating components were originally named 'delayed rectifiers' because of the Idelayed' change in membrane conductance that followed a voltage step (see this entry). Early definitions (based on electrophysiological criteria alone) did not anticipate the extraordinary diversity of K+ channel protein subunits that have since been shown capable of contributing to Idelayed rectifying' K+ currents (IK ) in vivo. Work on native cell types continues, both with and without reference to the Imolecular constitution' of the underlying channel(s). With some notable exceptions, direct Imatching' of native cell current characteristics to Icloned' channel properties has proven difficult or misleading (see discussions in Gene family under VLC K A -T, 44-05, and Channel designation under VLC K Kvl-Shak, 48-03). Information about certain native 11K' components such as IK,r (the rapidly activating component of the cardiac delayed rectifier, probably encoded by the erg gene) is cross-referenced in entry 46, VLG K eag/elk/erg. Similarly, further description of IK,s (the Islow' component of the cardiac delayed rectifier, possibly determined by KvLQTl gene product apparently activated by minK protein) is described within entry 54, VLG K minK. These Icardiac IK ' components may not be resolved into Ifast' and Islow' components in some studies. The task of Irelating' native currents to protein subunits that support them has begun, but it is complex and largely incomplete; inclusion of this entry can therefore include reports where the subunit composition remains undefined. This difficulty of specifying channel subunit composition in native cells has meant no extensive descriptions ofindividual current properties are included in this entry (these, however, have been extensively reviewed - see references in Related sources and reviews, 45-56). Coverage here emphasizes references describing specific physiological functions, cell type distribution patterns, receptor/transducer interactions and channel modulation. It is clear that the use of the terms Idelayed rectifier' (this entry) and lA-type' (entry 44) will assume less significance in channel classification (see Subtype classifications, 45-06). Defined hetero-oligomeric arrangements of Kv alpha subunits (channel-forming, entries 48-52 inclusive) conzplexed with Kv beta subunits (see VLC K Kv-beta, entry 47) may be able to account for many observed inactivation properties at the level of specified native cell types. Thus overlapping distributions of specific channel-forming and accessory subunits Imapped' to identified cell types within defined developmental lineages is likely to form a more stable framework for indexing native electrophysiological and pharlnacological behaviours (for details, see the CSN website, wwwJe.ac.uk/csn).

NOMENCLATURES

Abstract/general description 45-01-01: 'Delayed rectifier' potassium channels in native cells (this entry, denoted as I K or various current component designations) were originally named because of the 'delayed' or 'sigmoidal' change in membrane conductance they induced following application of voltage steps (see next

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paragraph and Trivial names, 45-07). In contrast to native 'A-type' potassium channels (see VLC K A -T [native], entry 44) current through delayed rectifiers persists while the depolarization is maintained, i.e. they lack a fast-inactivation process. Thus, delayed rectifiers typically have inactivating time constants of > 1 s (in comparison to 'A-type' currents, which usually inactivate in
nerve and muscle cells is repolarization of action potentials propagated by Ca2+ and Na+ influx. In the Hodgkin-Huxley model for electrical propagation in the squid giant axon (INa depolarizing, I K repolarizing) activation of IK was 'slow' (delayed) relative to the INa component. In consequence, the delayed rectifier component is frequently considered as the 'final' voltage-gated conductance to be activated following depolarization of native cells. Activity of delayed rectifiers can therefore regulate the degree of Ca2+ influx with each action potential and hence the action potential duration. In excitable cells, neurotransmitter modulation of I K can markedly affect pre-synaptic neurotransmitter release thresholds and/or development of post-synaptic potentials and muscular tone. I K components

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also contribute to the refractory period t between action potentials. Examples of delayed rectifier modulation regulating secretion coupling in non-excitable cells have been described (see Phenotypic expression, 45-14). 45-01-05: Uncertainty in specifying the protein subtypes underlying native 1K components may limit the value of phosphomodulation data unless the cell or tissue type is well-defined. Significantly, defined phosphomodulations appear capable of 'converting' rapidly inactivating currents to non-inactivating delayed rectifier currents (see Protein phosphorylation, 45-32). Receptorcoupled modulatory effects of 1K components probably exist in all cell types and several examples of these drawn from a range of cell preparations are listed in Table 8 under Receptor/transducer interactions, 45-49. 45-01-06: The ambiguity of subunit composition and their diversity in native cells has also slowed the development of highly selective blockers (or other pharmacological modulators). Despite this, much fundamental knowledge about ion channel function has been derived by studying mechanisms of block using relatively non-selective pharmacological tools (see listings under Blockers, 45-43). These studies have been complemented by approaches using highly potent (though still subtype non-selective) peptide toxin blockers (ibid.). A wide variety of current modulatory agents have been reported, although many of these studies fail to specify sites or mechanisms of action (see Channel modulation, 45-44).

Category (sortcode) 45-02-01: VLG K DR [native], i.e. voltage-gated K+ currents in native t cells generally referred to as 'delayed rectifiers' (for background, see Abstract/ general description, 45-01 and Current designation, 45-04). Note: UEIs (unique embedded identifiers) for this entry will be derived from specified gene family designations only in updates (see this field under the VLC K Kv series, entries 47 to 51).

Channel designation 45-03-01: Numerous designations have been used for channels underlying delayed rectifiers with inactivating time constants > 1 s (reviewed in ref.l and in Hille, 1992, see Related sources and reviews, 45-56); these include K(v), K(dr), K(DR) or DRK, the first three normally signifying the subscripted forms, Le. KV1 Km, KDR- The: terms K-DRI and K-DR2 channels have been used for multiple distinguishable subtypes of delayed rectifier in circular smooth muscle of canine colon2 . 'I-channels' or 'I-type' channels are used in ref. 3 to describe delayed rectifiers in peripheral myelinated axons from Xenopus laevis. Dorsal root ganglion neurone (DRG) channel subtypes distinguishable on the basis of their single-channel conductances, kinetics and sensitivity to external tetraethylammonium ion have been named DRF1 DR11 DR21 DR34. Note also the term 'delayed rectifier' has been applied to proposed channel activator proteins such as 1sK (minK) -

for clarification, see VLC (K) minK, entlY 54.

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Current designation Most designations in use do not make reference to the underlying subunit composition 45-04-01: Generally, I K; also variants IK(dr); IK(dK); IK(v) (i.e. the 'd' subscript representing gelayed outward K+current); Ix (in cardiac muscle; see next paragraph). Multiple components of the native delayed rectifier current 1dK in canine colonic myocytes have been described5 with protocols which separate the current into three distinct components that differ in their kinetics and pharmacology (I-dKft I-dKs and I-dKn ). In embryonic chick dorsal root ganglion (DRG) neurones 6 InRI< also passes Cs+ and Rb+; under conditions where intracellular [K+ h is reduced to <30 mM and an earlier study 7 in DRG neurones designated I eat as a 'Na+ current passing through a delayed rectifier K+ channel' (see Selectivity, 45-40). With some exceptions (see paragraph, 45-04-02) most designations for native 'delayed rectifying K+ currents' do not make any reference to the composition or arrangement of the underlying channel protein subunits (see also cross-references under Gene family, 45-05).

Designations commonly used for different kinetic components of cardiac I K 45-04-02: A large literature specifically defines the '!apidly activating' component of the cardiac delayed rectifier K+ current as IK,re More recent work has established a high similarity between the characteristics of native IK,r and those supported by heterologously expressed genes in the erg subfamily (e.g. HERG) (for further clarification and examples, see h-erg under VLG K Kv eag/elk/erg, entry 46). Similarly, the '~low-activating' component of the cardiac delayed rectifier K+ current is referred to as IK,se Many papers have described activities of the IsK (minK) protein as 'supporting' currents in heterologous cells with high similarity to native IK,s. Compelling evidence for an 'activator' function for minK protein eliciting 'IK,s-like' current has appeared (when heterologously co-expressed with subunits encoded by the KvLQTl gene); these and other findings are summarized in VLC [K] minK, entry 54. The 'ultrarapid' delayed rectifier K+ current (IKur ) in human atrial cells is similar to current conducted by heterologously expressed Kv1.5 channels (see VLC K Kvl-Shak, entry 48). Other 'ultrarapid' delayed rectifier designations include I sus and IK,p. A listing of these current designations as described for heart, with further source references and comparative biophysical data appears in the review by Boyett et al. (1996)8.

Gene family 'Matching' native currents with channel subunits 45-05-01: Since there is a great diversity of candidate K+ channel subunit arrangements (and modulatory conditions) that could contribute to native I K components, the task of 'matching' native currents to clones is not straightforward (for discussion/cross-references, see Gene family under VLC K A-T [native], 44-05). Simple descriptions of the 'type' of current

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_5_

supported by different Kv subunits in heterologous expression systems are listed under the field Current type in the entry covering the appropriate gene subfamily (e.g. fields 48-34, Shaker-related channels; 49-34, Shabrelated channels; 50-34, Shaw-related channels; 51-34, Shal-related channels). Comparative note: The major delayed rectifier in Drosophila neurones and muscle has been determined to be encoded by the Shab 10cus t9 (see VLC K Kv-Shab, entry 49).

Subtype classifications The term tdelayed rectifier' is inadequate for subtype classification 45-06-01: The term 'delayed rectifier' for classification of native K+ currents is now considered inadequate, largely because it does not discriminate between K+ channel subunit heterogeneity known to be expressed in native cells (see Cene family, 45-05). Channels that have been described as 'delayed rectifiers' could variously (conceivably) include channels formed from (i) Kva subunits alone (see Current type, field 34 under entries 48 to 52), (ii) Kva/{3 subunit complexes under oxidative conditions (see Channel modulation under VLC K Kv{3, 47-44), (iii) eag- and erg-subfamily K+ channels (see VLC K Kv eag/ elk/erg, entry 46), (iv) effector channels activated by minK/lsK-type subunits (see VLC K minK, entry 54) or (v) channels underlying muscarinic-inhibited K+ currents (see VLC K M-i [native], entry 53). This list is not exhaustive, and it is also conceivable that further (undiscovered) subunit heterogeneity beyond these 'classes' exists. Since non-inactivating, voltage-gated K+ channels are commonly observed in native cell preparations (see Cell-type expression index, 45-08) the term 'delayed rectifier' will probably continue to be used as a convenient (albeit limited) description for these (or for 'cloned' channel subtypes which do not undergo 'conversion' or 'switching' to A-type currents following co-expression with modulatory subunits (see Channel modulation under VLC K Kvf3, 47-44). For listings of 'candidate'

subunits associated with specific currents in native cell types of 'classical' electrophysiological preparations, see th,e appropriate fields in the 'cloned' K+ channel entries (as above, this volume).

Cells may express a multiplicity of delayed rectifier currents 45-06-02: Many cells contain more than one 'separable' type of delayed rectifying current (IK ). For example, the single 'type' of 'classical' delayed rectifier expressed in frog node of Ranvier has been resolved into at least three kinetically and pharmacologically distinguishable types of K+ current10. A large number of separate genes which encode K+ channels with delayed rectifying properties (see previous paragraph) can contribute to this heterogeneity. For example, up to nine distinct K+ channel genes have been demonstrated to be co-expressed in single phaeochromocytoma (PCI2) cells by RT-PCRt techniques 11 (see also Developmental regulation, 45-11).

Published tsurveys' of native K+ curlBnts 45-06-03: Understanding the complexity of native K+ current expression and its relation to functional 'role' has been the subject of a number of reviews

1__e_n_t_ry_45

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which have listed or classified subtypes described in specific tissues or cell types. These references are generally listed under Current designation (field 04), Cell-type expression index (field 08) and/or Related sources and reviews (field 56) of appropriate entries.

Trivial names 45-07-01: The 'classical' (Hodgkin-Huxley) delayed rectifier; 'slow-activating' voltage-dependent K+ channels (e.g. in the original model for repolarizing K+ currents in the squid giant axon12 the 'slow activation' was relative to that of voltage-gated Na+ channels). The use of the term 'delayed rectifier' is ambiguous as nearly all subsequently characterized native K+ channels also change membrane conductance with a delay (i.e. A-current and KCa current may also participate in action potential repolarization). Note: The term 'rectification' can be defined as 'non-ohmic' behaviour in conductance-voltage relations; in trivial descriptions of delayed rectifiers the less strict sense of rectification as 're-establishment' of a transient change in conductance has been used.

EXPRESSION For a background to the subunit diversity and expression patterns supporting 'delayed rectifier' currents, see the VLC K Kv series, entries 47 to 51.

Cell-type expression index Ubiquity of delayed rectifiers in excitable and non-excitable tissues 45-08-01: As discussed in Gene family, 45-05 and Subtype classifications, 4506, there is a subunit heterogeneity and multiplicity of current subtypes which contribute to 'lK' components in native cells. For example, native

neurones, muscle and epithelial/secretory cell types express a large and highly differentiated 'repertoire' of voltage- and ligand-gated channel proteins, and 'phenotypic' analyses (field 14) must take account of these. The ubiquity and complexity of current interactions in native cells is such that it is likely to require cell type modelling (e.g. of electrical and pharmacological parameters) to define their relationships to known ion channel gene product distributions. Table 1 lists selected references to tissue slice, enzymatically dispersed and cell culture preparations in which a 'delayed rectifier' current has been characterized as a substantial part of the work. Although not exhaustive, the source references may provide a link to extensive descriptions of current properties in a specific cell type/preparation of interest. Note that wherever lK components have been specifically associated with known gene products or distinct currents they have been listed under the most appropriate entry (e.g. lK,r !"'.Jh-erg, VLC K Kv eag/elk/ erg, entry 46, lK,s !"'.JKvLQTl/minK in VLC [K] minK, entry 54, lK,M !"'.Jnative M-current in VLC [K] K M-i [native], entry 53). See also references listed describing specific preparations under Related sources and reviews, 45-56.

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Table 1. Sample cell type preparations and source references where a delayed rectifier current has been characterized as a substantial part of the work (excluding IK,r, IK,s and IK,M) (From 45-08-1) Adrenal gland Astrocytes

Zona glomerulosa (ZG) cells; rat and bovine, two current types 13 Hippocampal, acutely isolated, rat 14; spinal cord astrocytes, cultured (2-13 days in vitro) (e.g. see ref. 15 under INR K [native], entry 32)

Blood cells

B lymphocytes (and precursors)16; B lymphocytes, mouse16; platelets ('resembles I K of nerve, muscle and T lymphocyte'); human, rabbit, rat17; T lymphocytes, malignant, rat Nb2 cells, distinguishable from those in normallymphocytes 18; T lymphocytes, human (see Kv1.3, Kv3.1 and Phenotypic expression, 45-14)

Ear, inner

Cochlea, apical and basal regions, chick embryo19; utricle hair cells, types I and IT, mouse, acutely excised20 (see note 5); hair cells, vestibular type IT, isolated from cristae ampullares of guinea-pii1 hair cells from utricle, saccule and lagena, basolateral surface, pigeon (see note 3); cochlear hair cells, chick221'23 Growth plate cartilage, cultured, chick24

Chondrocytes Epithelium

Eye

e.g. in alveoli, rat25; type IT alveolar epithelium, cultured, rat26; choroid plexus, rat27; cultured cells from retinal pigment epithelium (RPE) of rat28, ibid. human and monker9, ibid. bullfrog RPE30; epithelial intermediate portion, endolymphatic sac31 Corneal keratocytes32 (see Developmental

regulation, 45-11) Glia

Hepatocytes

RetinallViiiller cells, rabbit33; immature astrocytes or 'complex' cells, CAl stratum radiatum, brain slice, rat34; oligodendrocyte progenitor cells, optic nerve, rat35; oligodendrocytes, cortical progenitor (O-2A) cells (see Developmental regulation, 45-11) Guinea-pig36 (Ca 2+ -sensitive', see Channel

modulation, 45-44) Melanoma

Cell line (IGR 1, non-excitable t ), human37 (see Developmental regulation, 45-11)

1__e_n_t_ry_45

----I_

Table 1. Continued Muscle, cardiac

For specific references to cardiac IK,r components, see th-erg' under VLC K Kv eag/ elk/erg, entry 46; for cardiac IK,s components, see tKvLQT1' under VLC [K] minK, entry 54. Atrium, human38; ibid. (IKur ) (see Receptor/transducer interactions, 45-49); comparison of ventricular versus atrial cell IK components (rabbit, dissociated)39, cultured40; atrium, bullfrog41; guinea-pig atrium42; cardiac atrioventricular node, rabbit43; cardiac embryonic tissue (7-10 day), chick44; cardiac embryonic atrium (6-11 day), chick45; neonatal, mouse, primary cultured46; cardiac sinoatrial node, rabbit (major conductance is IK - see note 4)43,47,48; cardiac ventricle, guinea_pig49,50; cat55; cardiomyocytes, frog 52

Muscle, skeletal (see note 8)

Vesicle preparations, frog 53; skeletal, frog 54; ibid. normal and mdx mice 55; myoball preparation, rat56; ascidian larval muscle57. See also fibroblasts, embryonic lung, human, cultured (uninfected and infected with human cytomegalovirus) (see

Developmental regulation, 45-11) Muscle, smooth

e.g in airways (review5 8 ); airway, canine59; airway, human60; airway, porcine and canine61 ; trachealis (airway) smooth muscle, ferret 62; aorta, rabbit63; arterial, basilar artery; arterial, cerebral, rabbit64; colon, canine (multiple components, I-dKf , I-dKs and I-dKn , - see Current designation, 45-04); colonic circular, phasic (conducting electrical slow waves)65; coronary artery vascular, human66; coronary artery, left descending, rabbit89,186,187; gastrointestinal, proximal colon circular (phasic), canine67 (see Receptor/transducer interactions, 45-49); jejunal circular, canine and human68; portal vein, rabbit69- 72 (see Receptor/transducer interactions, 45-49); pulmonary arterial cells, rat 73,74; pulmonary artery, cultured, human 75; pulmonary artery76, 'oxygen-sensing' (see Phenotypic expression, 45-14); pulmonary myocytes, rat 77 (see arachidonate under Channel modulation, 45-44); renal artery78;

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_S___

Table 1. Continued small mesenteric resistance artery, rat (two I K components)79 vascular smooth muscle, cerebral blood vessels (1995 review)Bo; ureter,

guinea_pigB1 ; urinary bladder, ileal longitudinal strips, guinea-pigB2; vascular, tail artery, rat B3

Neurones

e.g in auditory cortex, pyramidal and nonpyramidal cells, rat B4; autonomic, ciliary ganglion, chickB5; axon segments (various, see Kv1.4 under entry 48); bag cells of Aplysia (j' classical' preparation); cerebellar granule cells, cultured, rat B6- BB; cerebellar Purkinje cells (for models, see Phenotypic expressio.n, 14-45); luteinizing hormonereleasing hormone (LHRH)-containing neurones, embryonic; ibid., in explant cultures derived from olfactory pit regions of embryonic mice 90; dissociated embryonic rat hypothalamus, cultured in 'synaptically coupled' networks 91; dorsal root ganglion (DRG)4; ibid. acutely isolated from 12-dayold embryos, mouse 92; ibid. chick (also Icat )7; ibid. chick embryo (Cs+ - and Rb+ -permeant I K )6; globus pallidus, external segment (Gpe), rat 93; hippocampal CAl, rat 94; ibid. cultured, rat 95; hippocampal dentate gyrus granule cells 96; dentate gyrus (neurofilamentpositive, probable interneurones or projection neurones of the dentate hilus in dissociated cell culture, rat)97; hippocampal pyramidal cells of rat and guinea-pig; hippocam.pal, CA3 pyramidal cells, rat, in Vitro; two types - I D and IKslow (see note 6); hippocampal, in culture, e.g. mouse9B; identified glutamatergic pre-synaptic terminals (e.g. rat calyx of Held 99 (see Subcellular locations, 45-16); locus coeruleus 10o; major pelvic ganglion (MPG), acutely dissociated, rat101; motoneurones (1995 review)102; neocortex, layer I neurones and layer II/III pyramidal cells 103; neostriatal, grafted104;: olfactory bulb neurones (OB) output (m.itral/tufted), rat105; periglomerular cells, olfactory bulb, frog, in vitro 106; olfactory receptor (ORNs), zebrafish107; ibid. channel catfish10B; sensory neurones,

l_e_n_t_ry_4S

-----'_

Table 1. Continued rat 109,110; spinal cord, sympathetic preganglionic, neonatal rat 111; ibid. embryonic, Xenopus (two types of IK )112; superior cervical sympathetic ganglia, dissociated, cultured, rat l13; sympathetic ganglion, bullfrogl14; sympathetic, coeliac-superior mesenteric ganglia (C-SMG), adult rat, acutely dispersedl15; supraoptic neurones, rat l16; visual cortex, superior colliculusprojecting (SCP) (identified)117 - see also nerve fibres, axolemmal membranes of the medial giant fibre of the common earthworm, Lumbricus terrestris l18; myelinated axons from Xenopus laevis 3 Neurone-like

Oesophagus Osteoclasts Pancreatic beta cells

Neuroblastoma cell line 4lA3, murine, clones NI and N7119; neuroblastoma, SHSYSY cell line, differentiated120; neuroblastoma cells, mouse121-123 (see ceruloplasmin under Channel modulation, 45-44). Neuroblastoma x glioma (NGI08-IS) hybrid cells 124 Tunica muscularis mucosal cells, rabbit 125 Rabbit 126 (see extracellular [Ca 2+] under Channel modulation, 45-44) Mouse 127

Phaeochromocytoma (PC12) cells

Nerve growth factor-differentiated, rat 128

Photoreceptors, rod bipolar cells

Mouse129 (similar to Kvl.l, with contributions of Kvl.2 or Kvl.3 - see VLC K Kvl-Shak, entry 48); photoreceptors, type B in Hermissenda 130 ('classical' preparation). See also retinal horizontal cells, solitary from cat131 Adult rat 132; chick85

Pineal cells Pituitary somatotrophs

Schwann cells

Primary cultured, rat 133; pituitary, anterior, chick85; ibid., growth hormone-secreting cells 134; pituitary cell line G14C t 135; pituitary clonal cell line GH3, rat 136 Sciatic nerve, cultured, neonatal rabbit 137 (citation comprises a classification of delayed rectifiers in Schwann cells) (see also Phenotypic expression, 45-14 and Blockers, 45-43)

_L.-

en_t_ry_4_5_

Table 1. Continued Taste receptor cells

Posterior, rat138 (see also note 7); olfactory receptor neurones (ORNs), zebrafish107 (see also ILG CAT cAM~ entry 21)

Testis

Leydig cells, rat. Note: 'resembles delayed rectifier of T lymphocytes'139

Thyroid

Calcitonin-secreting C-cellline, 'TT' human140

Notes: 1. The purpose of this table is to exemplify studies of native delayed rectifier currents; citations contain further reference lists for studies in the same or similar preparations. 2. The term 'cell type expression' is normally used to describe distribution patterns of specified genes or gene products (as opposed to ionic currents of uncertain origin). 3. This conductance258 is very similar to a delayed rectifier characterized previously in semicircular canal hair cells as 'IKI ' (do not confuse with 'IKI ' as in INR K [native], entry 32). 4. Cardiac sinoatrial node (SAN) possesses a delayed rectifier as the major K+ conductance; 'classical' phosphorylation-regulated inward rectifier K+ channels (as described in entry 32) have not been reported in SAN preparations. 5. 'Type I' cells have an additional delayed rectifier conductance (gK,L) with an activation range that is 'unusually negative and variable' and 'significantly permeable to Cs+ (Pcs+/PK+f'V 0.31)20. The role of I K in shaping voltage responses of type I hair cells is outlined in ref. 141 . 6. Iv, a slowly inactivating conductance, is often seen following hyperpolarizing pulses at a holding potential of approximately -50mV. IKsl ow , a slowly inactivating, voltage-dependent potassium current in CA3 pyramidal cells of rat hippocampus in vitro, appears to determine 'discharge onset' after a period of membrane hyperpolarization142 (see also Receptor/ transducer interactions, 45-49). 7. In isolated taste cells, the intensely bitter tastant denatonium reduces voltage-activated K+ currents of the delayed rectifier type and activates Ca2+ influx143 through a receptor-mediated mechanism l44 . 'Sweet' tastants have also been described closing K+ channels following activation of adenylate cyclase and channel phosphorylation-dependent process. Ion channels (including K+ channels) coupling to taste transduction mechanisms are reviewed in refs145-148. 8. In cell-attached patches of extensor: digitorum longus skeletal muscle from mice, the delayed rectifier is the most commonly observed class of channel, with 'a density of roughly 8 channels/Jlm2 '55.

---J_

1~_e_n_t_ry_4_5 Channel density

Internodal regions have a low density of K+ channels Voltage-dependent potassium-selective conductances cannot usually be recorded at large mammalian nodes of Ranvier. Processes of axon myelination appear to 'redistribute' K+ channels that contribute to the action potential repolarization and maintenance of the resting potential (see also this field, and Protein interactions, field 31, in the VLG K Kv series, entries 47 to 51).

45-09-01:

Developmental regulation Special note: Assessing changes in density or Itypes' of delayed rectifier potassium currents in developing embryonic or post-natal tissue may be difficult or ambiguous when measured by electrophysiological criteria alone. Factors contributing to this ambiguity may include (i) the subunit composition of the underlying channels not being deducible from current properties (ii) in situ modulatory factors (themselves under developmental control) may profoundly change current properties, possibly giving rise to incorrect assumptions that the Ichannel type' has altered during development and/or (iii) imprecise Imapping' of developmental expression to the same cells or time-points between studies (an exception is morphologically identified cells or Icompartments' in rigorously defined developmental lineages). With these caveats, distribution studies of native ion channels can provide direct evidence for functional expression of channel subtypes that are difficult to imply from molecular distributions alone. Some studies (e.g. ref. 149, see field 49-11) have begun to employ in situ recording combined with molecular subtype distributions to study developmental changes in delayed rectifier currents/channels. For further background, see this field (Developmental regulation) under the Icloned'

channel entries and use of ion channel gene expression databases on the CSN website (www.le.ac.uk/csn/).

Regulation of spontaneous electrical activity during development 45-11-01: As for native A-currents, many observations have been reported

describing changes in native delayed rectifier current densities in specified cell or tissue types (e.g. during organismal development, in the presence/ absence of various developmental cues and/or during the progression of disease). To exemplify these, Table 2 lists a number of developmental studies involving I K (for use of molecular probes to map specific developmental changes, see this field under entries 47 to 53). Developmental regulation of ion channel activities may involve control of protein abundance/density, subunit stoichiometry, co-expression with distinct gene products, subcellular localization and time of emergence and disappearance. Although different ion channel and receptor complexes may only be transiently expressed in the course of development, they may influence subsequent differentiation events. 'Sequential activation' of different sets of ion channel genes requires complex interactions between multiple sets of stage- and cell type-specific transcription factors t. 'Programmed' changes

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_5_

Table 2. Developmental regulation studies citing native delayed rectifier currents (From 45-11-01) Feature/property or cell/tissue type

Descriptions and cross-references

Developmental studies of native lK in specified cell or tissue types

45-11-02: T lymphocytes (see Table 3, this field). See also development of native lK in retinal glial cells33; immature astrocytes or 'complex' cells (i.e. identified glial cells) in the CAl stratum radiatum in thin brain slices obtained from P5-35 rats34; ascidian larval development57. 45-11-03: Changes in densities and kinetics of delayed rectifier K+ currents have been frequently observed during neuronal differentiation (e.g. young and mature spinal neurones from Xenopus embryos, refs 155'-157 - see also this field under VLC K Kv1-Shak, 48-11); developing cerebellar granule cells (cultured in the presence/absence of high [K+] and N:MDA) including 'activity-dependent' processes regulating lK and lA expression within 'critical periods' of development86; cultured embryonic hypothalamic neurones in 'synaptically coupled' networks (similar to adult hypothalamic neurones)91; acutely isolated rat hippocampal pyramidal cells (areas CAl and CA3) during post-natal development (P6-8, 9-14 and 26_29)158.

Changes in native lK following or accompanying pathophysiological processes

45-11-04: Hypertrophied t versus non-hypertrophied cardiac left ventricle (feline)159.

Changes in native lK accompanying anatomical or morphological changes

45-11-06: Changes in electrophysiological properties of guinea-pig ventricular myocytes between pre- and post-natal development (e.g. action potential duration) have been interpreted in terms of altered densities of lK and lea currents 160.

45-11-05: A delayed rectifier K+ current is present in 100 % of normal corneal keratocytes (cells functional in repair of corneal stromal matrix following injury or infection). In freeze-wounded cells, the lK is found in 91 % of cells isolated from the corneal rim and 33 % of cells isolated from the wound region itself 32 •

45-11-07: Changes in lK expression co-incident with developmental changes in neuroblastoma x glioma (NGI08-15) hybrid cell morphology are described under Developmental expression in VLC K M-i

[native], 53-11.

l_e_n_t_ry_45

----'_

Table 2. Continued Feature/property or cell/tissue type

Descriptions and cross-references

Expression of viral limmediate-early/ early' genes affecting lK expression

45-11-08: Following infection with human cytomegalovirust (CMV) the proportion of human embryonic lung (HEL) fibroblasts expressing a delayed rectifier potassium current 'gradually increases' from 80/0 (in non-infected controls) to 200/0 (at 18-24h post-infection) to 100% (at 48h and 72h)161. Phosphonoformate (a CMV DNA polymerase inhibitor) induces 950/0 block of expression of CMV 'late' proteins in infected cells but can not prevent the increased expression of the potassium current (or the elimination of a native sodium current also observed in these experiments over the same time course)161. On the basis of these and other results 161, expression of CMV limmediateearly' or 'early' proteins was associated with the 'upregulation' of lK and the 'down-regulation' of lNa,v in HEL cells.

Differentiationsensitive lK following monocyte ~ macrophage transition

45-11-09: THP-l human leukaemia cells have been extensively employed as a model of monocyte-to-macrophage differentiationt and in studies of apoptosis r. Patch-clamp techniques have identified five ion channel types in undifferentiatedt THP-l monocytes, including a native delayed rectifying K+ current, designated IDR162. Profound changes in ion channel expression that occur during differentiation of THP-l cells have been described163. Following their induction to differentiate into 'macrophage-like' cells by the phorbol ester PMA (phorbol-12myristate-13-acetate) the following changes take place: (i) lOR (present in almost all undifferentiated THP-l monocytes) is undetectable in differentiated macrophagesj (ii) two 'new' K+ currents are observed in THP-l macrophages (a) a 'classical' inwardly rectifying K+ current (1m '" 30pS, Kir) and (b) a Ca2+-activated channel (lBK '" 200-250pS, maxi-K). Note: THP-l cells contain mRNA encoding Kvl.3 (see VLC K KvlShak, entry 48) and IRKl (Kir2.1, see lNR K [subunits], entry 33); mRNA abundance for these channel subunits also varied following PMAinduced differentiation (IRKl mRNA increasing 'at least 5-fold' and Kvl.3 mRNA decreasing 'on average 7_fold,)163.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_5-----J

Table 2. Continued Feature/property or cell/tissue type

Descriptions and cross-references

Cell proliferation control in melanoma and breast cancer cells

45-11-10: Delayed rectifier potassium channels have been suggested to be involved in control of melanoma cell proliferation37 (cited as analogous to similar studies on T lymphocytes and human breast carcinoma cells). In non-excitable cells, these K+ channels control the driving force for calcium-influx events critical for Ca2+-dependent cell cycle control proteins and/or secretory events. In the case of melanoma cells, block of I K prevents cells entering the S phase in the cell division cycle37 (see also lantiproliferative effect of K+ channel blockers in T cells' in this field under VLC K Kv1-Shak, 48-11).

'De-differentiation' exemplified by human retinal pigment epithelial cells in culture

45-11-11: In retinal pigment epithelial (RPE) cells,

'Selective' effect of transcriptional inhibition on native K+ current development

45-11-12: Transcriptional inhibitors t applied during

Channel 'partitioning'into sibling cells

45-11-13: 'Symnletrical segregation' of channels underlying IK and IK.IR between sibling cells at cytokinesis t has been described164 (see Developmental ~regulation under INR K [native],

most foetal cells do not express the delayed rectifier or inward rectifier of adult cells, whereas A-current (entry 44) is absent in freshly isolated adult human cells. The occurrence of the A-current in cultured adult cells indicates that under culture conditions, RPE cells undergo a change to the less-differentiated, more 'neuroepithelial-like' phenotype29. an early period of development in culture can selectively and irreversibly block delayed rectifier current without affecting the development of an inactivating K+A-current. 156

32-11).

Developmental control/channel regulation of repetitive firing in Aplysia bag cell neurones (comparative note)

45-11-14: In Aplysia , adult bag cell neurones exhibit

an afterdischarge, consisting of prolonged depolarization and an ability to undergo repetitive firing (which couples to peptide release and induces egg-laying behaviour)165. Juvenile neurones do not exhibit afterdischarge in response to pleuralabdominal connective shock or TEA+, but do exhibit prolonged depolarizations in the presence of TEA+. A number of changes in the native electrical properties occur during the developmental period in which these neurones acquire the capacity to fire repetitively: (i) three K+ currents decrease - IKca (entry 27) and two components of IKv, (ii) IA

1__e_n_t_ry_4_S

----l_

Table 2. Continued Feature/property or cell/tissue type

Descriptions and cross-references increases, and (iii) two lea components increase ('basal' and 'protein kinase C-activated'). These patterns are consistent with increases in the ability to fire repetitively observed during bag cell maturation165

such as these appear to underlie development of critical phases including 'commital' to particular developmental pathways and the generation of 'spontaneous' electrical activities. Through receptor-coupled mechanisms, therefore, regulated ionic flux may mediate genomic (Le. transcriptional) and cytosolic (Le. post-translational) events in cellular growth and developmental control (for reviews, see refs150-154 and ILG Ca InsP3, entry 19).

Glutamate regulation of 0-2A stage oligodendrogenesis by I K modulation 45-11-15: Proliferating cortical oligodendrocyte progenitor (O-2A) cells express functional delayed rectifier K+ channels (KOR ), which are absent in

pro-oligodendroblasts166 . Glutamate and AMPA-subtype selective agonists (see entry 07) inhibit proliferation of 0-2A cells cultured with different mitogens. Furthermore, both glutamate receptor agonists and tetraethylammonium ions strongly inhibit KOR and arrest 0-2A proliferation (as judged by conventional cell proliferation assays. These and other results 166 have been taken to suggest that oligodendrocyte progenitor-cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel inhibition.

T cells as a model for stable developmental regulation of multiple IK subtypes 45-11-16: Regulated expression of multiple voltage-dependent K+ channel genes in a single cell type is exemplified by T lymphocytes, where three distinct 'types' have been characterized using electrophysiological, and latterly, molecular criteria (types n, n' and 1, see Table 3, see also this field under ILG K Ca, 27-11). In general, the pattern of K+ channel expression/ modulation is determined by the state of cellular differentiation and mitogenic activation (for reviews, see refs167-172). Thus, the appearance of these channels/currents varies consistently with the developmental state and correlates with commonly used cell surface antigenic markers (Table 3). These channels/currents play integrated roles in the physiology of lymphocytes, including the re~lation of membrane potential, cell volume, calcium signalling, lymphokine t secretion and mitogenic activation177 (see also Kv1.3 function sustaining IL-2 secretion in activated T cells briefly described under Phenotypic expression of VLG K Kv1-Shak, 48-14).

III

II

Table 3. Basic distinctions between T lymphocyte native K+ channels (From 45-11-16)

Type n voltagegated K+ channel

Type n' voltagegated K+ channel

Type 1 voltagegated K+ channel

Commonly observed in

Predominates in normal immature (proliferating) T lymphocytes and mature-phenotype CD4+CD8thymocytes (precursors to helper T lymphocytes)

Sparingly in CD4-CD8- Thy1+ T cell subset; these contain precursors to cytotoxic and suppressor T cells

Sparingly in CD4-CD8- Thy1+ T cell subset containing precursors to cytotoxic and suppressor T cells and abundantly from mice with autoimmune disease

Activates at

Less depolarized potentials (> -40mV)

Similar potentials to the type n channel

More depolarized potentials (-10mV)

Inactivates

Slowly, during depolarizations lasting hundreds of milliseconds

Over a similar time course to the type n channel

Closes more rapidly than type n; use-dependent

Recovery from inactivation is

Very slow (30-60 s)

Very slow (30-60 s)

Faster than type n

Single-channel conductance

10-18pS

10-18 pS

25-30pS

Blockers

rv10mM TEA rv3 nM charybdotoxin

rv100mM TEA (i.e. more resistant than type n)

rvO.1 mM resistant to charybdotoxin (no block at 50 nM)

Gene not identified

Kv3.1 probably expressed as a homomultimeric t protein

Encoded by vertebrate gene

See entry:

Kv1.3; probably expressed as a homomultimeric t protein

VLC K Kv1-Shak, entry 48

('1)

=:s

f""t"

-

VLG K Kv3-Shaw, entry 50

~ ~ c.n

Il..--e_n_t _ry_4_5

---'_

Phenotypic expression Summary of general functions ascribed to delayed rectifier channels/ currents 45-14-01: The heterogeneity of channel protein subtypes underlying 'native' 1K currents has been described in Gene family, 45-05 and Subtype classifications, 45-06 and at the head of Developmental regulation, 45-11. In most circumstances, therefore, the 'subunit composition' of channels supporting native currents are undefined or unknown. Generally, delayed rectifier currents have characteristics that contribute to repolarization functions when membranes are depolarized by inward Na2+ and Ca2+

fluxes. 1K components that display a slower rate of activation would therefore tend to allow sufficient 'delay' for inward calcium currents to develop (in non-excitable cells, delayed rectifiers can prolong calcium-influx events linked to secretory control, e.g. type n [Kvl.3] channel openings sustaining IL-2 secretion in activated T lymphocytes - see Developmental regulation, 45-11). Beyond these generalities, the 'precise' contribution of 1K components to phenotype will largely be determined by the cell type (and in consequence, the types of co-expressed channel and receptor-transducer couplings facilitating neurotransmitter modulation). Neurotransmitter modulation of 1K can markedly alter the shape and timing of action potentials, thereby influencing pre-synaptic neurotransmitter release thresholds and/or development of post-synaptic potentials t. 1K also contributes to the refractory period t . Table 4 describes some physiological 'functions' ascribed to some native cell 1K components, exemplifying the specialization that occurs in certain cell types.

Subcellular locations 'Anchorage' dependence in 'subcellular targeting' of I K 45-16-01: 1K distributions within differentiated (post-mitotic) neuronal cells suggests anchorage to specific subcellular locations following transit from sites of protein synthesis in the cell body (for mechanisms underlying channel 'clustering' and intracelluar targeting by protein-protein interactions, see, for example, Subcellular locations, field 16 and Protein interactions, field 31 under the VLG K Kv series, entries 48-51). Delayed rectifiers in skeletal muscle appear to be 'preferentially located' in the surface membrane, while transverse tubular membranes exhibit an approximately threefold lower density of expression184 . In mouse motor nerve terminals, delayed rectifier K+ channels are concentrated at the distal regions of where they possibly compensate for the depolarizing effect of the local Ca2 + ion influx185; conversely, delayed rectifier K+ currents are sparse in mammalian nodes of Ranvier (expression can be 'induced' following treatments that 'lift' the myelin sheath from the paranodal regions of the axons 186, an action that presumably disrupts normal 'anchorage' interactions). 'Differential distribution' of 1K (this entry), 1A (entry 44) and 1m (entry 32) have been recorded among hair cells across the cochlea23; in this case, 1K and lIR were 'preferentially expressed' by the taller hair cells (positioned nearer to the neural side of the cochlea). In contrast, Ca2+-

e_n_try_4_S

_'---

-----l

Table 4. Examples of general physiological1functions' described for native IK components (From 48-14-01) Example/current

Descriptions and cross-references

Cardiac IK,r components

45-14-02: 'Rapid' component of the cardiac delayed rectifier current (see, for example, h-erg under VLC K eag/elk/erg, entry 46).

Cardiac IK,s components

45-14-03: 'Slow' component of the cardiac delayed rectifier current (see KvLQT1/minK/lsK under VLC

[K] minK, entry 54). Cardiac I K (general)

45-14-04: In cardiac ventricle, pharmacological block of 'IK' components prolongs action potential duration predominantly by lengthening phase 2 of the action potential, and the lengthening becomes more pronounced at longer cycles 174 - see Phenotypic expression under ·VLC K A-T [native], 44-14 and

sections describing mutants underlying congenital long QT syndrome. 45-14-05: Several computational models of cardiac ventricular 'excitation' for heart rate-dependent arrhythmias incorporating changes in the delayed rectifier current (IK ) have appeared (e.g. ref. 175). Compare role of native inward rectifier (IK1 ) in regulating excitability at long cycle lengths (see Phenotypic expression under INR K [native), 32-14). During the relatively short plateau of the atrial action potential'IK ' is not activated strongly and contributes a relatively small repolarizing current39 • 45-14-06: Several studies and reviews (e.g. refs 38,176-178) have directly compared properties of native cardiac K+ channels activating at membrane potentials corresponding to the plateau of the action potential to properties of 'cloned' K+ channels (see also the VLC K Kv series, entries 48 to 51). IK,M components

45-14-07: 'Muscarinic-inhibited' delayed rectifying currents (see VLC K M-i [native], entry 53).

Phenotype/function descriptions of native I K in specified cell or tissue types (excepting IK,r, IK,s, IK,M listed above and other cell type listings in this table)

45-14-08: Fibroblasts from patients with Alzheimer's disease lack a 113pS tetraethylammonium (TEA+)sensitive K+ channel179 (see Blockers, 45-43). A TEA+induced increase of [Ca2+Ji that depends on a functional 113 pS :K+ channel is also 'eliminated or markedly reduced} by addition of 100M ,a-amyloid protein in these fibroblasts 18o. 45-14-09: Pharmacological block of IK,v (but not SKca, entry 27) affects brown fat cell proliferation rate in culture181 .

_e_n_t_ry_4_5

------'_

Table 4. Continued Example/current

Descriptions and cross-references 45-14-10: Chondrocyte delayed-rectifying K+ channels may contribute to the 'unusually high' [K+] found in the extracellular fluid of growth plate cartilage24 .

I K phenotypes in

different types of smooth muscle

45-14-11: I K contributes to resting membrane potentials in pulmonary arterial smooth muscle cells and their activity can be modulated by nitric oxide and chronic hypoxia (see Channel modulation, 45-44).

45-14-12: Evidence for voltage-dependent K+ channels being involved in the regulation of membrane potential in small cerebral arteries (i.e. 100-300 ~m diameter) and responsive to intravascular pressure is outlined in ref. 64. 45-14-13: Delayed rectifier K+ channels maintain resting membrane potential in ferret trachealis (airway) smooth muscle62. In comparison, calcium-activated potassium channels (see ILC K Ca, entry 27) contribute a smaller component of the resting K+ conductance, and are thus less significant for defining the 'passive behaviour' of airway smooth muscle62 (see also review of delayed rectifier channels in airway smooth muscle cells58). Shab-like native current in cultured Schwann cells

45-14-14: Electrophysiological and pharmacological characteristics of the 'type II channel' studied in rabbit Schwann cells (cultured from neonatal sciatic nerve and lumbar/sacral spinal roots) are similar to those displayed by the mammalian Shab subfamily in heterologous expression systems 182 (see VLC K Kv2Shab, entry 49).

Channel-type 'specialization' in photoreceptors

45-14-15: As reviewed in ref. 183, delayed rectifier K+ channels in the rapidly responding photoreceptors of Ifast-flying' flies have activation ranges and dynamics which (i) 'match' light-induced signals, and (ii) enable rapid responses by reducing the membrane time constant r. In comparison, Islow-moving' flies have slowly responding photoreceptors that lack the delayed rectifier, but express inactivating K+ conductances that are 'metabolically less demanding'. See also 'diurnal modulation' under Channel modulation, 45-44. 45-14-16: Native delayed rectifier K+ currents in mouse rod photoreceptor bipolar cells function in the intact retina to allow complex modulation of retinal synaptic signals129 with most similarity to Kvl.l; by single-cell RT-PCR, sequences encoding Kvl.l Kvl.2 and Kvl.3 have been detected (see also VLC K Kvl-Shak, entry 48).

_'---

e_n_try_4_5_

activated potassium current (IKca , entry 27) does not apparently vary between cells of different shape or cross-cochlear position (for further details, see ref.23). Characterization of 'subcellular targeting' of specified ion channel proteins underlying I K components has begun using subtype-specific antibodies (for details, see field cross-references above).

Identified pre- and post-synaptic recording at a glutamatergic synapse in brainstem slices 45-16-02: Within in vitro brainstern slice preparations of the superior olivary complext , current-clampedt pre-synaptic terminals (the calyx of Heldt) generate trains of action potentials at frequencies up to 200 Hz with each action potential displaying an after-hyperpolarizationt lasting <2 ms 99 . By contrast, post-synaptic target neurones (i.e. the principal neurone of the medial nucleus of the trapezoid body or MNTB, note 1) respond to sustained depolarization with a single, short-latency action potential. Under voltage-clampt, step depolarizations of identified pre-synaptic terminals generate a tetrodotoxin-sensitive inward current (see Blockers under VLC Na, 55-43) followed by activation of an outward delayed rectifier K+ conductance showing (i) characteristically rapid activation at potentials more positive than -60mV; (ii) 'little inactivation' over 150ms depolarizing steps and (iii) partial block by 4-aminopyridine (approx. 630/0 ± 14.50/0 block of sustained current at OmV with 200 IlM 4_AP)99. In this study, confirmation of 'exclusive' recording from pre-synaptic or postsynaptic sites (e.g. in identifying calices enclosing single MNTB neuronal somata) was aided by microinjection of Lucifer Yellow dye in living tissue or the fluorescent stain DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) in fixed tissue. Notes: 1. The medial nucleus of the trapezoid body (MNTB) relays auditory information important for sound source localization. MNTB neurones 'faithfully follow' temporal patterning of action potentials occurring in their single giant input synapse, even at high frequencies. 2. Further characterization of post-synaptic K+ conductances, including a high-voltage activating fast delayed rectifier (enabling high-frequency action potential responses) has appeared187. See also VLC K Kv3-Shaw, entry 50 for ploperties and distribution of Kv3.1 protein as a 'high voltage activation threshold' channel that is enriched in nuclei/fast-firing neurones of the auditory brainstem (including calyx of Held, ref.188, field 50-16).

STRUCTURE AND FUNCTIONS

Protein phosphorylation 45-32-01: Uncertainty in specifying the protein subtypes underlying native I K components may limit the value of phosphomodulation data unless the cell or tissue type is well-defined. Note in particular that certain treatments may be capable of 'converting' rapidly inactivating currents to non-inactivating delayed rectifier currents (e.g. protein kinase C on Kv3.4 189, see Protein phosphorylation under VLC K Kv3-Shaw, 50-32). Table 5 outlines

e_n_t_ry_4_S

1_ _

_

Table 5. Examples of phosph om odulation of native delayed rectifier currents (From 45-32-01) Cell or tissue type

Descriptions and cross-references

Cardiac ventricular 1K (1K,r and 1K,s components - see Cell-type expression index, paragraph 45-08-01); protein kinases A and C

45-32-02: Numerous studies have demonstrated increased cardiac ventricular 1K by protein kinase A

(PKA) and protein kinase C (PKC) activation190-194. Modulation by PKC produces a relatively larger increase in 1K at positive voltages 1931'195 than that produced by PKA, which produces a larger increase at negative voltages. In keeping with these findings (see also this field for h-erg channels, 46-32 and Effects of 1sK/minK proteins, 54-32), 1K components have also phosphatase been characterized as being'activated' by ,B-agonists, Qinhibitors on 1K in agonists, cAMP, [Ca2+h elevations (lsK/minK), [K+]o frog cardiomyocytes elevations (HERG, 1K,r), Ca2+-calmodulin kinase (lsK/minK), phorbol esters, GAG and cell swelling (for further citations on each factor, see review, ref. 8). Inhibition of protein phosphatase t activity by okadaic acid (GA) and microcystin (MC) decreases amplitude of 1K in frog cardiomyocytes (an effect opposite to those produced by cAMP-dependent phosphorylation)52. Decreases in amplitude occur without effects on voltage-dependent activation or reversal potential t . Treatments that promote cellular ATP depletion 'totally abolish' 1K , suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier. Furthermore, peptide inhibition of protein kinase A (PKI(s-22)) and a range of other well-characterized protein kinase inhibitors show no effect on basal 1K (or basal I ca ,L)52. It has been suggested that native frog cardiac atrial delayed rectifier channels must be phosphorylated for basal channel function (possibly at the protein kinase A 'stimulatory' site) while a second mechanism inhibits channel activity and is revealed by inhibition of protein phosphatase activity (as above). Comparative note: In general, the activating effect of phosphatase inhibitors on L-type calcium channels expressed in frog cardiomyocytes are opposite to those on the delayed rectifier521'196 (for details, see Protein phosphorylation under VLC Ca, 42-32). The inhibitory effect of microcystin on 1K cannot be blocked by several commonly used kinase inhibitors, leading to the suggestion that the effect of microcystin may be due to a direct effect of microcystin on the 1K channel196.

_ L...----

entry 45 _

Table 5. Continued Cell or tissue type

Descriptions and cross-references

NeuronalIK (examples) Hippocampal pyramidal neuroneSj Xenopus spinal neurones regulated byPKC

45-32-03: Hippocampal pyramidal neurones express delayed rectifier-type currents which are inhibited by PKC activation197. 45-32-04: Mouse dorsal root ganglion neurones possess delayed rectifier currents which are decreased in amplitude by phorbol ester and forskolin treatments 198 . 45-32-05: In Xenopus spinal neurones, the IK component plays a dominant role in the 'developmental conversion' of calcium-dependent action potentials to sodium-dependent spikes 157. During maturation, I K undergoes a pronounced increase in rate of activation, dependent upon calcium influx through voltage-dependent channels and the action of PKC. For further examples of early I K kinetics modulation and associated changes in the ionic dependence of action potentials, see Developmental regulation, 45-11. 45-32-06: Comparative note: The delayed rectifier K+

current (IK ) in nerve growth factor-differentiated phaeochromocytoma (PCI2) cells is unaffected by PKC activators 128 . 45-32-07: PKC inhibits the delayed rectifier K+ current Smooth muscle I K in rabbit vascular smooth muscle derived from portal (examples) Vascular, portal vein vein 71 . Substitution of extracellular Ca2+ with Mg2+ (in the presence of 10 mM intracellular BAPTA) does not affect suppression, indicating the involvement of a Ca2+-independent isoform of PKC 71 .

Vascular, tail artery 45-32-08: In vascular smooth muscle cells (VSMCs) of rat tail artery, prostaglandin E2 (PGE 2 ) inhibits I K in a concentration- and G protein-dependent mechanism199. This PGE2-induced inhibition has been proposed to involve activation of both protein kinase A and protein kinase C 83 (compare cardiac modulation, this table, above). In artery, activation of PKC apparently potentiates the cAMP-PKA stimulation (conversely, however, the cAMP-PKA cascade did not seem to affect the PKC pathway). GH4 C 1 pituitary cellsj differential responses to cAMP analogues

45-31-09: Modulation of inactivation of voltagedependent potassium channels by cAMP has been studied in the GH4 C 1 pituitary cellline20o . A major component of voltage-dependent potassium current in these cells inactivates slowly, with a time constant t of

lL.--e_n_t_ry_4_5

----'_

Table 5. Continued Cell or tissue type

Descriptions and cross-references several seconds. Application of dibutyryl cAMP decreases this current at voltages positive to -10 mV and increases inactivation rate by approximately twofold. Single-channel recordings reveal two channel types whose voltage dependence and kinetics of inactivation 'match' those of the macroscopic current200 . The smaller conductance (7.5 pS) channel is sensitive to db-cAMP (decreasing the latencyt of the channel to enter a long-lasting inactivated state and increasing its rate of inactivation). Somatostatin, which activates a serine/threonine phosphatase, completely reverses the effect of dibutyryl cAMP (dibutyryl GMP is without effect). In contrast, the rate of inactivation of a larger conductance (approx. 19 pS) is not accelerated by db-cAMP in these cells 20o .

several examples of native current modulation by common enzymes (see also Receptor/transducer interactions, 45-49). For phosphoregulatory mechanisms determined for 'cloned' channel subunits, see Protein phosphorylation (field 44) in entries 47 to 51.

ELECTROPHYSIOLOGY For a background to the protein subunit diversity supporting 'delayed rectifier' currents, see VLC K eag/elk/erg (entry 46), the VLC K Kv series (entries 47-51) and VLC [K] minK (entry 54). As for native 'A-type' currents (entry 44), 'variable' properties have confounded attempts to 'classify' native delayed rectifying currents by biophysical properties alone. It is therefore emphasized that the content of the remaining sections is limited to general properties of native currents (for detailed comparisons, see the references under Related sources and reviews, 44-56).

Activation Original description of 'delayed' activation and general features of I K activation 45-33-01: In the Hodgkin-Huxley model12 for electrical propagation in the squid giant axon (depolarizing INa, repolarizing IK ) activation of IK was 'slow' (delayed) relative to the INa component. In consequence, the 'delayed rectifier' component is frequently considered as the 'final' voltage-gated conductance to be activated following depolarization of native cells. Various native delayed rectifier K+ currents also show (i) voltage- and timedependent activation with asymmetrical ion transport through the open

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_5_

pore; (ii) activation processes that are sigmoidal and can described by firstorder kinetics raised to a power; (iii) rise times t that decrease with increasing depolarization; greater repolarizing capacity may be available with large depolarizing stimuli when the starting membrane potential is more negative (i.e. when hyperpolarizing inhibitory tone is present - in some preparations, only half the channels are available for activation 'from rest' but 50-1000/0 of current is available by depolarizing from -85 mV). (iv) The I K activation threshold (the point at which channels first open) is 'characteristic' of a cell type/preparation, and will depend on the molecular subtype of K+ channel. 'Typical' I K activation thresholds may fall within a 'low-voltage' range (e.g. -50mV to -40mV) to a 'high-voltage' range (e.g. 'more positive than -20mV', see this field under VLC K Kv3Shaw, 50-33).

Current type 'Superficial' similarities in native delayed rectifying currents 45-34-01: 'Delayed-rectifier' type K+ currents observed in different native

cells may have broadly similar time courses of activation and inactivation (e.g. in remaining active over millisecond time-scales - see Inactivation, 45-37). These comparisons can be superficial since channel complexes supporting 'similar' I K components are now known to vary considerably in their subunit composition. The 'precise' arrangement and stoichiometry of constituent subunits are still largely undetermined in single identified neurones (for further background, see VLC K A-T [native], entry 44, VLC K Kv-beta, entry 47 and the VLC K Kv series, entries 48 to 51).

Current-voltage relation 45-35-01: Under physiological conditions, open-channel currents through

most delayed rectifiers are larger in the outward than the inward direction (cf. INR K [native], entry 32). This outward rectification is largely a consequence of higher intracellular K+ c.oncentration, leading to a greater outward K+ conductance and may not be an 'intrinsic' property of the underlying channels. Although in whole-cell I-V curves, rectification t of delayed rectifier-type channels is always outward (resulting from the voltage dependence of activation) single-·channel 1-V relations may show inward or outward rectification, even in symmetrical isotonic solutions. Notably, both the 'rapid' native cardiac I K component IK,r and the underlying h-erg channel display inward instantaneous t rectification201,2o2 by a fast-inactivation process, whereas the 'slow' IK,s does not exhibit rectification 201 (see also VLC K eag/elk/erg, entry 46 and VLC [K] minK, entry 54).

Inactivation See special note in this field under VLC K A-T [native], field 44-37 regarding influences of subunit composition and modulatory mechanisms on inactivation properties.

l_e_n_t_ry_4_5

_

Qualitative descriptions of inactivation time courses for delayed rectifiers 45-37-01: Inactivation properties of delayed rectifiers in native cells have been described as displaying 'slow inactivation' (i.e. an inactivation process lasting hundreds of milliseconds to > 10 s) or 'non-inactivating' at maintained depolarized potentials. Native delayed rectifier channels have thus been described as inactivating over periods in the order of 'seconds to minutes'

(e.g. refs203-206). For quantitative data on the inactivation properties of individual voltage-gated K+ channel subunits, see the VLC K Kv series, entries 47 to 51 inclusive. See also the unusual fast-inactivation process underlying the inward rectification characteristic of erg subfamily channels (field 46-37).

Kinetic model The 'classical' delayed rectifier

45-38-01: Since the 1952 Hodgkin-Huxley model12 describing electrical propagation in the squid giant axon (depolarizing INa, repolarizing I K ) delayed rectifier currents in various preparations have been subject to extensive kinetic modelling. Furthermore, I K components (mostly based on the HodgkinHuxley equations) are universally incorporated in computational models able to simulate, for example, tonic firing and oscillatory activities observed in 'real' neurones (e.g. refs.207-211). Within the 'silicon neurone' model (described in ref.208) an electronic 'analogue' of IK provides a repolarizing component as distinguished from analogues of IA (see VLC K A-T [native], entry 44) and I AHP (see ILC K Ca, entry 27) which control rate of impulse production. 45-38-02: As part of the slow conductance change as the 'operational definition' of a delayed rectifier (see Abstract/general description, 45-01) the 'delay' can be modelled as several intermediate steps or conformational changes that precede channel opening (see also Activation, 45-33).

Selectivity Delayed rectifier channels are generally highly selective for potassium ions 45-40-01: Generally, K+ -selective delayed rectifiers follow a permeability sequence similar to K+ > Rb+ > NHt »Na+. For example, the 'classical' delayed rectifier in frog node of Ranvier has ionic permeability ratios (Px / PK ) of TI+ (2.3):K+ (I.O):Rb+ (0.91):NHt (0.13):Cs+ «0.077):Li+ «0.018):Na+ «0.01):CH3NHt «0.021)212. Comparative note: Although Cs+ and Na+ are not permeant in delayed rectifiers in myelinated nerve (ref.213 and above), in T lymphocytes214 these ions show 'moderate' permeation through other K+ channels (for a comparative summary, see

this field under VLC K M-i [native], 53-40).

'Anomalous' permeation of Na+ through a delayed rectifier K+ channels 45-40-02: In the absence of external K+, the ion selectivity of a (TEA+-

insensitive) K+ channel in rat superior cervical ganglion neurones is

II

_'----

e_n_try_4_5_

'profoundly diminished'215. Under these conditions, Na+ traversing a K+ channel can generate an 'unanticipated' inward current. This 'unanticipated' current (Iu ) is reduced in the presence of external or internal K+. Interactions of external K+ with Na+ on the I u tail amplitude can be partly explained by invoking anomalous mole-fraction t behaviou~15. The channel supporting I u displays a permeability sequence to monovalent cations Rb+, K+, Cs+, Na+ and Li+ in the ratios 3.5: 2.5: 2: 1:0.5. Similarly, permeation of Na+ through a delayed rectifier K+ channel at reduced [K+ h has also been observed in chick dorsal root ganglion (DRG) neurones, where a conductance (designated as leat) has been described as a 'Na+ current passing through a delayed rectifier K+ channel' when intracellular [K+ h is reduced to <30mM 7. Otherwise, I eat exhibits the sanle voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in DRG neurones. Following removal of intracellular K+, I eat appears as a large, voltage-dependent inward tail current t. The high selectivity of these channels for K+ over Na+ (under physiological conditions) was due to the inability of Na+ to compete with K+ for an intracellular binding site (as opposed to a barrier excluding Na+ from entry into the channel or a selectivity filter preventing Na+ ions from permeating the channel) 7. Methodological note: 'Anomalous permeation' of Na+ may affect recordings of inward Ca2 + currents when recorded in the presence of external Na+ (see also paragraph 45-41-01).

Single-channel data 45-41-01: Single-channel conductances for delayed rectifiers have been

determined in many preparations, and many fall in the range "-115-20 pS (at symmetrical 150mM K+).

Voltage sensitivity 45-42-01: For details, see the VLC K Kv series, entries 47 to 51. Native

delayed rectifiers are prone to bursting t behaviour with depolarization normally increasing the frequency of bursts.

PHARMACOLOGY See notes relating lack of selectivity and interpretation of pharmacology applicable to native versus heterologous cell types at the head of this section under VLC K A-T [native], entry 44. For molecular characteristics of delayed rectifier-type channels formed by 'cloned' K+ channel subunits, see the VLC K Kv series, entries 47 to 51.

Blockers For further notes on blockers of cardiac IK,r components see 'h-erg' under VLC K Kv eag/elk/erg, entry 46; for cardiac IK,s blockers, see 'KvLQT1'

l_e_n_t_ry_4_5

_

under VLC [K] minK, entry 54 and references under Related sources and reviews, 45-56.

General considerations on blocker selectivities 45-43-01: As may be expected from the diversity of subunits underlying native 'delayed rectifier' K+ channels (see VLC Kv series, entries 47 to 51) simple ionic blockers (see Table 6) are generally not highly selective or potent in their action. Extensive biophysical analyses have been made of block produced by agents such as tetraethylammonium ions and 4-aminopyridine (ibid.) in addition to several toxins and clinical pharmacological compounds. Although many such studies have characterized block at the single-channel level, it is usually not possible to make reference to subunit composition in native tissues. However, many non-specific blockers of delayed rectifier potassium channels are routinely used in electrophysiological studies for 'pharmacologic separation' of IA and I K (e.g. ref.46) and also for eliminating K+ -selective channel currents when studying (for example) Na+ - and Ca 2+-selective channels. Incomplete block of 'IK' observed in native tissues (e.g. squid axon K+ currents blocked by 4_Ap216 ) can arise due to co-expression of multiple independent IK components (see also next paragraph). See also note 1 in Table 6 pertaining to inadequacy of 'tension assays' in characterizing mechanisms of K+ channel block in contraction coupling.

Blockers of cardiac K+ current components 45-43-02: Blockers of cardiac delayed rectifier K+channels are further described in this field under entries 46 and 54. These agents prolong the repolarization phase of the heart (i.e. they extend the myocardial refractory period) which constitutes the basis of their class III antiarrhythmic t action. 'Class ill' agents have demonstrated efficacy in suppressing re-entrant t atrial and ventricular arrhythmias in animal models and in their initial clinical application. For example, the class III antiarrhythmic agent E-4031 appears to block specifically a fast component of the delayed rectifier (IK,r, described under h-erg in VLC K eag/elk/erg, field 46-43). As described above, E-4031 significantly prolongs cardiac action potential durations at voltages of 0 m V and above, or when measured at APD so (action potential duration at 500/0 repolarization) or APD90 (action potential duration at 900/0 repolarization)217. Other novel class ill compounds block the slow component of the delayed rectifier (IK,s, described in VLC [K] minK, field 46-54) although most blockers are non-specific. Comparative note: Compounds which show high selectivity for native I K1 (inward rectifier) channel components in heart may also display 'pure class Ill' antiarrhythmic activity (see Blockers under INR K [native], 32-43).

Channel modulation 45-44-01: Studies citing 'modulatory' effects of endogenous and exogenous compounds, messengers, various ions and physical factors on native cell I K components are summarized in Table 7. For scope, see general note at the head of this section.

_~

e_n_try_4_5_

Table 6. IK blockers in native cells (From 45-43-01) Blocker

Descriptions and cross-references

Alkali cations

45-43-02: Internal application of the alkali cations Na+, Li+ and Cs+ produces a steeply voltage-dependent block of delayed rectifiers which can be relieved by raising the concentration of permeant and impermeant ions (e.g. in the squid axon218 and node of Ranvier preparations219 ). The mechanism of block appears similar to the block by TEA/QAs (this table, below) but lacks a measurable time dependence t and has a steep voltage dependence consistent with the alkali cation-binding site being farther into the electric field. 45-43-03: In primary cultured anterior pituitary cells from rat, extracellular Li+ (140mM) reversibly suppresses both the I K and ITO (entry 44) K+ currents to 71 and 69% of control, respectively134. 45-43-04: Internal Na+ block can be relieved by large depolarizations; under these conditions Na+ can become a charge carrier through the channel.

Other inorganic cations, Ca 2 +, Mg2+

45-43-05: Note: Delayed rectifiers may also be sensitive to block by Ca2 + and Ba2+ ions. Voltage and time-dependent 'blocking and unblocking' effects of internal and external Ca2 + and Mg2 + ions on gating of the sinoatrial node (SAN) I K have been described220 .

45-43-06: The functional significance of 'block' of delayed rectifier K+ channels by [Ca2 +h and the regulatory roles of Ca2 +-dependent ion channels in vascular and visceral smooth muscles has been reviewed221 .

Peptide toxin blockers active at voltage-gated K+ channels

45-43-07: A number of peptide toxins, notably noxioustoxin (NxTx), margatoxin (MgTx), charybdotoxin (ChTx) and kaliotoxin (:KTx) have been shown to block Kv channels in a human T cells222-224 and other cell types. In human and murine B lymphocytes, kaliotoxin behaves as a 'selective' voltage-dependent K+ channel blocker. Charybdotoxin is more frequently associated as a blocker of calcium-activated K+ channels (see Blockers under ILG K Ca, 27-43). 45-43-08: A report describing'selective' blocking action of tityustoxin-K alpha (TsTx-Ka) on I K but not IA or IKI in cultured hippocampal neurones and cerebellar granule cells has appeared (10-600/0 I K block over a 2.5-120nM range)225. 45-43-09: For patterns of non-specific dendrotoxin block of native delayed rectifiers see, for example Protein molecular weight (purified) under VLG K Kv-beta, 47-22.

I

entry 45

L...--

_

Table 6. Continued Blocker

-

Descriptions and cross-references 45-43-10: See also note 2, describing irreversible, voltageindependent inhibition of I K by the'classical' SKca channel blocker apamin.

General properties of block by internal TEA+ ions

45-43-11: Native delayed rectifiers are relatively sensitive to blockage by intracellular tetraethylammonium ions (TEA, rv6-10mM) but are relatively insensitive to 4aminopyridine (4-AP, rvl mM) (compare Blockers under VLC K A-T [native], 44-43).

45-43-12: Quaternary ammonium (QA) ion derivatives (such as TEA+) possess large hydrophobic groups, and prior to the molecular cloning of Kv channel genes, their patterns of block predicted a large hydrophobic vestibule as part of the internal 'TEA+ receptor'. According to the original hypothesis of Armstrong (reviewed in ref.226), the mechanism of block by TEA+, QAs and inorganic ions involved the blocker entering the diffusion pathway of the permeant ions and binding within the channel aqueous pore. Other QA derivatives in common use include tetraOther QA derivatives in use alkylammonium (TAA+), tetrapropylammonium (TPrA+), tetrabutylammonium (TBA+), tetrapentylammonium (TPeA+) and tetrahexylammonium (THA+) ion (e.g. in block of delayed rectifier K+ channels in mouse neuroblastoma cells 122. Patterns of intracellular TEA+ and other QA derivatives has been reviewed227. Analogous to internal block, external QA block appears to require one QA ion per channe1228, but occurs at a site outside the electrical field.

Examples of external TEA+ block

45-43-13: Unitaryt delayed rectifier channels in cultured rat (CAl) hippocampal neurones exposed to very low external concentrations of TEA+ ions display unitary currents that are reduced in amplitude (with increased open channel noise t) consistent with a relatively fast voltage-independent block of the channel by single TEA+ ions (Kd rv 53.4 JlM); both the blocking rate constant (approx. 350mM- I ms- I ) and the unblocking rate constant (20 ms- I ) are independent of voltage. TEA+-blocked channels also show reductions in mean open timet, consistent with a second, slower blocking reaction (with a Kd probably between 300 and 800 JlM)95. Channels blocked by single TEA+ ions appear able to close normally (i.e. without first requiring an 'unblocking' event)95. Delayed rectifiers in myelinated nerve, skeletal muscle/sarcolemma229 and molluscan neurones (but not in squid axon) are also blocked by external TEA+ application (largely independent of time and voltage).

_L..--

e_n_try_4_5_

Table 6. Continued Blocker

Descriptions and cross-references

Apparent inactivation

45-43-14: Certain TEA analogues cause the delayed rectifier to exhibit rapid apparent inactivation similar to that of the Na+ channel or the A-type channel230 due to the block occurring only after channel opening.

4-Aminopyridine block (see also note 1 pertaining to inadequacy of tension assays for characterizing K+ channel blockers in muscular contraction coupling)

45-43-15: A detailed analysis of mechanisms involved in the 4-AP-induced block of I K in rabbit coronary arterial vascular smooth muscle cells has appeared231 .

Heterogeneity of 4-AP-sensitive K+ current/ subtypes in Schwann cells (an example using 4-AP sensitivities to discriminate native currents)

45-43-17: A 'classification' of native delayed rectifier K+ currents in rabbit Schwann cells cultured from neonatal rabbit sciatic nerves has appeared, suggesting an underlying heterogeneity of protein subtype137. Under whole-cell patch-clampJ' depolarizing voltage steps (40 ms duration) activate two types of K+ current: Type I currents (i) have an apparent activation threshold of approx. -60mV (half-maximal conductance at -40 ± 1 mY); (ii) are reversibly blocked by a-dendrotoxin (a-DTx, Kd 1.3 nM); (iii) activate rapidly and then show a much slower 'fade', which becomes more marked with larger depolarizations 137. Type. II currents (i) have an apparent activation threshold of about -25 mV (half-maximal conductance at + 11 ± 1 mV)j (ii) are unaffected by a-DTx up to 500 nMj (iii) activate more slowly than than type I and depend less steeply on voltage. The longest activation time constant for type II gating is more than twice the corresponding time constant for type Ij however, the time constants determined from tail current decays (at potentials more negative than -60 mV) are shorter for the type IT currents than for the type I currents 137. Type I and IT currents show differential sensitivity to to 4-AP, with the type II Kd for 4-AP block (630JlM at pH 7.2, Em == -10mV) being approx. sixfold higher than for type I current. Whereas most cells exhibit both type I or IT currents, some cells display only type I or type IT. Additionally, type III currents are generated in most cells at positive membrane potentials and are insensitive to millimolar concentrations of 4_Apl~J7.

45-43-16: In general, block of I K with 4-AP in vascular smooth muscle preparations leads to contraction and an enhanced myogenic response to increased intravascular pressure. Signal transduction mechanisms involving vasoactive agonists activating cAMP-dependent protein kinase (PKA) or protein kinase C (PKC) and their effects on smooth muscle I K components have been reviewed232 .

f"..J

e_n_t_ry_4_S

_

1_ _

Table 6. Continued Blocker

Descriptions and cross-references

Capsaicin block of I K in rabbit Schwann cells

45-43-18: The lipophilic alkaloid capsaicin has been shown to block type I delayed rectifier K+ currents in rabbit Schwann cells in culture when applied by superfusion (Kd approx. 8.7 JlM)233. The kinetics of capsaicin block resembles an 'inactivation', where the rate of blockade increases with increasing concentrations of capsaicin over the range 1-100 JlM. In contrast to internal TEA+ ions (see this field) capsaicin reduces both inward and outward type I current by the same proportion. Capsaicin block can be relieved by hyperpolarization (subsequent to depolarization/opening/block) which is consistent with capsaicin having to first 'unbind' to permit channel closure233 •

Other agents 45-43-19: The resistance of I K components to Ca2 + and non-selective for Na+ channel antagonists (see Blockers under VLC Ca and K+ channels VLC Na, 42-43 and 55-53) generally permit Verapamil pharmacological separation of these currents in native cells. Notably, however, a Cs+ - and Rb+ -permeant delayed rectifier in embryonic chick dorsal root ganglion (DRG) neurones (IDRK ) is 'effectively suppressed' by external verapamil6 (apparent dissociation constant of approx. 4 JlM). The time course, kinetic scheme and frequency dependence of block indicate that verapamil can bind to the channel only when it is in the open state 6 (see note 4). Phencyclidine

45-43-20: Block of a colonic smooth-muscle delayed rectifier channel by phencyclidine have been noted234 •

Caffeine and IBMX (low potency)

45-43-21: In excised outside-out 'maxi patches' of chick autonomic ganglion neurones, chick sympathetic neurones, chick pineal cells and rat anterior pituitary cells, caffeine (O.1-10mM) produces a 'robust blockade' of delayed rectifier K+ currents 85 . Caffeine block of I K is rapid in onset and concentration and voltage dependent (greater inhibition with larger depolarizing voltage pulses). Application of 1 mM IBMX (3-isobutyl-1-methylxanthine, a structural analogue of caffeine) also inhibits I K and I A in chick ciliary ganglion neurones 85 .

Doxorubicin See also note 5

45-43-22: The anticancer drug doxorubicin which has also been shown to release Ca2 + from cardiac sarcoplasmic reticulum prolongs action potential duration and blocks guinea-pig ventricular I K • The enhancing effect of IKblock on cardiac excitation-contraction coupling has been proposed to be due in part to increased Ca2+ entering and

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_5_

Table 6. Continued

Blocker

Descriptions and cross-references accumulating in the cytosol (as long as depolarization is maintained) leading to relatively greater activation of contractile proteins235 (see also fluoxetine under Channel modulation, 45-44).

Notes: 1. As discussed in an article by Halliday et al. (1995)63 the pharmacological properties of 'selective' K+ current blockers may be of little practical value in isolating individual channel contributions to 'contraction-coupling' functions. For example, in rabbit aorta preparations, several compounds in clinical or experimental use (imipramine, phencyclidine, sotalol and amitriptyline) fail to block selectively any of the components of K+ current63 . Additionally, 'classical' K+ channel blockers such as TEA and 4-AP (by themselves) induce 'significant increases in tension' and are more effective when applied together. Overall, there may be 'no correlation' between the effects of the drugs tested by tension assay and their actions on currents recorded from isolated myocytes. It was concluded63 that (i) tension studies are an inappropriate means of investigating the mechanism of action of these drugs, and (ii) studies on ionic currents in isolated myocytes cannot easily predict drug actions on intact tissues. 2. I K components in whole-cell patch-clamped guinea-pig ventricular myocytes can be partially inhibited by bath application of apamin, a peptide toxin normally used to inhibit small-conductance Kca channels (see Blockers under ILG K Ca, 27-43). Apamin inhibits IK irreversibly at all voltages tested, but in a concentration-dependent manner (Ki approx. 34.4 OM and a Hill coefficient of 1.2)236. In contrast, the activation curve (P-infinity curve) of I K is not shifted by apamin, suggesting that the voltage dependence of I K activation (or their kinetics) are unaffected. Partial apamin block of an 'apamin-sensitive subpopulation' of cardiac IK may also underlie apamin's effect in prolongation of action potential duration236 . 3. The relative non-selectivity of caffeine and its analogues for ion channels is further described under ILG Ca (Ca) RyR-Caf, entry 17. 4. A detailed account of the mechanisms by which phenylalkylamines (e.g. verapamil, D600 and related compounds) block inactivating delayed rectifier K+ currents in rat alveolar epithelial cells has appeared25 . 5. Information on a series of 4-(alkylamino)-I,4-dihydroquinoline compounds describe~37 as novel inhibitors of voltage-activated n-type K+ channels in human T lymphocytes is outlined under ~vl.3 in Blockers, VLG K Kv1-Shak, 48-43. General note on claimed tselectivities' of K+ channel blockers.

1"--_e_n_t_ry_4_5

----'_

Table 7. Modulatory effects on native I K components (From 45-44-01) Modulator

Descriptions and cross-references

Arachidionic acid

45-44-02: In rat pulmonary myocytes, externally applied arachidonic acid (AA, 50 ~M) induces a membrane depolarization (average 16mV, n == 6)77. Application of between 1 and 50 ~M AA (i) accelerates the rate of I K activation (i.e. increasing current amplitude when measured at the beginning of the voltage stepi this enhancement of early I K was mimicked by a protein kinase C activator) 77 and (ii) accelerates the rate of current decay (i.e. reducing I K amplitude when measured toward the end of the depolarizing steps). The magnitude of the inhibitory effects on IK was correlated with the number of double bonds but was independent of tail length in fatty acids containing between 14 and 22 carbons77.

Cadmium ion modulation (cardiac - see note 1)

45-44-03: In the presence of cadmium ion (Cd2+, 0.2mM), peak tail currents of feline cardiac ventricular myocyte I K increase from a control value of 85 ± 12 to 125 ± 18pA (n == 4)51. Cd2 + shifts the voltage dependence of activation to more positive potentials by 16.4 2.0 mVand increases the slope factor of the activation curve from 6.1 ± 0.2 to 6.9 ± 0.2mV51 .

Ceruloplasmin as an endogenous neuronal depolarizing factor

45-44-04: The activity of ceruloplasmin (previously

Cytochrome p 450 inhibitors

Diurnal modulation of K+ current 'type'

Ca2 +, extracellular (cardiac - see note 1)

characterized as a copper carrier and an antioxidant) inducing a 'rapid and sustained membrane depolarization' in neuroblastoma cells has been ascribed to its significant inhibition of a TEA-sensitive delayed rectifier K+ channel123 . This activity is abolished when (i) the copper is removed from ceruloplasmin or (ii) ceruloplasmin is heat-inactivated. 45-44-05: Modulatory effects on I Kv ,

lKA and l KATP of a number of cytochrome P450 inhibitors (proadifen, clotrimazole and 17-octadecynoic acid) in rat portal vein have been described238 . 45-44-06: As reviewed in ref. 183, membranes of locust

photoreceptors can exhibit delayed rectifier-type conductances during the day while exhibiting inactivating K+ currents at night (see also Phenotypic expression, 45-14 and VLC K Kv-beta, entry 47 for other examples of tinterconversion' of inactivating to noninactivating channels). 45-4-07: Removal of [Ca2+]o and [K+]o from the solution

bathing guinea-pig ventricular myocytes frequently induces a leak t conductance, but does not affect the ionic selectivity or time-dependent activation and

_i-..

e_n_try_4_5_

Table 7. Continued Modulator

Descriptions and cross-references deactivation t properties of I K • Lowering [K+]o from 4 to OmM increases the magnitude of IK,s (entry 54, note 1), as expected from the change in driving force t for K+, but decreases IK,r (entry 46, note 1)239. 45-44-08: In rabbit osteoclasts126, elevation of extracellular [Ca2+] from 1.8 to 18 mM (i) shifts the halfpoint for I K activation by + 11.5 mVj (ii) shifts the voltage dependence of inactivation by +9.7mVj (iii) slows the rate of I K activation and deactivationj (iv) increases the voltage-dependent kinetics of I K inactivation (at all potentials tested). Effects (i) to (iii) but not (iv) are consistent with screening of cell surface negative charge as predicted by simple surface charge theory. Effect (iv) suggests an additional, regulatory role for [Ca2+]0 in the gating of I K channels126.

Intracellular Ca2+-release agents, e.g. ryanodine

45-44-09: Negative chronotropic t and other electrophysiological actions of ryanodine on single rabbit sinoatrial nodal cells, including inhibition of the delayed rectifier K+ current (this entry, see note 1) and If (entry 34) have been described240 (see also ILG Ca (Ca) RyRCal, entry 17 and Blockers, 45-43).

I K modulation by

45-44-10: The serotonin uptake inhibitor fluoxetine is a commonly prescribed antidepressant (e.g. as Prozac™) with frequent gastrointestinal side-effects. In relation to this, one study has shown fluoxetine to have 'direct effects' on canine and human jejunal circular smooth muscle68: Relatively low concentrations of fluoxetine (100nM, 1 JlM and 10 JlM) markedly inhibited canine delayed rectifier potassium current (120/0 ±30/0, 27% ± 120/0, and 37% ±3%, respectively) and depolarized the membrane potential (e.g. by 9.7 ± 1.8 mV at 10 j.t:\1:)68. Higher concentrations (100 Jll\i to 1 mM fluoxetine) activated KCa channels (by 88% ± 40% and 4750/0 ± 2700/0, respectively). Human jejunal IK was also inhibited by the drug (260/0 ± 4% at 1 JlM fluoxetine) while the Kca was increased (1340/0 ± 220/0 at 100 JlM fluoxetine)68.

fluoxetine may partly explain its frequent gastrointestinal side-effects

Hypoxia inducing membrane depolarization

45-44-11: Vascular smooth muscle cell types that are 'dominated' by the delayed rectifier ('K-DR cell types'241) can be morphologically distinguished and occur in resistance arteries at relatively high density. Hypoxia depolarizes K-DR cells by 'rapidly and reversibly inhibiting' one or more of the tonically active K-DR channels (including a 37pS channel) that control resting membrane potential. (See also note 2.)

'---e_n_t_ry_4_5

_

Table 7. Continued Modulator

Descriptions and cross-references

Role of Ca 2+ inhibition of IK in hypoxia

45-44-12: In canine pulmonary arterial cells an early key

event in hypoxic pulmonary vasoconstriction is release of Ca2 + ions from caffeine-sensitive intracellular Ca2+ stores (see entry 17). Hypoxia (induced by the O 2 scavenger, sodium dithionite) reduces macroscopic K+ currents, an effect that can be prevented by strong intracellular buffering of [Ca2+Ji. Ca2 +-inhibition of delayed rectifier K+ channels has thus been proposed as a mechanism promoting membrane depolarization and further Ca2+ entrr42 (see also next paragraph).

Persistent depolarization correlated with chronic hypoxic modulation of I K

45-44-13: Isolated smooth muscle cells from small pulmonary artery preparations derived from chronically hypoxic rats show (i) marked (40-500/0) reductions in I K amplitude; (ii) resting potentials significantly more positive (-43.5 ± 2mV) than that of cells from normoxic t animals (-54.3 ± 2mV)243.

Native I K in loxygen sensing' within pulmonary smooth muscle

45-44-14: A role for delayed rectifier K+ channels as

Lanthanum ions (trivalent cation modulation)

oxygen sensors in pulmonary artery smooth muscle (PASM) cells has been described 76; 'striking similarities' between PASM and other oxygen-sensing cells (carotid body type I cell, neuroepithelial body) are also discussed 76 . Furthermore, closure of the ductus arteriosus at birth (essential for post-natal adaptation), is initiated by an increase in 02 tension. Further work has demonstrated oxygen-induced membrane depolarization and constriction of rabbit ductus arteriosus occurs following inhibition of a 58 pS voltage-sensitive delayed rectifying K+ channel sensitive to 4-aminopyridine244 . (See also notes 4 and 5). 45-44-15: A shift of 10mV (in the depolarizing direction)

has been observed for the activation curve of the delayed rectifier current in isolated rat cerebellar granule neurones in the presence of 10 ~M La3 + ions 88 (see also Channel modulation under VLC K A-T [native], 44-44). 45-44-16: In guinea-pig cardiac ventricular myocytes, the Kf delayed rectifier current consists of (i) a La3 + -sensitive component activating rapidly with moderate depolarizations and (ii) a La3+ -resistant current slowly activating at more positive potentials245 (for significance, see note 1).

Nitric oxide in pulmonary vasodilatation

45-44-17: Nitric oxide opens native voltage-gated

channels in pulmonary arterial smooth muscle cells, resulting in membrane hyperpolarization and suppression of Ca2 + -dependent action potentials246 .

_L...-

e_n_try_4_5_

Table 7. Continued

Modulator

Descriptions and cross-references

Rubidium ions

45-44-18: Effects on gating of delayed rectifier K+ channels in frog skeletal muscle when Rb+ is the charge carrier are discussed in ref. 54.

Singlet oxygen

45-44-19: Singlet oxygen ('reactive oxygen' or 01 2 ), generated by illuminating the photosensitizer rose bengal, suppresses the delayed rectifier potassium current in single frog atrial cells247. Despite the short half-life of singlet oxygen, 'tens of seconds' are often required for I K to reach a new steady state following suppression. This 'progression' of the modulation (referring to the component of current modification which occurs after cessation of illumination) has been proposed to result from the kinetics of K+ channel state transitions (as opposed to a long-lived reactive intermediate produced during the initial 01 2 exposure)247.

(cardiac - see note 1)

Oxygen free radicals

(cardiac - see note 1) Effects of sulphydryl group-specific reagents on gating

45-44-20: Oxygen free radicals (OFRs) non-specifically suppress guinea-pig cardiac ventricular delayed rectifier current in addition to L-type calcium current (entry 42) and a native inward rectifier K+ current (entry 32). (See also note 3). 45-44-21: Chemical modification of sulphydryl (-SH) groups on delayed rectifier K+ channels using the organic mercurial compound p-hydroxymercuriphenylsulphonic acid (PUMPS) have been characterized within internally dialysed voltage-clamped squid axon preparations248 . In this study, intracellular or extracellular addition of PHMPS changed the magnitude and kinetic parameters of I K including: (i) a marked slowing of macroscopic current activation kinetics; (ii) simultaneous reductions in IK amplitude; (iii) increased delay of the macroscopic current at various pre-pulse potentials ('the Cole-Moore shift t ') and (iv) a sharp decrease in singlechannel Po pen (4- to 5-fold, with small reductions in unitary conductance of approx.. 200/0)248. Generally, PHMPS induces (i) a reduction in the voltage dependence of the activation process; (ii) a shift of the charge/voltage relationship (towards rnore positive potentials) and (iii) an increase in the mean open time (each suggesting a destabilization of the open state). Notably, all effects of PHMPS are partially ~~versed by the sulphydryl group reducing agents dithiothreitol or ,a-mercaptoethano1248 .

(See also significant lo~~idoreductive' modulation of K+ current inactivation properties under Channel modulation of VLC K Kv-beta, 47-44).

l_e_n_t_ry_4_S

----'_

Table 7. Continued Modulator

Descriptions and cross-references

Taurine modulation (cardiac - see note 1)

45-44-22: Taurine modulation (suppression) of the delayed rectifier and enhancement of the transient outward K+ current in embryonic chick cardiomyocytes have been described249.

Tetraethylammonium (TEA+) analogues

45-44-23: In the presence of TEA+ analogues (see Blockers, 45-43) delayed rectifiers may exhibit rapid inactivation similar to that of the Na+ channel or the A-channeI230.

Notes: 1. Many reports do not resolve cardiac I K in terms of IK,r (the rapid component of IK associated with h-erg subunits - see VLC K eag, entry 46) and IK,s (the slow component of IK,s which has some similarities with currents seen following heterologous expression of the KvLQT1/minK/IsK gene products - see VLC K minK, entry 54). 2. 'K-DR-enriched' resistance vessels constrict to hypoxia, whereas conduit arteries have a biphasic response predominated by relaxation. Nitric oxide (this table) is effective at relaxing vessels in both segments (by a cGMPdependent mechanism) although it relaxes conduit rings to a greater degree than resistance rings 241 . 3. Comparative note: Oxygen free radicals significantly increase the open probability of ATP-sensitive K+ channels (KATP , see entry 30) at a relatively narrow range of ATP concentrations (0.2-2 mM)250. 4. The ductus arteriosus permits blood ejected from the right ventricle to bypass the pulmonary circulation in utero. 5. In systemic arteries, hyperpolarization of the smooth muscle cell membrane (leading to vasodilatation) is associated with hypoxia in opening native KATP channels (INR K ATP-i [native], entry 30) and/or Ca2+dependent K+ channels (ILC K Ca, entry 27). Redox modulation of Kvj1 subunits in association with Kvo: subunits may play an important role in oxygen sensing coupled to electrical responses (for details, see VLC K Kvbeta, entry 47).

Receptor/transducer interactions I K modulation following G protein-linked receptor signalling 45-49-01: Protein kinase/phosphatase-dependent phosphorylation/dephosphorylation of native K+ channels constitutes a basic mechanism for control of cellular excitation (see this field under VLC K A-T [native], 4449). Receptor-coupled modulatory effects of I K components probably exist in all cell types and several examples of these drawn from a range of cell preparations are listed in Table 8 (see also Channel modulation, 44-44). 'Phosphomodulatory' effects on single-channel 'types' in native cells can rarely be interpreted in isolation, and individual reports of receptor/

en_t_ry_4_5

_'--

----l

Table 8. Examples of receptor-coupled modulation of native delayed rectifier currents (From 45-49-01) Receptor class

Descriptions and cross-references

Angiotensin II (see note 2 for clarification of effects on cardiac I K,s versus I K,f components)

45-49-02: Angiotensin II induces inhibition of I K in canine renal artery 'within 15 seconds' of application, an effect that is reversible upon washout78. 45-49-03: Angiotensin II (O.l-lOOM) induces substantial inhibition of delayed rectifier (and inward rectifier) K+ channel activities in rat and bovine adrenal zona glomerulosa (ZG) cells251 . In these (cell-attached patch-clamp) experiments, angiotensin II could not interact directly with the extracellular sides of the membranes, leading to the suggestion that the inhibitory effect was via an indirect (cytosolic) transduction path""ray. Functional note: Angiotensin II-induced ZG cell membrane depolarization following K+ channel inhibition is likely to activate voltagedependent Ca2+ channels, thus stimulating aldosterone secretion. 45-49-04: Application of angiotensin II (100 OM) induces a reversible inhibition of I Kv in isolated rabbit portal vein smooth muscle cells, an effect that appears consistent with Ca2+-independent protein kinase C (PKC) activation 72 . Note: A PKC isoenzyme-specific antibody panel used in this study was able to detect PKC alpha, epsilon and zeta isoenzymes (but not beta, gamma, delta and eta isoenzymes) in this preparation 72 . 45-49-05: Angiotensin II (lOOOM) elicits an AT2 receptor-mediated stimulation in delayed rectifier outward K+ current in neurones co-cultured from rat hypothalamus and brainstem. The stimulation effect is abolished by pre-treatment of cultures with pertussis toxin (PTxj 200ng/rnl) and by anti-Goi antibodies (other antibodies, such as anti-Goo, anti-Gq,ll were ineffectual). The angiotensin II effect could also be inhibited by the serine/threonine phosphatase inhibitor okadaic acid (l-lOrlM) or by anti-type 2A protein phosphatase (PP2A) antibodies, but not by the tyrosine phosphatase inhibitor sodium orthovanadate (1 mM)252,253. Later results 254 suggested that AT2 receptor modulation in this cell system increased the open probabilityt of a 56 pS delayed rectifier K+ channel.

{3-Adrenoceptors (see note 3) and a-adrenoceptors

45-49-06: Extensively studied in cardiac preparations (see note 1); in general IK amplitude is increased by {3-adrenergic agonists (for further details, see example

l"---e_n_t_ry_4_5

----'_

Table 8. Continued Receptor class

Descriptions and cross-references refs 2551'256 and listings under Protein phosphorylation, 45-32, Related sources and reviews, 45-56 and VLG K eag/elk/erg, entry 46 and VLG [K] minK, entry 54). 45-49-07: {1-Adrenergic stimulation enhances and

a-adrenergic stimulation has been reported to inhibit the 'ultrarapid' delayed rectifier K+ current (IKur ) in human atrial cells257 (see IKur under Subtype classifications, 45-06). These actions appear to be mediated by protein kinase A and protein kinase C, respectively. 45-49-08: Stimulation of {1-adrenoceptors in rabbit vascular smooth-muscle cells (derived from portal vein) leads to an enhancement of the I K component by a mechanism involving activation of adenylate cyclase and protein kinase A 70 . See also the review of functional modulation of I K in vascular smooth muscle by {1- adrenoceptors and their transducer/effector enzymes232 .

Basic fibroblast growth factor Endothelin

45-49-09: Reductions in cardiac delayed rectifier

current (see note 1) in response to basic fibroblast growth factor (BFGF) have been noted258 . 45-49-10: Inhibitory effects of endothelin on cardiac ventricular IK (see note 1) are mediated by the ET-l receptor subtype, phospholipase C and protein kinase C in rat pulmonary arterial myocytes 259 . 45-49-11: In cardiac myocytes (see note 1), the inhibitory effects of endothelin on I K and arachidonic acid on I A are additive, and both effects can be blocked by staurosporine26o . Endothelin has been shown to activate phospholipase A2 and release arachidonic acid in isolated rat hearts with inhibitory effects on IA (see ILG K AA [native], entry 45 and VLG K A-T [native], entry 44); endothelin also has a positive inotropic t effect in cardiac muscle, suggesting that endothelin increases Ca2+ influx or the amount of Ca2+ released from the sarcoplasmic reticulum (see below, ILG Ca Ca RyR-Caf, entry 17 and ILG Ca InsP3, entry 19).

Gustatory (tastant) receptors

45-49-12: In tongue epithelia, receptor-mediated

suppression of delayed rectifier current following application of the bitter tastant denatonium has been characterizedl44 - see also reviews on native ion channels (including K+ channels) in taste transduction mechanisms145-148.

_'---

e_n_try_4_5_

Table 8. Continued Receptor class

Descriptions and cross-references

Kappa opioid

45-49-13: Activation of kappa-opioid receptors on mossy fibre terminals in the hippocampus inhibits excitatory amino acid release. In CA3 pyramidal cells, presynaptic kappa-opioid receptors are coupled to activation of a Shaker-type native voltage-dependent potassium channel that can be inhibited by dendrotoxin (DTX) and mast cell degranulating peptide (MCDP)188. Note: An increase in presynaptic K+ conductance would repolarize action potentials faster, thereby limiting calcium influx and neurotransmitter release at the terrninal - see also the constitution of the 'DTX acceptor' in VLC K Kv-beta, entry 47 and VLC K Kv1-Shak, entry 48.

P2 purinoceptors

45-49-14: In guinea-pig cardiac atrial myocytes (see note 1), external application of ATP (50 JlM) moderately enhances lK (max. 2-fold). Although this effect appears to involve P2 -purinoceptors, the effect was demonstrated to be independent of protein kinase A or protein kinase C activation or intracellular free Ca2 + 261.

Parathyroid hormone

45-49-15: Effects of human parathyroid hormone (PTH) on cardiac ventricular myocytes (see note 1) include increases in peak amplitude of lK and the slow inward calcium current (lea)262. These results indicate that in ventricular muscle, hPTH prolongs the action potential duration (APD) and Q- T interval by a 'balance' of the prolongation effect on APD (by enhancing lea) and the shortening effect on APD (by enhancing lK).

Somatostatin

45-49-16: For effects of somatostatin on serine/ threonine phosphatase activities affecting voltagedependent potassium channels in native GH4 C 1 pituitary cells, see Protein phosphorylation, 45-32.

Vasoactive intestinal peptide

45-49-17: Vasoactive intestinal peptide (VIP, 1 JlM) inhibits phasic contractions (electrical slow waves) and tone of gastrointestinal smooth muscles. In canine proximal colon circular smooth muscle, VIP's action on electrical slow waves appers partly due to activation of a 4-AP-sensitive, delayed rectifier K+ channel67 (KDR1 , contributing to 'premature' slow wave repolarization, reduced Ca2+ entry, and inhibition of contractile force). Notably, VIP is ineffectual on two additionaI4-AP- and charybdotoxin-insensitive K+ channels (90 pS and <4pS) in this preparation67.

1'--_e_n_t_ry_4_5

_

Table 8. Continued Receptor class

Descriptions and cross-references

Vasopressin

45-49-18: In single guinea-pig cardiac ventricular myocytes (see note 1), arginine vasopressin (AVP, 0.01-

1 flM) has been reported to enhance IK in a concentration-dependent manney263. This effect can be abolished by the specific VI receptor antagonist OPC21268 (1 flM)263. VI receptor-dependent increases in I K were antagonized by inhibitors of protein kinase C (staurosporine, 1 nMi H-7, 10 flM). In isolated guinea-pig cardiac ventricular (papillary) muscles, AVP (0.1-3 flM) induced negative inotropy (i.e. decreased contractile force) in a concentration-dependent manner, an effect antagonized by OPC-21268 (10 flM) but not by a specific V2 receptor antagonist (OPC-31260, 10 flM)263.

Notes: 1. Many reports do not describe cardiac I K in terms of IK,r and IK,s (for meaning, see note 2) and thus coupling to specific receptor/transducer systems may be ambiguous. 2. Physiological concentrations of angiotensin IT appear to modulate two major outward potassium current components involved in cardiac repolarization, such that modulators of angiotensin IT may exhibit 'direct' cardiac electrophysiological effects264 : In guinea-pig cardiac ventricular myocytes, 'physiological' concentrations of angiotensin lIt (30 nM) increase tail current amplitudes of the rapid component IK,r by 13 ± 3 % following short (250 ms) depolarizing pulses to potentials> +IOmV. (IK,r is probably encoded by the h-erg gene - see VLC K eag/elk/erg, entry 46). In contrast, angiotensin IT (lOOnM) decreases tail current amplitudes of the slow component, IK,s by 19 ± 3% following long (5000 ms) depolarizing pulses (voltage independent over the range -10 to +SOmV)264. Note: IK,s has some similarities with currents seen following heterologous expression of the KvLQTl/minK/IsK gene products (for details, see VLC K minK, entry 54). 3. In 1972, the cardiac muscle delayed rectifier current (Ix - see Current designation, 45-04) was one of the first examples of cyclic AMP-dependent ion channel modulation in response to ,B-adrenergic agonists265 . Monobutyryl cAMP as well as the phosphodiesterase inhibitors theophylline and RO 7-2956 mimic the actions of adrenaline in increasing I K in cardiac Purkinje fibres. Intracellular injection of the catalytic subunit of protein kinase A (PKA) into ventricular muscle cells can also mimic the effects of adrenaline266 .

transducer interactions might not take account of subunit heterogeneities, effects on multiple channel effectors and 'cell type or species-dependent' observations. See also notes relating lack of selectivity and interpretation of pharmacology applicable to native versus heterologous cell types at the head of the PHARMACOLOCY section under VLC K A-T [native}, entry 44.

_'---

e_n_try_4_S-----'

INFORMATION RETRIEVAL Due to the heterogeneity of subunits supporting 'delayed rectifier' currents, see also this field under VLC K eag/e1k/erg, entry 46, the VLC K Kv series, entries 47 to 51 and VLC {K} minK, entry 54. For cardiac IK channel reviews in particular, see entries 46 and 54 (as above).

Related sources and reviews Selected reviews including descriptions of native delayed rectifier currents 45-56-01: Earlier reviews (including I K ) of native voltage-dependent currents of vertebrate neurones (1986, ref. 267j 1991, ref. 268 )j K+ currents in motoneurones (1995 review); comparisons of native cardiac K+ currents and/or comparisons with cloned channels (see also entries cross-referenced at the head of this section/field) ion channels in myocardial disease269 and normal cardiac physiology (1996 reviews)176,177j cell swelling and ion-transport pathways in cardiac myocytes270j modulation of native K+ channels by antiarrhythmic and antihypertensive drugs (1992 review)271 j descriptions of membrane current properties in cardiac pacemaker tissue, including I K 272 (see also INR K/Na Ifhq, entry 34); mechanisms of K+ channel regulation (1996 review)273 j the roles of ion channels in an inherited heart disease, especially molecular genetics of the long QT syndrome (1996 review, see also entries 46, 54 and 55); roles of potassium channels in cerebral blood vessels (1995 review, including delayed rectifiers)8o and airway smooth muscle 58j native ion channels (including K+ channels) in taste transduction mechanisms145-148j TEA+ and QA block227j diversity and ubiquity of K+ channels (1988 review, predominantly describing native channels)l.

Book references Adams, D. and Nonner, W. (1990) Voltage-dependent potassium channels: gating, permeation and block. In Potassium Channels: Structure, Classification, Function and Therapeutic Potential (ed. N.S. Cook), pp.40-69. Ellis Horwood, Chichester. Halliwell, J. (1990) K+ channels in the central nervous system. In Potassium Channels: Structure, Classification, Function and Therapeutic Potential (ed. N.S. Cook), pp.348-81. Ellis Horwood, Chichester. Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA.

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en_t_ry_4_5_1

K+ channels encoded by

VLG K eag/elk/erg Edward C. Conley

genes related to Drosophila eag (ether-a-go-go) (gene subfamilies eag, elk, erg) Entry 46

Note on coverage: Although entries are largely concerned with the properties of ion channels expressed in vertebrates, the central importance of studies in Drosophila for defining the eag/elk/erg gene subfamilies and their functional relationships justifies their integration into several fields of this entry. The symbols used to denote properties reported for different species and gene subfamiles are shown in Table 1 under Gene family, 46-05.

NOMENCLATURES

Abstract/general description 46-01-01: Drosophila ether-a-go-go (eag) originally referred to behavioural phenotypes that eag mutant flies displayed under ether anaesthesia, characterized by an abnormal leg-shaking phenotype. These effects were subsequently linked to increases in neuronal excitability and neurotransmitter release at the neuromuscular junction consistent wth defects in K+ channel conductances (see Phenotypic expression, 46-14). Chromosome walking and chromosome jumping techniques were originally used to identify cDNAs that corresponded to the eag transcript (see Isolation probe, 46-12). These cDNAs (spanning >35 kb at the genomic level) included the mutant sequences associated with four different eag mutant alleles (see Phenotypic expression, 46-14). A hybridization t probe based on the hydrophobic core sequences of Drosophila eag was subsequently used to retrieve further cDNA clones encoding eag and those encoding elk, the eag-like K+ channel from Drosophila head-specific cDNA libraries 1 . The isolation of cDNA clones encoding the Drosophila Eag K+ channel polypeptide (see Isolation probe, 46-12) formed a prototype for an extended gene family as a distinct branch of the voltage-gated channel gene superfamily. Subsequently isolated members have been placed in eag, elk and erg subfamilies based on their sequence similarity. Isolation of other clones (or species homologues of existing clones) in the gene family may clarify and extend the present classification. The eag designation has been retained for mammalian species homologues (see Table 1 under Gene family, 46-05). 46-01-02: Presently known eag, elk and erg subfamily members share at least 470/0 amino acid identity in their hydrophobic cores with all sequences possessing a segment homologous to a cyclic nucleotide-binding domain. Additional localized sequence comparisons indicate that members of this family are distantly related to certain 'six transmembrane domain' inwardly rectifying K+ channels of plant origin (e.g. clones KAT1, AKT1 and KST1, see descriptions under Domain conservation, 46-28). 46-01-03: Higher mammalian erg genes have undergone much functional adaptation from their likely common ancestral origin with present-day voltage-gated channel superfamily genes. For example, an erg subfamily cDNA originally isolated from a hippocampal cDNA library and designated

_ _ _ _ _ _ _ _ _ _.

en_t_ry_4_6_

as the human ~ag-!elated gene (h-erg or HERG (see Table 1 and Isolation probe, 46-12) displays strong K+ inward rectification following heterologous expression in oocytes due to the operation of a novel inactivation mechanism which prevents K+ efflux during depolarization2 (see Fig. 6 under Activation, 46-33). Otherwise, the biophysical properties of human erg subfamily channels expressed in Xenopus oocytes are very similar to the rapidly activating IK,r component in native cardiac myocytes (associated with initiation of terminal repolarization in the cardiac action potential). Unique fast-inactivation properties of erg subfamily channels which result in their characteristic inwardly rectifying property are described under Inactivation, 46-37. 46-01-04: Mutations in HERG are associated with the chromosome 7-linked form of long QT (LQT) syndrome. Gene linkage and physical mapping analyses have co-localized the type 2 LQT syndrome pedigree marker LQT2 and HERG to chromosome locus 7q35-36 (ibid. 3 ). Single-strand conformation polymorphism t (SSCP) and direct sequence analyses in type 2 LQT families have revealed a spectrum of mutations in HERG, including, to date, two intragenic deletions, one splice-donor mutation and three missense mutations (for further details and description of delayed repolarization arrhythmias, see Phenotypic predisposing to life-threatening card~iac expression, 46-14 and Chromosome location, 46-18). 46-01-05: The beneficial effects of /3-blockade in preventing development of arrhythmias may be due to cAMP-dependent regulation of channel subunits other than HERG, important candidates being KvLQT1, voltagegated Na+ channels and voltage-gated Ca2 + channels. Different LQT2 mutations may affect HERG function in different ways, and there appears to be a role for additional subunits in native HERG function. 46-01-06: Rat eag mRNA is expressed at high levels in brain, with no apparent signal outside the CNS, at least by relatively low-sensitivity Northern blots. In situ hybridization studies indicate that rat eag mRNA is most prominently expressed in the hippocampal formation and cerebellum. The highest levels of eag transcripts were detected in the granular cells of the dentate gyrus, in the CA3 pyramidal cells of the hippocampal formation and in the cerebellar granule cells. Rat eag is expressed at relatively low levels in the hippocampal CAl field, the caudate putamen, in the granule cell layer of the olfactory bulb and some neocortical layers. By similar techniques, erg transcripts show a 'wide tissue distribution' and are 'abundant' in heart as well as brain, retina, thymus, and adrenal gland and detectable in skeletal muscle, lung, and cornea mRNA pools (see mRNA distribution, 46-13). 46-01-07: In neurones, integrin-mediated neurite outgrowth in neuroblastoma cells appears to depend on the activation of HERG channels in situ (see Receptor/transducer interactions, 46-49). SH-SY5Y human neuroblastoma cells exposed to substrate-bound laminin or soluble laminin induce an early (5-15 min) activation of the HERG-like 1:lR in these cells (for further details, see Developmental regulation, 46-11). Specific inhibition of heterologously expressed rat eag channels occurs at the onset of maturation in Xenopus oocytes. This inhibition, indicated by a marked reduction in r-eag current

l_e_n_t_ry_4_6

_

amplitude, is mimicked when the mitosis-promoting factor (a complex of cyclin Band p34 (cdc2)) is injected into oocytes. 46-01-08: Drosophila eag (1174 amino acids) and elk (1284 amino acids)

encode relatively large polypeptides in comparison to those encoded by the Shaker family (approx. double their 'typical' length of 500-600 aa, excepting those encoded by Shab, 924 aa and drk, 853 aa). The eag and elk open reading frames are also considerably longer than those 'typical' of cyclic nucleotide-gated channels. Within the h-erg locus, several intron/ exon junctions have been characterized (see Gene organization, 46-20). 46-01-09: Noted similarities of eag/elk/erg family members with CNG and plant inward rectifier family sequences are described in Table 4 under Domain conservation, 46-28. Hydropathy analyses of eag, elk and erg subfamily polypeptides suggest a '81-86 + H5' transmembrane domain arrangement typical of those found in the voltage-gated K+ channel superfamily. The eag/elk/erg family proteins contain a positively charged amphipathic segment S4 and a P-region (ibid.). An N-glycosylation motif NX[S/T] is shared by the eag/elk/erg subfamilies located 12-17 aa upstream of the respective pore regions (see Sequence motifs, 46-24). A cross-family consensus site for phosphorylation by protein kinase C exists 2-4 aa downstream of transmembrane domain S6 (see Protein phosphorylation, 46-32).

46-01-10: A subunit interaction domain designated as NAB (HERG) has been localized at the hydrophilic cytoplasmic N-terminus of HERG (analogous to similar domains in the Shaker subfamily - see entry 48). NAB (HERG) is able to form a tetramer in the absence of the remainder of the HERG protein. Significantly, the HERG Ll1261 mutation associated with one long Q- T syndrome kindred (see Table 2 under Phenotypic expression, 46-14) results in a truncated protein that contains the subunit interaction domain and can explain observed effects on decreased channel expression. The minK protein (entry 54, initially proposed to co-assemble with KvLQTl to conduct native cardiac IK,s, ibid.) has also been shown to enhance functional expression of HERG-K+ currents in CHO cell co-transfection studies. 46-01-11: Injection of either Drosophila or rat eag mRNA into Xenopus oocytes gives rise to voltage-activated, non-inactivating outward K+currents of the 'delayed rectifier' type. In comparison to Drosophila eag, rat eag currents show a marked variation in activation kinetics according to the voltage pulse protocol used to elicit outward current (see Activation, 4633). Differences and similarities between vertebrate eag and erg subfamily channel currents, together with distinctions between heterologously expressed HERG currents, native inwardly rectifying cardiac IK,r and native IK1 are also summarized under Activation, 46-33 and Current-voltage relation, 46-35 and Inactivation, 46-37. The resemblance of h-erg inactivation to IC-type' inactivation (characterized as a 'slow inactivation' mechanism in other K+ channels such as Kv1.3 and involving a 'conformational switch' in the outer mouth of the channel) is described under Inactivation, 46-37.

_1-...

e_n_try_4_6_

46-01-12: Despite marked differences in the characteristics of eag and erg channel currents, initial analysis of the voltage-dependent gating transitions in these subfamilies is characteristic of most channels in the 54containing superfamily of ion channels. A schematic model of HERG channel gating modified from those developed for Sh channels is cited under Kinetic model, 46-38. 46-01-13: Some variation in ionic selectivity characteristics have been reported for heterologously expressed IJrosophila and mammalian eag and erg channel homologues (summarized ~in Table 5 under Selectivity, 46-40). Alignment of sequences corresponding to the pore-forming (P-domain) regions of several K+ -selective channels has revealed some distinguishing features in the eag/elk/erg subfamilies (summarized in Fig. 5, this entry). 46-01-14: A large number of studies have described block of native IK,r and heterologously expressed h-erg channels by class ill antiarrhythmic drugs, that act by slowing cardiac repolarization (phase 3) and prolong the duration of action potentials (see Blockers, 46-43). Agents that have been extensively investigated include E-4031, MK-499, sotalol, dofetilide, clofilium, quinidine, sematilide, L691,121 and WIN 61773-2 (ibid.). [3H]Dofetilide binds with high affinity to sites associated with the guinea-pig cardiac IK,r channel (see Ligands, 46-47). Quinidine and sotalol have been associated with an lacquired' or ldrug-induced' form of long QT syndrome (see Blockers, 46-43). Many other non-selective blockers of IK,r have been described (ibid.). 46-01-15: Rat eag channels expressed within HEK-293 cells have been described as 'rapidly and reversibly inhibited' by rises in [Ca2+]i between 30 and 300nM (mean IC so of 67nM in an I/O patch)4. Generation of intracellular calcium oscillations following muscarinic receptor activation appeared to induce a synchronous inhibition of r-eag-mediated outward current. These and other data led to the conclusion that r-eag channels are 'voltageactivated, calcium-inhibitable' channels, with the Ca2 +-inhibitory effects independent of calcium-dependent kinases and phosphatases4. There is some discussion relating these properties in rat eag to similarities and differences to native M-current (see entry 53 and notes 4, 5 and 6 in Table 6, this entry). 46-01-16: In voltage-clamped oocytes expressing Drosophila eag, bath application of membrane-permeable 8-Br-cAMP (8-bromoadenosine 3'5'cyclic monophosphate, 1 mM) or 8-Br-cGMP (8-bromoguanosine 3'5'-cyclic monophosphate, 1 mM) increase the amplitude of eag outward currents, while thresholds of activation are shifted to more negative potentials. Rapid application of cAMP (2 mM) to inside-out patches produces significant increases in outward current amplitudeS. Similar effects cannot be induced by cGMP in inside-out patches, indicating a direct modulation' of Drosophila eag channels by binding of cAMP to the channel protein. Cyclic nucleotide binding (see entries 21 and 22) has been excluded from a role in Drosophila, mouse or rat eag channel gating although a role for cyclic nucleotide-dependent phosphomodulation appears likely (see Channel modulation, 46-44). Notably, heterologously expressed HERG channel activity is unaffected by cyclic nucleotides (ibid.). I

lL..--e_n_t_ry_4_6

_

46-01-17: Extracellular K+ has a profound effect on increasing the amplitude of HERG current; under conditions of lowered extracellular K+ (i.e. in modest serum hypokalaemia t ) the effects of IK,r blockers tend to be exaggerated, lengthening of cardiac action potentials, and inleading to ~excessive' duction of torsade de pointes. Elevation of serum [K+] in hypokalaemic patients receiving medications which block IK,r or in individuals with the chromosome 7-linked LQT syndrome has been demonstrated to correct abnormalities of repolarization duration, T-wave morphology, QT/RR slope (slope of the relation between QT interval and cycle length), and QT dispersion in patients with chromosome 7-linked LQT syndrome (see Channel modulation, 46-44). Extracellular Mg2+ (at physiological concentrations) 'dramatically slows' activation of r-eag channels expressed in oocytes in a dose- and voltage-dependent manner (ibid.).

Category (sortcode) 46-02-01: VLG K eag/elk/erg, i.e. voltage-gated potassium channels encoded by genes in the subfamilies eag, elk and erg. The eag-related family is a distinct branch of the voltage-gated channel gene superfamily (for clarification, see Fig. 1 under Gene family, 46-05).

Information sorting/retrieval aided by designated gene family nomenclatures 46-02-02: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: eag or elk or erg. If a systematic nomenclature is established for the eag-related gene family, than this may take preference (see this field in other entries for examples).

Channel designation Designations distinguishing eag family member properties 46-03-01: At the time of compilation, no universal nomenclature has been proposed for eag, elk or erg channels. For convenience, properties reported for different species homologues of these subfamilies are designated in this entry by an underlined prefix indicating the species/subfamily combination (e.g. d-eag:, m-eag:, r-eag:, d-elk:, h-erg: etc. (see Table 1 under Gene family, 46-05). Note: Strictly, the capitalized name 'HERG' has been used to specify the gene encoding human erg (ether-a-go-go-related) subfamily channels (human eag-related gene, ibid.); for this reason references to HERG are italicized within this entry.

Current designation 46-04-01: d-eag: Native Drosophila currents 'affected by' mutations in eag are briefly described under Protein interactions, 46-31. h-erg: Evidence suggesting the human eag-related gene (HERG) encodes the channel protein subunit underlying human cardiac rapidly-activating delayed rectifier ~+ current IK,r (designated with the comma between the K and r in these entries) is

_ entry 46

'------------

summarized under Phenotypic expression, 46-14). In native cardiac ventricular preparations, IK,r has also been designated as I-Kr and/or 'E4031-sensitive repolarization current' (e.g. ref. 76, see also Blockers, 46-43). Compare also the distinctive properties of the Islow-activating' delayed rectifier current component in heart designated as IK,s under KvLQT1 in VLC [K] minK, entry 54.

Gene family eag, elk and erg define an extended family of K+ channel genes 46-05-01: The isolation of eDNA clones encoding the Drosophila Eag K+ channel polypeptide (see Isolation probe, 46-12) formed a prototype for an extended gene family, with subsequently isolated members placed in eag, elk and erg subfamilies based on their sequence similarity. Isolation of other clones (or species homologues of existing clones) in the gene family may clarify and extend the present classification. Table 1 therefore lists a partial nomenclature for genes related to Eag. Presently known eag, elk and erg subfamily members share at least 470/0 amino acid identity in their Table 1. Incomplete gene family nomenclature and Inon-systematic' names for ion channel genes/protein subunits showing relatedness to eag. cDNA Iclone' names (isolates) in use are indicated in bold. (From 46-05-01)

Subfamily Drosophila (see note 2) isoforms Eag

eag6 Denoted in this entry as d-eag: (see note1)

Elk

elk1 Denoted in this entry as d-elk: (see note 1)

Erg

Human isoforms

Rat isoforms

Mouse isoforms

rat eag7 m_eag1?8 Denoted in this Denoted in entry as this entry as r-eag: (see m-eag: (see note 1) note 1)

h-erg1 also in capitals, i.e. HERG Denoted in this entry as h-erg: (see noteland Channel designation, 46-03)

Notes: 1. The designation used to indicate species and subfamily data is underlined, which mostly appears at the beginning of paragraphs within this entry. 2. Amino acid sequence alignments for these channels are given in Fig. 2.

entry46

_

I" " ' - - - - - - - - - -

hydrophobic cores with all sequences possessing a segment homologous to a cyclic nucleotide-binding domain (see ref. 1, Domain conservation, 46-28, ILG CAT cAM~ entry 22 and ILG CAT cGM~ entry 23). Additional localized sequence comparisons indicate that members of this family are distantly related to certain 'six transmembrane domain' inwardly rectifying K+ channels of plant origin (e.g. clones KAT1, AKT1 and KST1, see descriptions under Domain conservation, 46-28).

Functional diversity within the eag/elk/erg gene subfamilies 46-05-02: Both Drosophila eag and the mouse species homologue (m-eag) encode voltage-gated, outwardly rectifying K+ channelss"s. In Drosophila, the eag gene encodes a polypeptide homologous to, but distinct from, the Shaker K+ channel subunits 9; electrophysiological studies have revealed allele t -specific effects of eag on four identified K+ currents in Drosophila larval muscles lo (for details, see Phenotypic expression, 46-14 and Protein interactions, 46-31).

An extended gene family tree including the Eag, Elk and Erg channel polypeptides 46-05-03: Estimates of amino acid identities and a dendrogram t representing the gene family relationships between known polypeptide sequences of eag, elk and erg subfamily members appears in Fig. 1 compared to various voltage-gated and cyclic-nucleotide-gated channel families. The dendrogram depicted in Fig. Ib indicates a common ancestral origin for present-day voltage-gated channel superfamily genes (see ref. 11). Note: Sequence alignments of eag, elk and erg subfamily members predicting a similar overall structure including a central hydrophobic core and substantial similarity in a putative cyclic nucleotide-binding domain are shown under Encoding, 46-19 (for descriptions of these features, see Domain conservation, 46-28).

Adaptive radiation of human erg genes 46-05-04: An Erg subfamily cDNA isolated from a hippocampal cDNA library and designated as the human ~ag-!elated gene (h-erg or HERG (see Table 1 and Isolation probe, 46-12) displays -strong K+-inward rectification following heterologous expression in oocytes under certain conditions2 (see Fig. 6 under Activation, 46-33). This characteristic is due to the operation of a novel inactivation mechanism which prevents K+ efflux during depolarization (for details, see Inactivation, 46-37). h-erg does not appear to be the human homologue of Drosophila eag - the latter shares only about 500/0 amino acid identity with h-erg and lacks the inactivation mechanism described above. Note: Isolation of a mammalian elk homologue has been noted in ref. 1; however no reports on the functional expression of elk subfamily proteins were found during compilation of this entry.

Subtype classifications 46-06-01: d-eag: Drosophila eag channels in Xenopus oocytes display properties characteristic of delayed rectifier-type voltage-gated K+ channels

II

100

eag

slo Shab

Shal Shaw

11 18

"42 47 41 100

13 19 39

43 100

14 21

46 100

11 19

100

14

100

68

100

100

11

14

13

11

15

ShB

cGMP

cAMP

AKT1

14

14

14

11

18

24

25

100

KAT1

13

12

11

11

19

24

23

62

100

elk

12

13

11

14

17

20

25

26

24

100

h-erg

13

17

15

15

16

23

24

27

24

49

100

m-eag

12

12

13

16

13

25

27

27

26

47

49

100

eag

10

13

12

versus

Shaw

Shal

ShB

17

slo

Shab

20

24

26

15

KAT1 AKT1 cAMP cGMP

25

elk

49

49

65

m-eag h-erg

(a) Amino acid identities, percent

I

I

I

I

I

I

~

I

I....-

I

n

I (b) Dendrogram

I

Elk

H-Erg

M-Eag

Eag

KAT1

AKT1

cAMP

cGMP

Sio

Shab

ShB

Shal

Shaw

I

I

I~

0\

I~~

("I)

Figure 1. (a) Percentage amino acid identities limited to the hydrophobic core (Sl-S6 domains) in representative members of eag, elk and erg subfamilies are shown in comparison to voltage- and cyclic nucleotide-gated channels. Sequences used for computation of these values included the eag family members eag, m-eag, h-eag and elk 1, the plant inward rectifiers KAT1 and AKT1 (see Domain conservation, 46-28), the rat olfactory cGMP-gated cation channel (CNG-2, see ILG CAT cAM~ entry 21), the bovine retinal cGMP-gated cation channel (CNG-1, see ILG CAT cGM~ entry 22), the Drosophila slo Ca 2+-activated K+ channel (dslo, see ILG K Ca, entry 27) and the voltage-gated K+ channel family members Kv4, Shaw, RK5, Shal, RCK1, ShB, DRK1, Shab, IK8 and K13 (see Database listings, field 53, under the VLG K Kv series, entries 48 to 52). (b) Dendrogram showing relatedness between the representatives of the voltage-gated gene superfamily members described in (a). Horizontal branch lengths are inversely proportional to the similarity between the sequences. Note: Multiple sequence comparisons when limited to the hydrophobic core (Sl-S6 domains) of these channels (as used to calculate the similarities here) indicate that the plant inwardly rectifying K+ channels AKT1 and KAT1 are more closely related to the eag family than they are to the Shaker family (for details, see Domain conservation, 46-28). (All data taken from Warmke and Ganetzky (1994) Proc Natl Acad Sci USA 91: 3438-42 using sequence comparison algorithms defined therein.) (From 46-05-03)

II

~

M"

~ ~

0'\

_L--

e_n_try_4_6_1

(see Voltage sensitivity, 46-42). These channels also show some properties reminiscent of some intracellular ligand-gated channels in that current amplitudes are 'directly' modulated by intracellular cyclic AMP (see Channel modulation, 46-44, JLG key facts, entry 14 and JLG CAT cAM~ entry 21). 46-06-02: h-erg: The biophysical properties of human erg subfamily channels expressed in Xenopus oocytes are very similar to the rapidly activating delayed rectifier current IK,r in cardiac myocytes 12. The unique properties of erg subfamily channels, particularly those mechanisms that can give rise to inwardly rectifying K+ currents, are described in the ELECTROPHYSIOLOGY section (fields 33 to 42).

Trivial names Retention of trivial name 46-07-01: d-eag: Drosophila ether-a-go-go originally referred to behavioural phenotypes of eag mutants shown under ether anaesthesia; the eag designation has been retained for mammalian species homologues (see also Gene family, 46-05, Channel designation, 46-03 and Phenotypic expression 46-14).

EXPRESSION

Cell-type expression index See Isolation probe, 46-12 and mRNA distribution, 46-13.

Studies characterizing IK,r in native cells 46-08-01: h-erg: The rapidly activating 'delayed rectifier' current IK,r (thought to be dependent on channel protein subunits encoded by HERG 12 ) has been characterized in various cardiac preparations including ventricular myocytes of guinea-pig13 and cat14, cultured atrial myocytes (cell line AT1)15 and in nodal cells of rabbit heart16 (for further details on IK,r, see Phenotypic expression, 46-14).

Cloning resource Stable HERG-like channel currents in cardiac and neurone-derived cell lines 46-10-01: cDNA and genomic library resources used for isolation of presently known eagfelkferg sequences are referred to under Isolation probe, 46-12 and references under Database listings, 46-53.. Note that atrial tumour myocytes derived from transgenic mice (AT-l cells) possess a current with similar characteristics to native cardiac IK,r 17. A constitutively expressed inwardly rectifying current described as 'HERG-type' has been extensively characterized in a rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid cell line (F-ll) (see also Blockers, 46-43).

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Developmental regulation Mitosis-promoting factor-mediated suppression of rat eag current in oocytes 46-11-01: Specific inhibition of heterologously expressed rat eag channels occurs at the onset of maturation in Xenopus oocytes. This inhibition, indicated by a marked reduction in r-eag current amplitude, is mimicked when the mitosis-promoting factor (a complex of cyclin Band p34 (cdc2)) is injected into oocytes 18 .

Integrin-mediated activation of HERG-like current associated with neurite extension 46-11-02: SH-SY5Y human neuroblastoma cells exposed to substrate-bound laminin or soluble laminin induce an early (5-15 min) activation of the HERG-like hR in these cells 19. In cells adherent to substrate-bound laminin, 1IR potentiation can persist for 90-120min accompanied by a similar, but transient, increase in cell leak conductance (GL ), shifting the resting potential from -12 to nearly -30 m V over approx. 120 min. In cells adherent to polylysine, exposure to soluble laminin induces hyperpolarization through a transient potentiation in 1m, without any effect on leak conductance 19. Notably, these effects of substrate-bound and soluble laminin can be mimicked by antibodies raised against the integrin {31 subunit (i.e. culture-dish substrate-bound antibodies simultaneously activate the hR and GL , whereas in soluble form they only activate 1m). Significantly, cells adherent to substrate-bound laminin undergo neurite extension, which can be inhibited by E-4031 and Cs+ (see Blockers, 46-43). Notes: 1. Integrin-independent substrates such as polylysine or bovine serum albumin have no effect on these cells. 2. Earlier studies showed activation of a HERG-like 1IR upon integrin-mediated adhesion to fibronectin or vitronectin in a subclone of a murine neuroblastoma cellline2o.

Isolation probe De novo sequence cloning of Drosophila eag 46-12-01: d-eag: Chromosome walkingt and chromosome jumping t techniques were originally used to identify cDNAs that corresponded to the eag transcript21 . These cDNAs (spanning >35 kb at the genomic level) included the mutant sequences associated with four different eag mutant alleles t (see Phenotypic expression, 46-14). A hybridization t probe based on the hydrophobic core sequences of Drosophila eag was subsequently used to retrieve further cDNA clones encoding eag and those encoding elk, the eag-like K+ channel from Drosophila head-specific cDNA libraries 1 .

Probes used to retrieve mammalian eag/erg subfamily homologues 46-12-02: m-eag/h-erg/r-eag: Degeneratet primerst based on the Drosophila eag peptide sequences ACIWY, TYCDL, ILGKGD were initially used to PCRamplify sequences at a low annealing temperature (42°C) using randomprimed cDNA from mouse skeletal muscle as template; products corresponding to putative eag homologues were identified by cross-hybridization to a

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_6--'

Drosophila elk probe by low-stringency cross-hybridization. Fragments within the range 750-950 bp were recovered from agarose gels and were reamplified with sense and antisense degenerate primers based on ACIWY and ILGKGD (see above), subcloned and sequenced. Low-stringency hybridization using Drosophila elk-hybridizing fragments did not result in retrieval of elk homologues from a mouse brain cDNA libraryl. Additional PCR analysis and hybridization screening of the same library isolated a sequence representing mouse eag (m-eag)l, which was subsequently used to probe a human hippocampal cDNA library to isolate a related sequence defining a third branch of the eag family, designated human eag-related gene (h-erg) (see Gene family, 46-05). The extreme 5' end of the h-erg sequence reported in ref. 1 was deduced from a composite of genomic and cDNA sequences (see 'Incomplete' cDNA sequences under Resource D - Diagnostic tests). In separate studies, a Drosophila eag cDNA probe (nt 1155-2329) was used in low-stringency t hybridizations to isolate a rat genomic library clone encompassing the predicted 5' and 3' terminal sequences of rat eag (r-eag)7. PCR screening of a rat cerebellum cDNA libraries retrieved two overlapping cDNA clones, yielding a combined cDNA of 4.6 kb and predicting an open reading frame of 962aa 7. Rat eag sequences were also amplified from hippocampal cDNA libraries.

mRNA distribution High-level expression of rat eag mRNA in brain 46-13-01: r-eag: Rat eag mRNA has been reported to be 'predominantly expressed'inthe central nervous system 7 with no apparent signal on Northern t blots of poly(A)+ RNA derived from rat heart, spleen, lung, liver, skeletal muscle, kidney and testes. In situ hybridization using 47-mer (nt 1738-1691) and 49-mer (nt 3126-3077) oligonucleotide probes 7 show that rat eag mRNA is most prominently expressed in the hippocampal formation and cerebellum. The highest levels of eag transcripts were detected in the granular cells of the dentate gyms, in the CA3 pyramidal cells of the hippocampal formation and in the cerebellar granule cells. Rat eag is expressed at relatively lower levels in the hippocampal CAl field, the caudate putamen, in the granule cell layer of the olfa.:tory bulb and some neocortical layers7. tHigh abundance and wide distribution' of h-erg transcripts 46-13-02: h-erg: In Northern blot analyses, a PCR probe (HERG nt 679-2239) shows strongest hybridization to human heart mRNAs (two bands, see Transcript size, 46-17), with weak signals in brain, liver and pancreas3 . RNAase protection assays using eDNA fragment probes of erg homologues from guinea-pig, rabbit, human, dog and rat have confirmed that erg message is 'expressed uniformly' throughout the heart of all five species22 . By these techniques, erg transcripts show a 'wide tissue distribution' and are 'abundant' in heart (estimated at '50% more abundant' than Kv4.3 message - see entry 51) as well as brain" retina, thymus, and adrenal gland and detectable in skeletal muscle, lung, and cornea mRNA pools22.

l_e_n_t_ry_4_6

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Phenotypic expression Background - ether-a-go-go (eag) mutants exhibit thyperexcitability' phenotypes 46-14-01: d-eag: Drosophila carrying the eag mutation display an abnormal legshaking phenotype23 . These effects were subsequently linked to increases in neuronal excitability and neurotransmitter release at the neuromuscular junction consistent wth defects in K+ channel conductances24~25. The mutations designated eag 1, eag4PM , eagX6 and eag24 reduce different currents in larval muscles, where they interact synergistically with Shaker mutations (see VLG K Kv1-Shak, entry 48) to enhance the behavioural and physiological phenotypes. Double mutants such as eaglSh S , eag 1Sh 120 , eag4PMSh120, eag24 Sh s and eag24 Sh 120 have a Ivigorous' leg-shaking phenotype under anaesthesia, sometimes coupled with a 'wings-down' phenotype26~27. These mutants are also characterized by high-frequency, spontaneous excitatory junction potentials (EJPs) of high amplitude at neuromuscular junctions24~28, with spontaneous discharges of EJPs or action potentials in adult flight muscles27. Transmitter release persists for an order of magnitude longer in eag Sh double mutants than in either single mutant, resulting in the characteristic 'plateau-shaped' synaptic potentials and long trains of action potentials in motor axons. For the types of current reduced in eag mutants, see Protein interactions, 46-31. Notes: 1. Roles for cAMP-modulation of eag-type channels in controlling synaptic efficiency in the central and peripheral nervous systems have been discussed5 (see Channel modulation, 46-44). 2. The possible role of calcium/calmodulin-dependent protein kinase II (CaMKII) in influencing synaptic plasticity phenotypes by functional modulation of Drosophila Eag!9 are summarized under Protein phosphorylation, 46-32. 3. The resemblance between the Drosophila EAG current and the mammalian M-current (entry 53) is briefly discussed under [Ca2+h ions under Blockers, 46-43.

HERG encodes the potassium channel underlying IK,r in heart 46-14-02: h-erg: Following its heterologous expression in Xenopus oocytes, the biophysical properties of the human eag-related gene product (HERG) have been reported12 as 'nearly identical' to the rapidly activating 'delayed rectifier' K+ current (IK,r) described in native in cardiac myocytes13- 16. IK,r is known to have an important role in the initiation of repolarization during cardiac action potentials (see also Phenotypic expression under INR K native, 32-14). Despite its name, the native cardiac delayed rectifier contributes exhibits profound inward rectification (see ref.3D and Tseng, 1995, under Related sources and reviews, 46-56). These similarities between HERG-induced current and IK,r extend to their strong inward rectification t properties due to operation of a novel inactivation mechanism limiting K+ efflux during depolarization (see Inactivation, 4637), activation by extracellular K+ (see Channel modulation, 46-44), blockage by lanthanum and cobalt ions (see Blockers, 46-43). Comparative note: The inward rectification displayed by IK,r is distinguishable from that induced by the'classical' I K1 inward rectifier, which contributes to the final phase of ventricular repolarization (for clarification, see Activation, 46-33 and associated figures under Phenotypic expression of INR K [native], 32-14).

II

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e_n_t_ry_46_ _1

Mutations in HERG are associated with the chromosome 7-1inked form of long QT syndrome 46-14-03: h-erg: Many studies aimed at identifying the molecular bases of human cardiac arrhythmias t have focused on the incidence of long QT (LQT) syndrome t, a relatively rare disorder that is characterized by prolongation of the QT interval f on electrocardiograms, blackouts (syncopall attacks), seizures t and sudden death (typically following a ventricular arrhythmia, torsade de pointes t - see also next paragraph). Diagnostic criteria for the various forms of LQT syndrome have been summarized31 ; these make important distinctions between congenital (familial) and sporadic t forms of the disorder (see also the reviews/commentaries in refs32-34). Gene markers t which segregate t with various forms of the disorder (within LQT pedigrees) have been mapped to chromosomes Ilp15.5 (LQT1), 7q35-36 (LQT2) and 3p21-24 (LQT3), (for details, see Chromosomal location, 46-18). Subsequent gene linkage and physical mapping analyses co-localized LQT2 and HERC to chromosome locus 7q35-36 (ibid. 3). Single-strand conformation polymorphism t (SSCP) and direct sequence analyses in LQT2 families have revealed. a spectrum of mutations in HERC, including two intragenic deletions, one splice-donor mutation and three missense mutations (for details, see Table 2). Strong expression of the HERC mRNA transcript in human heart (see mRNA distribution, 46-13) and the the loss of function in mutant channels (some by a dominant negative t effect that varies in severity), have led to the proposal that HERC is LQT2 and its expression provides a cellular basis for torsade de pointes3,35 (see paragraph 46-14-13). Comparative note: In separate studies, LQT3 has been linked to a three amino acid deletion in the seNSA gene encoding the human heart voltage-gated sodium channel Q subunit hHI (~KPQ, affecting the cytoplasmic linker between domains m' and IV). Heterologously expressed ~KPQ mutant channels show a small sustained inward current compared to the wild type, reflecting a defect in c.hannel inactivation36 (summarized in Phenotypic expression, Chromosomal location and Inactivation under VLC Na, 55-14, 55-18 and 55-37 respectively).

-

Delayed repolarization predisposing to life-threatening arrhythmias 46-14-13: h-erg: Mechanistic links between inherited forms of LQTs and the (more common) acquired forms of LQTs have been proposed by Sanguinetti et a1. 12 (see also Blockers, 46-43 and Channel modulation, 46-44). Direct support for hypotheses invoking ion channel mutation to explain certain forms of congenital LQTs comes from the equivalence of LQT2/HERG and LQT3/SCN5A (see paragraph 46-14-03, Tables 2 and 3). This 'myocellular hypothesis' is also indirectly supported by QT prolongation induced following pharmacological block of K+ channels in human and animal models (see ref.41 and Blockers, 46-43). Delays in myocyte repolarization (indicated by prolonged QT intervals) are known to induce reactivation of cardiac L-type Ca2+ channels (see VLC (~a, entry 42) resulting in secondary depolarizations42,43. In turn, these events have been suggested as a likely cellular mechanism for torsade de pointes, a sinusoidal twisting of the QRS axis around the isoelectric line on electrocardiograms44- 46 • Torsade de pointes can degenerate into ventricular fibrillation t prior to sudden death.

entry46

I-

_

- - - - - -

Table 2. Summary of gene rearrangements and point mutations at the HERG locus associated with the incidence of long QT syndrome3 . For genetic linkages to the incidence of different LQT phenotypes, see Chromosome location, 46-18 (From 46-14-03) Rearrangement or mutation

Description, incidence and functional implication (where known)

HERG/LQT2 intragenic deletions

46-14-04: h-erg: A panel of PCR primers designed to the published eDNA sequence of HERG (ref. l , see Database listings, 46-53) were used to identify three intronic sequences within the protein-coding region (see Gene organization, 46-20). sscpt analyses using selected primers identified an A to G polymorphism t within HERG (nt 1692 on the eDNA) and analysis of one pedigree (kindred 2287 or K2287) produced results consistent with a null allele t; in repeat experiments with flanking PCR primers, unaffected members of K2287 (and >200 other unaffected individuals) showed a single product of 170 bpi in LQTs-affected individuals of K2287, two products of 170 and 143 bp were detected. This feature indicated the presence of a 27bp deletion beginning at HERG nt position 1498 (M500-F508, predicted to disrupt the S3 putative membrane-spanning

~I500-F508

domain as indicated on the [PDTMj, Fig. 4).

~1261

46-14-05: h-erg: SSCP analysis in a different family (K2595) identified an aberrant conformert in LQTsaffected individuals which was not present in >200 unaffected individuals; sequencing of normal and aberrant conformers revealed a single base deletion at nt position 1261 (~1261, predicted to introduce a frameshift

mutation resulting in the introduction of a stop codon within 12 aa residues, as indicated on the [PDTMj, Fig. 4; this deletion has been determined to affect HERG intersubunit association and expression level (see ref. 37

under Protein interactions, 46-31). HERG/LQT2 point mutations N470D

A561V

46-14-06: h-erg: In K2596 an A to G substitution was identified at the eDNA nt position 1408 (N470D, i.e.

substitution of an aspartate residue for a conserved asparagine at codon 470, predicted to alter the S2 putative membrane-spanning domain as indicated on the [PDTMj, Fig. 4). 46-14-07: h-erg: Three aberrant SSCP conformers were identified in LQT-affected members of K1956, K2596 and K2015. Cloning and sequencing of normal and aberrant conformers from K1956 identified a C to T substitution at position 1682 (A561\l; i.e. substitution of valine for a highly conserved valine at codon 561, predicted to alter

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_6_

Table 2. Continued Rearrangement or mutation

Description, incidence and functional implication (where known)

the S5 putative membrane-spanning domain as indicated on the {PDTM}, Fig. 4). A561T

46-14-08: A missense G-to-A transition at position 1681 resulting in Ala561Thr in domain 85 is associated with a prolonged T wave of low amplitude on the surface ECG. Specifically, a distinctive biphasic T-wave pattern in the left precordial leads of all affected subjects has been associated with patients expressing the Ala561Thr mutation (regardless of age, gender and beta-blocking therapy)38.

I593R

46-14-09: A single nucleotide substitution of thymidine to guanine (TI961G, Ile593Arg) in the channel pore region associated with LQT syndrome has been reported39.

splice site error

46-14-10: h-erg: In K2015, a G to C substitution was identified, predicted to disrupt the splice-donor sequence of intron III (see Gene organization, 46-20) and in consequence, affecting the cyclic nucleotide-binding domain. None of the aberrant conformers found in LQTaffected individuals were found in >200 unaffected individuals. In a separate study, a G to A substitution producing Va1822Met: in the cyclic nucleotide-binding domain of HERG in LQT syndrome has also been characterized40 .

V822M

HERG/LQT2 de novo mutation (sporadic case; one kindred)

46-14-11: h-erg: An aberrant SSCP conformer present in one individual of K2269 (which was not identified in either parent or in >200 unaffected individuals) revealed a G-to-A substitution at nt 1882 (G628S, i. e. substitution of

a glycine residue for a highly conserved serine at codon 628, predicted to alter the pore-forming domain as

indicated on the {PDTM}, Fig. 4). The identification of this de novo mutation provided further evidence for the equivalence of HERG and LQT2. Predicted neuronal phenotypes

46-14-12: ComparativB note: HERG is also expressed in brain (see Isolation probe, 46-12) implying that mutant HERG channels may exert some (presently undefined) effect on neuronal function in LQT2-affected individuals. Notably, none of the presently identified LQT families show signs of congenital neural hearing loss (a finding associated with the rare autosomal recessive form of LQT) or other phenotypic abnormalities3. The spectrum of presently known mutations in HERG (this table, see also {PDTM}, Fig. 4) nlay alter channel function in different ways.

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Implications for tgene-specific' therapy of LQT syndrome 46-14-14: Initial studies showing differential responses of patients taking into account known LQT2 and LQT3 gene defects 47 have indicated that (i) LQT3 patients may be more likely to benefit from Na+ channel blockers and from cardiac pacing due to a higher risk of arrhythmia at slow heart rates; (ii) LQT2 patients appear to be at higher risk of developing syncope under stressful conditions because of the combined arrhythmogenic effect of catecholamines with the insufficient adaptation of Q-T interval when heart rate increases 47.

Earlier hypotheses for origin of LQT phenotypes: tautonomic imbalance' 46-14-15: Comparative note only: A second hypothesis for LQT phenotypes suggests that a predominance of left autonomic innervation causes abnormal cardiac repolarization and arrhythmias (see Schwartz et al., 1995, under Related sources and reviews, 46-56). This hypothesis is supported by (i) induction of arrhythmias in canine models following right stellate ganglionectomy and (ii) clinical observations which suggest that certain LQT patients respond favourably to ,B-adrenergic blockade and left stellate ganglionectomy procedures (ibid.). For further mechanisms linking altered autonomic activity and arrythmias in LQT, see Receptor/transducer interactions, 46-49 and Channel modulation, 46-44.

Transcript size Band doublets revealed by HERG probes may indicate further transcript diversity 46-17-01: h-erg: The relatively strong and selective expression of HERG in human heart poly(A)+ mRNA is detected as two hybridizing bands of 1"'V4.1 and 1"'V4.4kb by Northern t assays using a HERG peR probe (nt 679-2239)3. The size of either hybridizing band is consistent with the predicted size from the largest open reading frame of the HERG eDNA (see Encoding, 4619), but the different sizes may represent alternatively spliced t transcripts or the co-expression of closely related sequences3 .

SEQUENCE ANALYSES Note: The {PDTM} symbol denotes an illustrated feature on the channel protein domain topography model (Fig. 4).

Chromosomal location Mutations in HERG are associated with chromosome 7-linked forms of long QT syndrome 46-18-01: h-erg: The gene encoding the human ether-a-go-go-related gene (HERG) has been localized to human chromosome 7q35-36 by linkage analysist and physical mapping t3 . This finding placed HERG at the same position as LQT2, one of three genes associated with autosomal dominantt forms of the long QT (LQT) syndrome (see Phenotypic expression, 46-14). As summarized in Table 3 extensive genetic analysis in many LQTs

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Table 3. Comparative summary of genetic linkages to forms of inherited long QT syndrome (From 46-18-01) LQT form/gene

Chromosome localization and gene product where known

LQTl (=KVLQTl, comparative note only, see also VLG K minK, 54-14 and 54-31)

46-18-02: The dominant t LQT1 gene has been linked by positional cloningt to human chromosome Ilp15.5 and encodes a protein (KVLQTl) with the structural features of a voltage-gated K+ channel with approx. 30% sequence identity to Drosophila Shaker48 (for summary, see Phenotypic expression under VLG K Kv1-Shak, 48-14). Earlier studies reported a 'tight' linkage to a polymorphism t at the Harvey-ras gene in several LQT families at IlpI5.5 49,5o. H-ras was excluded as a candidate for LQTl by linkage51 and sequence analyses (cited as unpublished in ref.3). The primary sequence of KVLQTl (above) and linkage analysis 52 have also excluded mutations in other K+ channel genes on IIp as LQT1 candidates (including the KCNA4 gene encoding hKvl.4 and the KCNC1 product encoding hKv3.1 - for refs, see fields 48-18 and 50-18). To March 1995, seven autosonlal dominant LQT families have been linked to the Ilp15.5 marker3,49,5o,53 and mutations in the LQTl gene are the most common cause of inherited LQT (>500/0 of known incidences). For updates, see OMIM Entry: 192500 IVentricular Fibrillation With Prolonged QT Interval [Ward-Romano Syndrome; WRS; Long QT Syndrome, Type 1; LQT; LQT1}'.

LQT2 (=HERG, this entry)

46-18-03: The dominant LQT2 gene has been localized to human chromosome 7q35-36. Identification of (i) new LQT families all linking to the 7q35-36 marker; (ii) mapping of HERG to the same locus; (iii) demonstration of strong expression of HERG in human heart (see mRNA distribution, 46-13) and (i'1~ characterization of six mutations in HERG within LQT subjects have provided strong evidence for the equivalence of LQT2 and HERG3. A summary of HERG rearrangements and point mutations reported in this study is given in Table 2 under Phenotypic expression, 46-14. To March 1995, 14 autosomal dominant LQT families have been linked to the 7q35-36 marker3,49,50,53. For updates, see OMIM Entry: 152427 ILong QT Syndrome, Type 2 {LQT2;HERGJ'. 46-18-04: Comparative note: Other genes co-localizing

to 7q35-36, including C1CN1, encoding a skeletal muscle chloride channel and CHRM2, encoding cardiac muscarinic M2 receptors were excluded as LQT2

entry46

_

I' - - - - - - - - - Table 3. Continued LQT form/gene

Chromosome localization and gene product where known candidates following linkage analysis (Wang et a1., cited in ref. 3 ).

LQT3 (== SeNSA; comparative note only - see VLG Na, entry 55)

46-18-05: The dominant LQT3 gene has been linked by positional cloning to human chromosome 3p21-24. The incidence of LQT3 has been associated with a three amino acid deletion in the SCN5A gene encoding the human heart type V voltage-gated sodium channel Q subunit hHI (LlKPQ, affecting the cytoplasmic linker between domains ITI and IV). Heterologously expressed LlKPQ mutant channels show a small sustained inward current compared to the wild type, reflecting a defect in channel inactivation36 (for further details see Phenotypic expression, Chromosomal location and Inactivation under the entry VLC Na, 55-14, 55-18 and 55-37 respectively). To March 1995, three autosomal dominant LQT families have been linked to the 3p21-24 marker3 ,49,5o,53. For updates, see OMIM Entry: 600163 'Long QT Syndrome, Type 3 [LQT3; SCN5A)'.

Other forms, including LQT4

46-18-06: Notes: 1. There is presently little information

on genetic linkage associated with the recessive form of LQT known as Jervell-Lange-Nielson (JLN) syndrome (see OMIM 220400). 2. Significantly, mutation of the HERC/LQT2 gene has been associated with one case of sporadic LQT (the commonest form, and by definition not heritable) (see Table 2 and the [PDTM), Fig. 4). 3. Hypothetically, distinct forms of LQT may be associated with mutations in genes encoding ion channels, carriers or pumps contributing to cardiac action potential shape/ duration and/or its modulation in situ. (see also Channel modulation, 46-44). To March 1995, three autosomal dominant LQT families remain unlinked to LQT1, LQT2 or LQT3 3 ,49,50,53. Schott et a1. 54 used linkage analysis to map the LQT4 locus in a 65-member family in which the LQT syndrome was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Positive linkage was obtained for markers located on 4q25-q27; maximum lod score t == 7.05 for marker D4S402. For updates, see OMIM Entry: 600919 'Long QT Syndrome, Type 4 [LQT4; Long QT Syndrome with Sinus Bradycardia}'.

Note: For general features of the long QT syndrome, see Phenotypic expression, 46-14 and references to OMIM (Online Mendelian Inheritance in Man) in table.

II

EAg II-EAg H-Erg Elk EAg II-EAg H-Erg Elk EAg II-EAg H-Erg Elk EAg II-EAg H-Erg Elk

108 10. 102 102

~~~~~~~~~~~~~~~~~~~~~~:HI

. . . . . . . . . . . . . . . GSCFLC~DV . . . . . . . . . . . . . . . O~~C~DI

VK

EiQ~;E~;~:~;~::::::::::::::::::::::::::::::::::::::::: E

GA RD

III

ILM~EVVIIEKDMVGSPAHDTMHRGPPTSWLAPGRAKTFRLKLPALLALTARESSVRSGGAGGA~APGAVVVD SHKQIEHT~IILEIIMVMEECD

: : : : : : : : ~ : : :Ii· GL::~::ffi:::~::

VDLTPAAPSSES · . . . . . . . . . . .

L

~

~

~

~

:8

: : : : : : : :: : : : : : : : : : : : : : : : : : :: : : : : : : : : : : : : : EVTAIIDMHVAGLGPAEERRALVGPGSPPRSAPGQLPSPRAHSLMPDASGSSCSLART G L P o(I;Jo G P A A S D G D T E. . . . . . . . . . . . . . . . . . . . . . . AGE G MN L D V P A G C MMG R R

~~~~~~~~~~~~~~~~~:~~~~~:~~~

.

185 162 307 210

S :: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

SR SCASVRRASSADDIEAMRAGVLPPPPRHASTGA SR..................................

- - - - - - - $1--I WD V~C

PT/ilQS. MrLlAH MM~SA~~

· iFSAHLPTLKD . . . . . . . . . . . . . . . . . . . . . . . GVLQ LAPSVQKGEM

168 U6 197 169

:~::~~~~~~@~:::::::

SVFALTAALLGARFRAGSNA~M

. M~Q~RQEA PKTPPHJilLmYCA~ Q KQEAPKTPPH~I HYCV

VH~HS.~AEVL~GS~.L

~:~:~~~i::~~~~~~~~~~~~~~~~~~~~~:~~:~:~:~~;:H~~~AP;

~ K~g:~

KT

I~:~m~~HY~~

WD

KOI:D

II I

H5 226 416 276

I

t t

EAg II-EAg H-Erg Elk

333 3H 513 363

EAg II-EAg H-Erg Elk

.22 .32 598 .51

EAg II-Eag H-Erg "Elk

532 5.2 703 5U

Ea; II-Eag H-Erg Elk

812 657

Eag M-Eag H-Erg Elk

639

6t9

· KDSAVGiAAM RA LTYCD LH ~K~~~ . . . . . . . . . . . . . . . KEATLA SCAMVRALTYCDLHVIK~LQKVL

EVL

ViTSNGQ"TiTTNSiGQ~~~~K~:~~~KA~:~~D~HC~:M~G~

:~~

F~S

F

~~NS~ARM~'

T~:HS

SRN~

~ :E~~~~

T mRH . vm .. I " LRK

::~~~HD~~~

RL I FRRVA DVK~:KfEJLA RIVFRKISDVKIiJ:~ERMK

ER

R/ilN!SP

RI!JN~A

~:~:YE:~~=;~~:~~~~~;~~~~~~N~i:~~~~~~~~~M~

717 727 901 767

Eag M-Eag H-Erg Elk

QLPQNQDHL~~IFSKFRR"PQVQAGSKELVGGSGQSDVEKGDGEVER~V

Eag M-Eag H-Erg Elk

SRVv GWAR

Eag M-Eag H-Erg Elk

. . . . . . . . LA Q R D~V A T vfL1t> MKV DV R L E L QR MQ Q R. . . . . . . . . . . . . . I G R lED LfLl<; E L VK RfL]A P. . GAS saG. . . . . . . . . . . . . . . . . . . . . . IPEQTLQAT~EVKYELKEDIKAL AK MTSIEKQ~EILRI~SRGSAQSPQ FMACEELPPGAPELPQEGPTRRLSLPGQLOAL SQPLHRHGSDPGS FPTFERFVLANPRL

Eag M-Eag Elk

~AGCGAGGGGTPTTQAPPTSAVTSPVDTVITISSpaASGSGSGTGAGAGSAVAGAGGAGLLDPGATVVSSAGGNGLOPLMLKKRRSKSGKAPAPP£QTLASTAGTATAAP

IFGAS PEEQPPISILPVDATPAPSVQEVRSSKRSIRKSTSGSNSSLSSSSSSSNSCLVSQSTGNLTTTNASVHCSNSSQSVASVATTRRASWKLQHSRSGEYRRLSEATAEYSPP

1063 989 1 ~ 0"

AGVAGSGMTSSAPASADQQQQHQSAADQSPTTPOAELLHLRLLEEDFTAAQLPSTSSGGAGGGGGSGSGATPTTPPPTIAGGSGSGTPTSTTATTTPTGSGTATRGKL~FL

117-a

A

l~IH

Eag Elk

LPK~PKL

PLILPPDH~~~LFQRFRQQKEARLAAERGORDLDDLDVEKOMALTDH~SANHSLVK~SVVTVRESPATPVSF

EVDSSPPSRD

QA~~~LARQDTIDEGG

.. QAA~~~MSDHAKLHAPG~CLGPKAVSCDPAKRK

:::~~~~:~~HN~::::~::~:~:~~::~::::~:~:~:::~~~~~::~=;:;~~~:~:~~~~:;~:::~~~~~~~~GK~;=~~;~~~~~~~~QDviQLSA~~:;~

877

, .

887 916 1113 987

NAP 0 Nrs! S G QT T P G D £ I

953 984 11 ~9 1097

IEOAA VSS~VGPSPP.VATTSSAA~AaVSGGPOSaGTV~IVTKADRNLALERERQIEMAS~RATT~TYDTGLRETPPT FKDACGKaEDWNKVSK~SIIETLP.ERTKAPOE~LKKTDSCDSG ITKSDLRLD NVGETR~PQDR~PIL.AEVKHSFYP

~:~~g~~~;~:;::~::~;~=~:~~:~::~~:~~~;:;~~~~~~~:~~~~~::~~i~~~;;~:~:it~~:~~~~~;~:~=;@i:~~:~~:~=~;;~;~;~;;;~~~~

LG~GIEPAIKNEMDL~QKQTLQISPLNTIDECVSPSDHN~SSKER~ITSSAVPTPGRIYPPLDDENSNDFRWTMKHSASHH~CCKSTDALLS

KTPLPVAGVSYGGDEEESVELLOPRRNSRPILLGVSQNQGQGQAMNFRFSAGDADKLEKGLRGLPSTRSLRDPSSK

802 835 1003

.1.

. . . .

ETG£I~RPQSPES~RD

('t

='

t"1"

~ ~

0\

Figure 2. Alignment of predicted amino acid sequences determined for representative members of the eag/elk/erg subfamilies. Positions with identical amino acids in at least three of the four sequences are boxed. The seven hydrophobic domains and the putative nucleotide-binding domain (cNBD) are overlined. lower case residues in the m-eag sequence denote an alternative spliced exon identified by comparison of different cDNAs. (Reproduced with permission from Warmke and Ganetzky (1994) Proc Natl Acad Sci USA 91: 3438-42.) (From 46-19-02)

II

('t)

::s

t"'t"

~ ~

0\

_"--

e_n_try_4_6---J

families have identified several linkage groupst segregatingt with different clinical forms of the disorder.

Drosophila chromosome mapping studies 46-18-07: d-eag; d-elk: The eag locus in Drosophila has been mapped to chromosome 1_4823~24. The elk locus has been mapped by in situ hybridization to polytene chromosome 2 region 54F-55A2 (right arm). Note: Flies heterozygous for deletions of this region do not show any locomotor defects l .

Encoding Relative lengths of Drosophila eag and elk polypeptides 46-19-01: d-eag: Drosophila eag (1174aa) and elk (1284aa) encode relatively large polypeptides in comparison to those encoded by the Shaker family (approx. double their 'typical' length of 500-600 aa, excepting those encoded by Shab, 924 aa and drk, 853 aa). The eag and elk open reading framest are also considerably longer than those 'typical' of cyclic nucleotide-gated channels (see Table 1 under Gene family in ILG CAT cGMP, 22-05). Drosophila eag forms a K+ -selective channel following heterologous expression in Xenopus oocytes 9 consistent with several conserved structural features with Shaker family channels (but see Note 1 under Table 5 under Selectivity, 46-40 and also the localized sequence

alignments of pore-forming regions under Domain conservation, 46-28). The predicted amino acid sequence encoded by the rat eag homologue 7 is 212 aa shorter than that predicted for Drosophila eag (compare r-eag, 962 aaj d-eag, 1174 aa, m-eag, 989 aa; d-elk, 1284 aa, h-erg, 1159 aa).

Amino acid sequence alignment of eag family members 46-19-02: d-eag/m-eag/d-elk/h-erg: A published alignment of predicted amino acid sequences determined for representative members of the eag/elk/erg subfamilies appears in Fig. 2 (from ref. l , using sequence comparison algorithms defined therein). Note: In comparison to the first published eDNA sequence of Drosophila eag9 , a subsequent reportS recorded a number of consistent variations in PCR-amplified eag cDNA sequences, including (i) absence of nt 665-670, resulting in an ORFt 2 aa residues shorter than previously reported; (ii) four different nucleotide changes: A-;.G at nt 2174 (encoding Ala571), C ~ Tat nt 2768 (encoding Leu769), A~ C; at nt 3263 (encoding Ser967) and G ~ A at nt 3608 (encoding Lysl049); (iii) different nucleotides at two additional positions (C1411 T and T1980C), neither of which altered the eag amino acid sequence. These authors concluded that the differences were due to fly-strain polymorphism t (Canton-S versus Oregon-R) as opposed to PCR amplification errors.

Gene organization A partial genomic structure for HERG 46-20-01: h-erg: A panel of PCR primers designed to the published cDNA sequence of HERG (ref. 1, see Database listings, 46-53) have been used to

l'---e_n_t_ry_46

----'_

Intran I -625 bp 5'-AGGAGgtgggg-3'

5'-ccccagCTGATC-3'

5'-ccccagCCCTC-3'

5'·TGGCTgtgagt-3'

NBD - - - . (split) Intran III -1100 bp 5'-CCTGGgtatgg-3'

5'-ctccagGGAAG-3'

Figure 3. Partial genomic structure for HERG. (Reproduced with permission from Curran et a1. (1995) Cell 80: 795-803.) (From 46-20-01)

initially identify three intronic sequences within the protein-coding region. Figure 3 indicates the consensus sequences found at each exon-intron boundary together with the approximate size and location of the introns relative to the predicted transmembrane domains (SI-S6) and the putative nucleotide-binding domain (NBD). Note: Fig. 3 is a partial structure and makes no specific reference to sequences 5' to those encoding Sl, sequences intermediate to those encoding S6 and the NBD or those 3' to the sequences encoding the NBD.

Homologous isoforms 46-21-01: d-eag/r-eag: The predicted Drosophila and rat eag polypeptides share >670 amino acids, with a sequence identity of 61 0/0, exhibiting similar N-terminal regions, hydrophobic cores, pore-forming (P) region and a potential cyclic nucleotide-binding site 7 - see Figure 1 for further

comparisons.

Protein molecular weight (calc.) 46-23-01: r-eag: The longest open reading frame t of the rat eag homologue (see Isolation probe, 46-12) encodes a protein of calculated molecular weight

rvl08kDa 7•

Sequence motifs N-Glycosylation motifs shared by the eag/elk/erg subfamilies 46-24-01: A motif conforming to the N-glycosylation consensus NX[S/T] is

shared by Drosophila eag (Asn424), m-eag (Asn433), elk (Asn444) and h-erg

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_---J1 6

(Asn598, see {PDTM}, Fig. 4) located 12-17 aa upstream of the pore region (according to the numbering1 in Fig. 2 under Encoding, 46-19).

STRUCTURE AND FUNCTIONS

Amino acid composition Hydropathy analyses of eag family sequences 46-26-01: Hydropathyt analyses of eag, elk and erg subfamily polypeptides suggest an '81-86 + H5' transmembrane domain arrangement typical of those found in the voltage-gated K+ channel family (VLC key facts, entry 41; compare the {PDTM}, Fig. 4 this entry, with that under VLC K Kv1Shak, entry 48). The eag/elk/erg subfamily proteins contain a positively charged amphipathic segment 84 and a P-region (ibid.). Each hydrophobic domain of eag, elk and erg shares sequence similarity with the corresponding domain in voltage-gated K+ channels (fo.r further details, see the multiple sequence alignment in Fig. 2 under Encoding, 46-19 and Table 4 under Domain conservation, 46-28).

Domain arrangement 46-27-01: See Amino acid composition, 46-26 and Domain conservation, 46-28.

Domain conservation Conserved residues between eag/elk/erg subfamilies and other cloned channels 46-28-01: Multiple sequence comparisons indicate a structural relationship between eag/elk/erg subfamily members to both cyclic nucleotide-gated channels and voltage-gated K+ channels1,9,21,55. Further similarities have been described between eag family members and certain hyperpolarizationactivated, inwardly rectifying K+ channels of plant origin unrelated to the Kir channel family (e.g. KATI 56- 59, AKT1 60 and KSTI 61 ). Although homologies between the AKTI/KATl/K8Tl channels and those of the Sh and eNG channel families were pointed out first (see refs 56,58, 60, 62 and entries 21, 22, 48-51 inclusive) the hydrophobic core of AKTI has only 38 aa residues in common with the corresponding region of ShB (compare to 58 identical residues between AKTI and bovine CNG-l and 68 residues between AKTI and eag)l. Notably, these similarities are distributed throughout the plant inward rectifier and the eag/elk/erg subfamily 'core' sequences1 . Furthermore, the proximal portion of the P-region is particularly well-conserved between KAT-I, AKT-l and the eag/elk/erg subfamilies (including aa 438-445 as denoted in Fig. 5). Table 4 summarizes several

~

1261 (frameshift) (K2595) introducing stop codon a-Erg

G628S (K2269) de novo mutation affecting pore-forming domain

GTCATCTACACGGCTGTCTTCACACCCTACTCGGCTGCCTTCCTGCTG

a-Erg

V:I

1t2595 1t2595

V:I

Y

T

AV

P'

T

P

Y

S

AA

P'

L

L

SC

L

H-Erg H-Erg K2269 K2269

GTCATCTACCGGCTGTCTTCACACCCTACTCGGCTGCCTTCCTGC~

Y

R

L

S

S

R

P

T

R

P

('b

G N AGC

V

S

= ~

N

V

C!

0\

r-t

GGC

S

V

G

F

S

V

G

F

S

~

Asn-598

Key

Extracellular

C Consensus site for protein kinase C phosphorylation 2-4 aa downstream of 56 at Thr 670 (h-erg) and shared by Drosophila eag (Thr-499), m-eag (Thr-509) and elk (5er-513).

r

Intracellular

Homology to cyclic nucleotidebinding domains

"

Key

T

Motif conforming to the N-glycosylation consensus: NX[SfT] in h-erg (Asn-598, as shown) located 12-17 aa upstream of the pore region and shared by Drosophila eag (Asn-424), m-eag (Asn-433) and elk (Asn-444).

I I I I I

Missense N470D (K2596)

S3

I

L

I

N

F

R

P'

L

:I

D

M

V

A

A

:I

P

GAC D

I

L

I

D

eOOH 881159

(HERG)

F

R

Deleted segment underlined

P'

D

G to C in splice-donor sequence of intron 11/ (see Gene organization, 46-20)

Missense A561 V (K1956)

TGGTTC CTCATC GAC ATGGTGGCC GCC ATCCCCTTCGACCTGCTC

w K2596 K2596

S5

Deletion Lll500 to F508 (K2287)

AAe D

aa 763-836 (HERG)

Splice error/truncation point (K2015)

S2

H-Erg a-Erg

(monomeric, 'unfolded')

L

a-Erg a-Erg

L

K1956 K1956

L

Channel symbol K

I

GCG A H

W

L

I

GTG V a

w

L

L

;: .:~GJ 1J~]: i{~i ;:

NOTE: All relative positions of motifs, domain shapes and sizes are diagrammatic and are SUbject to re-interpretation.

II

Figure 4. Schematic monomeric protein domain topography model {PDTM} for a human erg subfamily channel Q subunit (HER G). (Amino acid numbering from Warmke and Ganetzky (1994) Proc Natl Acad Sci USA 91: 3438-42.) (From 46-26-01)

II

Subfamily/cIon. (.ouree)

440

445

453

458

{entry c::IUSS-reference]

.ag (Drosophila) ~, this entry] .ag (mou•• ) [~, this entry] eag (rat) (~, this a'ltryJ erg (human) [~, this a'ltry] elk (Drosophila) [d-elk, this entry]

KATl (Arabidop.i.) [fields 28 arr1 55, this €!'ltry] A1tTl (Arabidop.i.) [fields 28 arr1 55, this €!'ltry] CNG-3 (rat) [II.13 ~ cPMP, €!'lay 211 CNG-l (bovine)

Y

V

TAL

Y

F

T

M

T

C

M

~

!

Y g

~

g

B y

A

A

E

T

D

N

E

YISSLYI'TMYSLTSVGFGNIAPSTDIE

ISS

SLY

F

T

M

T

S

L

T

S

V

G

F

G

N

I

A

PST

DIE

YVTALYFTFSSLYSVarGRVSPNTNSE YSTALYFTFTSLTSVGFGNVSANTTAE

Y

V

TAL

Y

W SIT

T

L

T

T

T

G

Y

G

D

FHA

E

N

PRE

YVTSMYWSITTLTTVQYGDLHPVNTKE

Y

I

Y

C

Y

W

S

I'

L

T

L

TTl

G

-

E

T

P

P

P

V

K

DEE

Y

V

Y

SLY

W

S

T

L

T

L

TTl

G

-

E

T

P

P

P

V

R

D

Y

W

T

C

V

Y

P

L

I

V

T

M

S

T

V

G

Y

G

D

V

Y

C

B

T

V

L

G

I

P

D

A

F

W

W A

V

V

T

•

T

T

V

0

Y

G

D

M

T

P

V

G

V

W

G

Sbab (JDaJIIIDa1ian) [VLG K IW2-~, a'ltry 49 J Shaw (JDaJIIIDal ian) {VLG K IW2-Eh:Iw, a'ltry 50]

I

PEA

F

W

WAG

I

T

M, T

T

V

G

Y

Q

D

I

CPT

I

P

L

W

W A

V

T

M

T

V

Q

Y

Q

D

MAP

8ha1 (JDaJIIIDa1 ian) [VLG K IW4-~, €!'ltIy 51J

IPAAFWYTIVTMTTVGYGDMVPBTIAG

ltirl.l (rat)

MTSAFLPSLETQVTIGYGYGFRFVTEQ

{II.13 ~

L

S

E

cG1P, €!'lay 22J

.10 (Drosopbila) [II.13 K Q!, €!'ltry 27J

ShB (Drosophila) [VLG K Kvl-£hak, entry 48]

[~

K

submi ts, a'ltIy 33 J

ltir2.l (mou•• ) [~

K

submits, a'ltIy 33J

ltir6.l (rat) [IM K

G

L

lET

T

F

T

A

A

F

L

.,

S

F

P

S

A

F

L

P

FIE

TEA

LTG

A

F

L

P

S

S

Q

TTl

K

T

G

T

G

Q

Y

G

F

R

C

V

I

Q

Y

0

Y

R

Y

lTD

TTl

Q

Y

Q

FRY

Y

I

DEC

P

submits, a'ltIy 33J

lCir3.1 (rat) [Im. K

L

TAL

submits, a'ltIy 33J

lCir5.1 (rat) [IM K sutunits, a'ltry 33J ltir6.l (rat) {Im. K sutunits, a'ltry 33J

L

E

Q

T

I

K

C

P

SEE

C

P

FTAAFLPSLETQTTIOYQYRCVTEECS F

T

S

A

F

L

PSI

E

V

Q

V

T

I

Q

.,

Q

G

R

M

M

TEE

C

P

g ~ ~

0\

(D

Figure 5. Local amino acid sequence alignments including P-regions from eag/elk/erg subfamily members and various K+ and cation-selective channels. Cross-references to other entries are indicated. The underlined stretch of amino acids (residues 452459 of Drosophila eag) show only one conservative substitution across the presently known eag/elk/erg subfamily members. The shaded portions indicate residues identical to those found in one or more of the aligned eag/elk/erg subfamily sequences (Drosophila, mouse or rat). The GFG motif (residues 454-456, top line) highlights a variation from the common GYG motif seen in K+ -selective voltage-gated and inwardly rectifying K+ channel subfamily members (except Kir6.1, see Domain conservation under INR K subunits, 33-28 and VLG K Kv1-Shak, 48-28). The numbered residues Ser453 and Asn458 in Drosophila eag further distinguish the pore of the eag/elk/erg subfamilies from those of the other subfamilies shown. Vertebrate cyclic-nucleotide-gated (CNG) channels, which are cation non-selective (or 'entirely Ca 2 + selective,119 with ~ 3mM[Ca 2 +]o) also show some distinguishing features in their pore-forming region (see ILG CAT cAM~ entry 21 and ILG CAT cGM~ entry 22). (From 46-28-01)

II

~

~ ~

0\

_L..-

,

e_n_t_ry_4_6_

Table 4. Summary of reported similarities and differences within specified regions of eag, elk and erg channel subfamilies (and other channel families, where noted) (From 46-28-01) Region

Similarities, implications and cross-references

Central hydrophobic core

46-28-02: Eag/elk/erg subfamiliy proteins share a similar overall domain arrangement to those typical of the Sh family of voltage-activated K+ channels (see the VLC K Kv series, entries 48-51 and rows below). These similarities include possession of a central hydrophobic core containing seven hydrophobic domains corresponding to the putative transmembrane a-helices (SI-S6). Similarities in amino acid sequences between Drosophila eag and cyclic nucleotide-gated channels have been specifically discussed55 .

SI-S2 loop (extracellular)

46-28-03: The SI-S2 segment (encoding an extracellular segment) is variable in length and composition within the eag/elk/erg subfamilies in a similar manner to the variability seen in the corresponding region of Sh proteins.

S2-S3 loop (intracellular)

46-28-04: The S2-S3 segment has the same length and shows considerable sequence similarity within the eag/ elk/erg subfamilies. 46-28-05: Variable in length and composition (as for

S3-S4100p (extracellular) S4 voltage sensor

Sl-S2 loop, above). 46-28-06: Eag/elk/erg subfamily proteins possess the characteristic S4 motif, with the positively charged amino acids 'near-identical' in the segment (exceptions being an Arg to GIn substitution at position 386 in the Drosophila elk polypeptide and an Arg to His substitution at position 370 in the m-eag polypeptide1

(see alignment under Encoding, 46-19). S4-S5100p (intracellular)

46-28-07: The S4-S5 segment has the same length and shows considerable sequence similarity within the eag/ elk/erg subfamilies.

S5 transmembrane 46-28-08: d-eagj m-eag: A distinctive hydrophobic segment occurs in S5 of the eag subfamily (see Encoding, domain 46-19). The segment has at least ten amino acid identities and no more than one conservative substitution within the presently known subfamily members with little similarity with the corresponding region of Sh proteins. P-region

II

46-28-09: Eag/elk/erg subfamily proteins possess a highly conserved pore (H5) region with strong similarity to various members of the voltage-gated K+ channel family (for details, see the local sequence alignment in

1"--_e_n_t_ry_4_6

_

Table 4. Continued Region

Similarities, implications and cross-references Fig. 5). Comparative note: D - t N substitutions in Shaker subfamily channels at positions homologous to the N458 residue of eag (see Fig. 5) have been reported to abolish K+ channel activity63, but this N-residue is conserved amongst the presently known eag/elk/erg subfamily members.

N-terminal, including the cNBD domain homologies

46-28-10: Although the N-terminal segment is of variable length amongst the eag/elk/erg subfamilies, its first 150 amino acid residues are well-conserved. 46-28-11: eag/elk/erg polypeptides contain a highly conserved segment that is related to regions forming cyclic nucleotide-binding domains (cNBD) within the eNG channel and cyclic-nucleotide-dependent protein kinase families (see ILG CAT cAM~ entry 21 and ILG CAT cGM~ entry 22 and the sequence alignment under Encoding, 46-19). Eag/elk/erg subfamily proteins show little similarity within N-terminal sequences downstream of the cNBD. For reported effects of cyclic nucleotides on eag family channels, see Channel

modulation, 46-44. 46-28-12: d-eag: Comparative note: A stretch of seven amino acids found near the N-terminal of Shaker family polypeptides conforming to the consensus NEYFFDR is absent from eag 9 .

(See also Sequence motifs, 46-24 and Protein phosphorylation, 46-32.)

other specific comparative features reflecting domain conservation which have appeared in the literature.

Predicted protein topography Expected assembly pattern of eag/elk/erg monomers into functional channels 46-30-01: By analogy to other voltage-gated K+ channels, the 'SI-S6 + H5' domain arrangement shown by eag, elk and erg subfamily proteins suggests that tetrameric assemblies of Q subunit channels are formed in native cells. No reports of association with accessory subunits (analogous to the Kv channel f3 subunits, see VLG K Kv beta, entry 47) have appeared to date, but see Protein interactions, 46-31. Preliminary models comparing HERG, AKTl, Kir, TWIK, and cyclic nucleotide-gated channel structures have appeared64 . In press update: These models should be compared to the first crystal structure derived for a K+ -selective channel120.

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_6_

Protein interactions tModulatory' hypotheses for Drosophila eag subunits in heteromultimeric K+ channels 46-31-01: d-eag: Whereas Shaker mutations eliminate the fast transient IA 65,66, voltage-clamp studies 10,67 of Drosophila larval muscle expressing mutant eag subunits have shown reductions in four identified potassium currents including (i) IA; (ii) IK (a slow, non-inactivating voltage-gated K+ current); (iii) ICF (a fast Ca2+-activated K+ current, see Drosophila slo under ILG K Ca, entry 27); (iv) Ics (a slow Ca2+-activated K+ current). The mechanisms by which eag affects all of these currents has been the subject of continuing investigation68 . Although none of the eag mutations (including alleles producing truncated mRNAs) eliminate any of the four K+currents, effects of the mutations exhibit strong temperature dependence. Notably, both a Ca2+/calmodulin antagonist (W7, N-(6aminohexyl)-5-chloro-l-naphthalenesulphonamide) and cGMP analogues (8Br-cGMP, see Resource C - Compounds and proteins) both modulate native K+ currents with actions that are altered or abolished by eag mutations68 . Patterns of allele t -specific interaction between eag and Sh have been taken to indicate a close association between the Sh and eag subunits within the fA channel heteromultimers t in Drosophila larval muscle membranes 68, consistent with the eag locus encoding a subunit common to different K+ channels. These and other observations have formed the basis of a hypothesis 68 proposing a role for the eag subunit in modulating channels (as opposed to gating, selectivity, and conductance properties of Sh subunits; but see Note 1 of Table 5 under Selectivity, 4640). Supplementary note: Although Shaker mutant strains show relative insensitivity to heat shock anaesthesia induced by volatile isoflurane, eag, para and slo mutants do not differ significantly from the wild-type in similar behavioural tests69.

Deletion mutations affecting subunit interaction domains 46-31-02: h-erg: A subunit interaction domain designated as NAB (HERG) has been localized at the hydrophilic cytoplasmic N-terminus of HERG37. NAB (HERG) is able to form a tetramer in the absence of the remainder of the HERG protein. Significantly, the HERG ~1261 mutation associated with one long Q-T syndrome kindred (see Table 2 under Phenotypic expression, 46-14) results in a truncated protein that contains the subunit interaction domain and can explain observed effects on decreased channel expression37• Note: There is an ongoing debate relating to whether 70 or not 71 'intersubfamily' heteromultimeric K+ channels can form following co-assembly of Drosophila eag and Shaker subunits in Xenopus oocytes.

Heteromultimer formation in h-erg channels 46-31-03: h-erg: Although biophysical studies indicate that HERG proteins contribute to the native cardiac current IK,r, additional subunit interactions may be required to reproduce sensitivity to known blockers of IK,r (see ref.12 and Blockers, 46-43). Furthermore, since mutations in HERG are the molecular mechanism for chromosome 7-linked long QT syndrome (see

III

1'--_e_n_t_ry_4_6

---J_

Phenotypic expression, 46-14 and Chromosomal location, 46-18) combination of normal and mutant HERG channels (i.e. 'heteromultimers' of wild-type and mutant a subunits) may underlie the phenotypic effects (ibid.). The types of mutations identified in affected families are consistent with such a dominant-negative t mechanism3 (for details see Table 2 under Phenotypic expression, 46-14). Thus mutations that result in premature stop codons t and truncated proteins (e.g. ~ 1261, the splice-donor mutation, the in-frame deletion ~I500-F508, ibid.) and mutations that alter conserved amino acids in the transmembrane domains (e.g. missense mutations A561V in 55, N470D in 52, ibid.) may impair HERG function through induced changes in secondary, tertiary or quaternary channel structure (even when co-assembled with wild-type subunits). Mutations that disrupt the P-region or the putative nucleotide-binding domain (see Table 2 and the {PDTM}, Fig. 4) could similarly alter HERG function by altering K+ -selectivity or disruption of an endogenous channel-modulatory mechanism (see Domain functions, 46-29 and Channel modulation, 46-44).

Indirect evidence for interactions between channels supporting IK,r and IK,s 46-31-04: AT-l cells (a cardiac atrial tumour cell line expressing an IK,r) exposed to antisense t oligonucleotides (targeting the 5' translation start site of the minK cDNA cloned from an AT-l library) exhibit a 'significantly reduced' I Kr amplitude (for further description, see Protein interactions, 5431). Interestingly, the minK protein (entry 54, initially proposed to coassemble with KvLQT1 to conduct native cardiac IK,s, ibid.) has also been shown to enhance functional expression of HERG-K+ currents in CHO cell co-transfection studies 73.

Protein phosphorylation Protein kinase A consensus site 46-32-01: d-eag: A single consensus site for cAMP-dependent phosphorylation with a presumed cytoplasmic location is found at amino acid position 1039 (see Resource G - Consensus sites and motifs, entry 62). Note: d-eag: 8-BrcAMP (but not 8-Br-cGMP) increases eag current amplitudes and induces hyperpolarizing shifts in voltage activation thresholds; these effects persist in the presence of the non-specific protein kinase inhibitor H-7 (for details, see Channel modulation, 46-44).

Shared PKC consensus sites across eag/elk/erg subfamilies 46-32-02: d-eagjm-eagjelkjh-erg: Ten sites conforming to the consensus site for phosphorylation by protein kinase C (PKC, Le. [5,T)X[R,K), see Resource G - Consensus sites and motifs, entry 62) were reported for Drosophila eag with presumptive intracellular locations 9 . A later analysis 1 noted that eag, m-eag, elk and h-erg polypeptides share one consensus site for PKC phosphorylation 2-4 aa downstream of transmembrane domain 56 at Thr499 (eag), Thr509 (m-eag), 5er513 (elk) and Thr670 (h-erg) (according to the numbering shown in the alignment under Encoding, 46-19).

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_6_

CaMKII may influence synaptic plasticity phenotypes in Drosophila 46-32-03: d-eag: Similar defects in both synaptic transmission and associative learning are produced in Drosophila melanogaster by (i) transgenic t inhibition of calcium/calmodulin-dependent protein kinase n (CaMKll) or (ii) mutations in the eag subunit gene29. These behavioural and synaptic defects are 'non-additive' in flies ca:rrying both an eag mutation and the CaMKll inhibitory peptide transgene t ala under the control of a heat-shock promoter, suggesting a common effector pathway29. Significantly, a segment of the putative cytoplasmic domain of Drosophila Eag acts as a substrate of CaMKll (Thr655), and there is evidence for in vitro interaction of the CaMKll and Eag molecules, suggesting that CaMKII modulation of eag function is an important determinant of synaptic plasticity in vivo. Comparative note: Transgenic disruption of the mouse a-CaMKII gene abolishes long-term potentiationt and severely impairs (but does not abolish) spatial learning ability (see Protein interactions under ELG CAT GLU NMDA, 08-31).

ELECTROPHYSIOLOGY

Activation Variations in rat eag activation kinetics dependent upon prepulse potential 46-33-01: d-eag/r-eag: Like Drosophila eag, injection of rat eag mRNA into Xenopus oocytes gives rise to voltage-activated, non-inactivating outward K+ currents of the delayed rectifier type 7 (see Voltage sensitivity, 46-42). In comparison to Drosophila eag, rat eag currents show a marked variation in activation kinetics according to the voltage pulse protocol used to elicit outward current. This novel activation behaviour depends strongly on the pre-pulse potential: Depolarizing pre-pulses (between a VH of -lOOmV and a test pulse of +40mV) accelerate the kinetics of activation 7, while hyperpolarizing pre-pulses (between a VH of -lOOmV and a test pulse of +40mV) slow down the kinetics of activation.

Differences and similarities between vertebrate eag and erg subfamily channel currents

46-33-02: m-eagfh-erg: Direct comparison2 of m-eag and HERG channels expressed in Xenopus oocytes under two electrode voltage-clamp reveal markedly different responses to depolarizing and hyperpolarizing protocols in elevated [K+]o (summarized in Fig. 6). Under specified expression conditions (suppressing the development of an outward component, see Fig.6e) HERG channels display strong inward rectification, attributable to an inactivation mechanism that attenuates K+ efflux during depolarization (see Inactivation, 46-37). Otherwise, HERG channels display gating properties consistent with eag and other K+ -selective, outwardly rectifying, voltage-gated channels containing an S4 voltage-sensor domain (see Kinetic model, 46-38 and Voltage-sensitivity, 46-42).

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Distinctions between of IK,r and

----'_

I K1

46-33-03: h-erg: The native cardiac rapidly activating 'delayed rectifier' current IK,r (encoded by HERG, see Phenotypic expression, 46-14) exhibits 'strongJ K+ inward rectification t properties and activation by extracellular K+ under certain conditions (for details, see Fig. 6, Inactivation, 46-37 and Channel modulation, 46-44). These properties are shared by the extensively characterized current IKI (encoded by genes in the Kir family, see INR K subunits, entry 33 and INR K [native], entry 32). I K1 is activated 'almost instantlyJ by membrane hyperpolarization whereas heterologously expressed HERG channels and native IK,r are activated relatively slowly by depolarization and are not activated by hyperpolarization alone 12. Note: The 'HERG-like J IIR in F-ll neuroblastoma cells (see Cloning resource, 46-10) shares voltage-dependent gating properties with native cardiac IK,r and I HERG although the F-ll hR outward currents are 'negligible in comparison J74 .

Current type 46-34-01: For 'typical J characteristics of eag and erg subfamily currents under specified recording conditions J see Subtype classifications, 46-06, Activation, 46-33, Inactivation, 46-36 and Channel modulation, 46-44.

Current-voltage relation Functional consequences of strong inward rectification by h-erg/native IK,r 46-35-01: In heterologous expression systems J human-derived (h-erg) channels display strong inward-going rectification due to an inactivation mechanism that attenuates potassium efflux during depolarization (for details see Inactivation, 46-37). Consistent with h-erg underlying native IK,rJ several studies in different species have described inward rectification of IK,r in native cardiac preparations. It has been concluded 75 that inward rectification of native guinea-pig ventricular myocyte IK,r is due to a voltage-dependent gating mechanism and does not result from block of the channel by intracellular Mg2 + (compare Blockers under INR K [native], 3243). HoweverJ intracellular Mg2 + does appear to determine the specificity of E-4031 block of native guinea-pig I K,r 75 (see Blockers, 46-43). In single rabbit ventricular myocytesJ I Kr has been described as 'essentially nil J at +30m~ despite the channel opening rapidly in this voltage range76. Note: This result is consistent with the ineffectiveness of E-4031 (see Blockers, 46-43) on the early part of the plateau phase of the action potential 76 . See Fig. 6 under Activation, 46-33 for an illustration of the inward-going rectification of the HERG current.

Inactivation tpartial inactivation' of Drosophila eag in excised oocyte patches 46-37-01: d-eag/m-eag: The 'partly inactivating' current observed in insideout membrane patches of oocytes expressing Drosophila eag 5 has fast and

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____________________ en_t_ry_4_6_

(a) mouse eag 'Normal' Ringer's in bath: 96 mM NaCl, 2 mM KCI, 1.8 mM CaCI2, 1.0 mM MgCI2, 5 mM HEPES (pH7.4).

M·EAG

~

5 mM HEPES (pH7.4) (Electrodes: 2 M KCI)

M-EAG

60 -105

-80

(b) mouse eag 'High-K' Ringer's in bath: 100 mM KCI, 1.8 mM CaCI2, 1.0 mM MgCI2,

Voltage protocol: Depolarizing, as shown Vertical bar: 1 J.1A; horizontal bar: 100 ms

80 _80-• • •1L:-~10~5~ Voltage protocol: Depolarizing, as shown Vertical bar: 1 J.1A; horizontal bar: 100 ms

(c) human erg

(d) human erg

'Normal' Ringer's in bath

'High-K' Ringer's in bath:

HERG

b+J----~!

....~ . .--'-

L-J~~~~l \)1/

60

-80

--1;;;;;;;;;;;;= -105

11\/

'{

60 ·130

Voltage protocol: Depolarizing, as shown Vertical bar: 1 J1A; horizontal bar: 100 ms

Voltage protocol: 200 ms prepulse at +60 mV before the hyperpolarizing steps as shown Vertical bar: 1 JlA; horizontal bar: 100 ms

(e) human erg

(f) human erg

'Normal' Ringer's in bath

'High-K' Ringer's in bath: I-V relation from record (D)

at approx. 10-fold higher erg expression levels to oocyte (D) as judged by support of> 100 J1A inward currents in high K Ringer's (not shown)

V(mV) ·150 -125 -100

-75

-50

. . . ••. .

-25

o·

l2

25

:.1·· · · .'

50

-2

-4 -6

IblA)

-8 -10

·12 -14

-80 -Jiiiiiiiiiii-.:1Q

Voltage protocol: Depolarizing, as shown Vertical bar: 1 J.1A; horizontal bar: 100 ms

Filled squares: HERG inward current Filled circles: water-injected control

Figure 6. Comparison of mouse eag and human erg channel currents following heterologous expression in Xenopus oocytes. Under two-microelectrode voltage-clamp, oocytes were bathed in either lnormal' Ringer's solution or lhigh-K+' Ringer's solution with composition as indicated. Voltage-recording and current-passing electrodes (tip resistance 0.5-1 MO) were filled with 2 M KCl throughout. (a) Large outward current mediated by m-eag following a series of depolarizing voltage steps (from -80mV,

l_e_n_t_ry_46

----'_

stepping to +60mV as shown 2 ). (b) Large m-eag current in high K+ Ringer's. (c) Small inward current mediated by HERG under the same voltage protocol as in (b). This small current was not observed in water-injected oocyte controls under the same voltage protocol. (d) Large inward currents mediated by HERG in elevated [K+]o following hyperpolarizing steps with a depolarizing pre-pulse (stepping to -130mV with a +60mV pre-pulse for 200ms as shown. Note the rapid activation of inward current (e.g. for hyperpolarization to -105 m V, time-to-peak was 27 ± 4.6 ms; mean ± SD; n == 7). (e) In tnormal' Ringer's, when HERG expression is increased by t10-fold or more' over levels used to study the inward component, an temerging outward component', exhibiting kinetics consistent with native cardiac IK,r appears. (I) Current-voltage relation from data in (d) illustrating strong inward rectification due to attenuation of K+ efflux by channel inactivation following depolarization. For further details see ref. 2 , Phenotypic expression, 46-14; Inactivation, 46-37; Kinetic model, 46-38; Blockers, 46-43 and Openers, 46-48. (Data compiled from different experiments reported in Trudeau et al. (1995) Science 269: 92-5.) (From 46-33-02)

slow components, with time constants of lS.2ms and 470ms at a test potential of +40mV. The fast component represents approx 200/0 of total current amplitude. In direct comparison, d-eag and m-eag are both voltage dependent and outwardly rectifying, but m-eag currents are generally sustained for the duration of an activating voltage command 77. See also Rundown, 46-39 and Table 5 under Selectivity, 46-40.

Rapid channel inactivation reduces conductance at positive voltages 46-37-02: h-erg: Unlike other members of the Eag family of voltage-gated,

outwardly rectifying potassium channels, the channel encoded by the human eag-related gene (HERG) displays an inwardly rectifying potassium channel under specified conditions 2 (see Activation, 46-33). HERG channels display typical gating properties of eag-related and other outwardly rectifying, S4-containing potassium channels, but with the addition of a voltage-dependent inactivation process that attenuates potassium efflux during depolarization. This feature of HERG channel function appears critical to the maintenance of normal cardiac rhythmicity (i.e. in the physiological suppression of arrhythmias, specifically extra premature afterbeats)78 (see Phenotypic expression, 46-14).

Inactivating gating mechanisms 46-37-03: h-erg: Ie-type' inactivation, considered to be a 'slow inactivation'

mechanism in other K+ channels such as Kvl.3, appears to involve a 'conformational switch' in the outer mouth of a channel (see Inactivation under VLG K Kv1-Shak, 48-37). N-type inactivation (or any mechanism relying on an intracellular pore blocker) was 'ruled out' by studies on HERG channels using (i) intracellular TEA+ ion (which inhibits N-type inactivationt) and (ii) N-terminal deletions (HERG ~2-37379, but see notes 1 and 2 below). Since extracellular TEA+ did interfere with inactivation, some resemblance to

II

_'---

.

en_t_ry_4_6-----1

features of IC-type' inactivation was postulated for h_erg78~79. Measurements of the instantaneous t current-voltage relationship for h-erg channels (in the saponin-permeabilizedt cut-open oocyte clamp t preparation)8o have determined the rate of inactivation to be strongly voltage-dependent at depolarized potentials (i.e. in contrast to C-type inactivation in other voltage-gated K+ channels). The voltage-dependence could be modulated independently of activation by increasing [K+]o from 2 mM to 98 mM, suggesting that inactivation of h-erg has its own intrinsic voltage sensor80. Notes: 1. Ntype processes would compete with TEA+ binding to the intracellular mouth and hence predict a slower inactivation in the presence of TEA+, which is not observed. 2. Another study81 has reported that truncation of the Nterminal region of HERG did shift the voltage dependence of activation and inactivation by +20 to +30mV, with the rate of deactivation of the truncated channel being 'much faster' than that of wild-type HERG81 .

Kinetic model Gating models developed for other voltage-gated channels applied to HERG 46-38-01: h-erg: Despite marked differences in the characteristics of m-eag and h-erg channel currents (e.g. see Fig. 6 under Activation, 46-33) initial analysis of the voltage-dependent gating transitions in these subfamilies2 is characteristic of most channels in the 54-containing superfamily of ion channels. A schematic model of HERG c.hannel gating modified from those developed for Sh channels has been represented as follows: Cl f==! C2 f==! C n

f=::::!

0

f==!

I(B)

According to the model2, HERG channels are 'at rest' in the closed state (Cd. Following depolarization HERG channels undergo voltage-dependent transitions leading sequentially to the open state (0) and to the inactivated or blocked state, I(B). Since only very small outward currents pass through HERG channels at the onset of depolarization (see Fig. 6), the forward rate for the 0 ~ I(B) transition must be very fast 2. During (subsequent) hyperpolarization, the inactivation is removed and channels enter the 0 state, resulting in inward current flow. The HERG current is transient (see Fig. 6d) as channels make the 0 ~ C transition, albeit at a slower rate than m-eag channels (see panels A and B in Fig. 6). HERG rectification properties resulting from rapid inactivation (particularly in its comparison with native cardiac IK,r) are further analysed in ref. 12. Note: The 'HERGtype' hR described in the rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid cell line (F-ll) and native cardiac IK,r have been compared by means of a unique kinetic model 74 .

Kinetic modelling of external divalent modulation processes 46-38-02: r-eag: A kinetic model for rat eag activation has been developed from data indicating that all four r-eag channel subunits undergo extracellular Mg2+ -dependent conformational transitions prior to final channel activation82 (see Channel modulation, 46-44).

l'---e_n_t_ry_4_6

--'_

Rundown Bag channel rundown can be reversed by 'patch cramming' 46-39-01: As part of the comparative study of Drosophila versus mouse eag K+ current properties expressed in oocytes 77 (see Table 5 under Selectivity, 46-40) d-eag currents 'rundown' more rapidly than do m-eag currents in

excised macropatchest. Rundown is generally reversible by inserting the patch into the interior of the oocyte, a feature generally taken to indicate that an undetermined 'cytosolic factor' regulates channel activity or stability. Note: Cyclic nucleotide binding (see entries 21 and 22) has been excluded from a role in d-eag or m-eag channel gating 77 although a role for cyclic nucleotide-dependent phosphomodulation appears likely (see also Gene family, 46-05 and Channel modulation, 46-44).

Selectivity Selectivity comparisons between eag species homologues 46-40-01: A summary of selectivity characteristics for heterologously expressed Drosophila and mammalian eag and erg channel homologues (compiled from refs. s,7) appears in Table 5. The table records reported differences 7 between Drosophila, rat and mouse eag channel selectivity expressed in oocytes. Alignment of sequences corresponding to the pore-forming (P-domain) regions of several K+ -selective channels has revealed some distinguishing features in the eagJelkJerg subfamilies (summarized in Fig. 5, this entry).

Single-channel data 46-41-01: d-eag: A single-channel conductance of 4.9 pS determined by nonstationary noise analysist was reported for Drosophila eag heterologously expressed in oocytes s. h-erg: Elementaryt properties of HERG-induced current in oocytes and native IK,r have been directly compared83 : In common with other studies, single-channel conductance was dependent on the extracellular potassium concentration ([K+]o). At physiological [K+]o, 'Y = 2pS, and at 100mM [K+]o, 'Y = lOpS. Single-channel openings occur in bursts with mean duration 26ms at -100 mY; in oocytes, h-erg channel mean open time was 3.2ms and closed times were 1.0 and 26ms.

Voltage sensitivity Steep voltage dependence of heterologously expressed eag channels 46-42-01: d-eag: Drosophila eag mRNA injected into oocytes under conventional two-electrode voltage clamp supports the expression of depolarization-activated, non-inactivating outward currents with activation thresholdst in the range -40 to -30mYs . Outward currents in this recording configuration are biphasic, showing an initial fast-rising phase followed by a slower second component. I- V relationships for Drosophila eag measured in inside-out membrane patches show a broadly similar I- V relation, but (i) a 'partly inactivating' current is observed and (ii) the biphasic rising phase is absent. Notes: 1. A Ca2+ -activated CI- channel (endogenous to the oocyte, see ILG Cl Ca, 46-25) may contribute to this

_'--

e_n_try_4_6

--J

Table 5. Selectivity of eag and erg channels following heterologous expression (From 46-40-01) Species/subfamily Reported properties/comparative notes (data from references as indicated)

Drosophila eag

46-40-02: d-eag: Generally, selective for K+ over Na+, and 'unexpectedly' permeable to NHt (ref.5, see also note 1

for apparent Ca 2+ permeability). The relative permeabilities of a series of test ions (note 2) follows the series K+ (1) > Rb+ (0.75 ±0.11) > NHt (0.25 ±0.08) > Cs+ (0.42 ± 0.14»> Na+ (0.11 ± 0.06) > Li+ (0.08 ± 0.06) (in the ratios shown in brackets; corresponding to Eisenman's seriest IV)5. Rat eag

46-40-03: r-eag: Strongly selective for K+ over Na+. Permeability ratios (note 3) follow the series K+ (1) > Rb+ (0.72±0.26»NHt (0.13 ±0.04) »> Na+ «0.01), Li+

«0.01), Cs+ «0.01). Note: Rat eag channels are not permeable to Cs+ (compare with Drosophila eag, above). Human erg

46-40-04: h-erg: Nernstiant relations confirm h-erg

channels are potassium selective (compare the GFG motif conservation described in Fig. 5 under Domain conservation, 46-28).

Notes: 1. Results consistent with simultaneous K+ efflux and Ca 2+ influx through Drosophila eag channels expressed in oocytes was reported and discussed in ref. 5 . This unusual property may have suggested mechanisms for modulation of synaptic efficiency in vivo and an alternative explanation for effects of eag mutants on multiple native K+ currents (including IK,ca types - see Protein interactions, 46-31). Notably, however, a comparative study of Drosophila versus mouse eag K+ current properties expressed in oocytes has appeared 77; this countered the previous reportS by finding no evidence for Ca2+ flux through eag channels, concluding instead that both d-eag and m-eag channels were 'highly selective' for K+ over Na+ ions. See also Rundown, 46-39 and Selectivity, 46-40. 2. As determined by measuring reversal potentials under bi-ionic conditions, with 115 mM of the chloride salt of the test ion and 1.8 mM EGTA outside versus 100mM KCI and 10mM EGTA inside; outside-out patches, pH7.2 both sides). 3. As obtained from reversal potential determined in two-electrode voltage clamp of oocytes (bath solutions contained 115 mM of the test cation as the chloride salt, 1.8mM CaCl2 and 10mM HEPES, pH 7.2).

'biphasic' activation property. 2. Rat eag channels expressed in the same system do not display biphasic activation, which has been suggested to be due to inherent differences in Ca2+ permeability 7 (see note 1 in Table 5 under Selectivity, 46-40).

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l_e_n_t_ry_46

_

PHARMACOLOGY

Blockers Block of native IK,r and HERG channels by class III antiarrhythmic drugs 46-43-01: A large literature exists describing patterns of IK,r block by drugs that act by slowing cardiac repolarization (phase 3) and prolong the duration of action potentials (described as 'class III' antiarrhythmic agents). Class ill drugs such as E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonylamidobenzoyl)piperidine, a sotalol derivative) and MK-499 are often described as potent and relatively specific blockers of IK,r in cardiac myocytes, although some studies report that these agents block other channel types. Many studies have described patterns of block of native IK,r or erg subfamily channels by dofetilide, clofilium, quinidine and sematilide (see also compounds listed in Table 6, this field).

Acquired LQT following therapy with cardiac K+ channel blockers 46-43-02: h-erg: Certain antiarrhymic drugs, such as quinidine and sotalol that include actions which block the cardiac rapidly activating delayed rectifier current (generally designated as IK,r) have been associated with an lacquired' or ldrug-induced' form of long QT (LQT) syndrome (see Phenotypic expression, 46-14). These similarities extend to the induction of torsade de pointes that is observed in familial LQT syndrome (ibid.). The cloning of HERG, its marked similarities of functional properties to IK,r following expression in oocytes (this entry) and the demonstration of mutations in HERG associated with the chromosome 7-linked LQT syndrome (see Chromosomal location, 46-18) thus provide a 'mechanistic link' between inherited and acquired forms of LQT syndrome 12. Pharmacological agents such as sotalol and dofetilide probably exert their antiarrhythmic effects by 'modest' lengthening of cardiac action potentials, thereby suppressing reentrant arrhythmias t (see also Channel modulation, 46-44 for application of elevated [K+]o for correction of LQT anomalies).

Channel modulation Heterologously expressed Drosophila eag ldirectly' modulated by cAMP 46-44-01: d-eag/r-eag: In voltage-clamped oocytes expressing Drosophila eag, bath application of membrane-permeable 8-Br-cAMP (8-bromoadenosine 3'5'cyclic monophosphate, 1 mM) or 8-Br-cGMP (1 mM) increase the amplitude of eag outward currents, while thresholds of activation are shifted to more negative potentials. These effects of 8-Br-cAMP (but not 8-Br-cGMP) persist in the presence of the non-specific protein kinase inhibitor H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine)5. Following its heterologous expression in Xenopus oocytes, rapid application of cAMP (2 mM) to inside-out patches produces 'significant increases' (typically 10-15 0/0) in outward current amplitude5. This increase is rapidly reversible on perfusion with intracellular bathing solution lacking cAMP. Similar effects cannot be induced by cGMP

_

•

entry 46 '----------------

Table 6. Reported patterns of ionic and pharmacological block of native IK,r or heterologously expressed eag and erg subfamily channels (From 46-43-01) Blocker

Species/homologue, characteristics and references

Small ionic blockers Calcium ions (intracellular)

46-43-03: r-eag: Rat eag channels expressed within HEK-293 cells have been described as 'rapidly and reversibly inhibited' by rises in [Ca2+]i between 30 and 300nM (mean ICso of 67nM in an I/O patch)4. Generation of intracellular calcium oscillations following muscarinic receptor activation appeared to induce a synchronous inhibition of r-eag mediated outward current. These and other data led to the conclusion that r-eag channels are 'voltage-activated, calcium-inhibitable' channels, with the Ca2 +inhibitory effects independent of calcium-dependent kinases and phosphatases4. See also notes 4, 5 and 6 for refs. discussing similarities and differences to Mcurrent.

Cadmium ions

46-43-04: d-eag: C:admium ions at 1 mM do not affect amplitudes of Drosophila eag-mediated outward currents in Xenopus oocytes. Note: This concentration blocks voltage-gated Ca 2 + channels endogenous to the oocyte5 .

Lanthanum ions

46-43-05: h-erg: HERG currents in oocytes can be blocked by lanthanum ions 12.

Barium ions

46-43-06: h-erg: When applied in the bath solution, Ba2+ ions inhibit peak inward HERG current in twoelectrode voltage-elamped oocytes at an IC so (note 1) ~O.6 mM (Fig. 7a2 );: the block is not apparently voltage dependent as observed with 'classical' inward rectifiers (compare Blockers under INR K native, 33-43).

Caesium ions

46-43-07: h-erg: Inhibition of peak inward HERG current in oocytes by Cs+ ions (1 mM in bath solutions) is more effective at negative voltages (Fig. 7b) and appears to reflect Cs+ entering the pore from the outside and interfering with K+ permeation (ibid. 2 ). 46-43-08: r-eag: Relatively impermeable to Cs+ ions 7 (see Selectivity, 46-40).

Large organic ion blockers TEA+

46-43-09: d-eag: I(:so (note 1)5: TEA: 33 ± 11 mM; 4AP >100mM (i.e. resistant; compare to most Sh-type voltage-gated K+ channels under the VLG K entry series). 46-43-10: r-eag: IC so (note 1)7: TEA: 28 ± 13 mM; 4-AP >100mM.--

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_

Table 6. Continued Blocker

Species/homologue, characteristics and references

Pharmacological agents (specifically citing IK,r block) E-4031; voltagedependence

46-43-11: Open-channel blockt of IK,r and HERG by methanesulfonanilides such as dofetilide, E-4031 and MK-499 has been characterized in detail 84 . MK-499 preferentially blocks open HERG channels (steadystate block is half-maximal at 123 ± 1211M at a test potential of -20mV. MK-499 (150nM) does not affect the voltage dependence of activation and rectification nor the kinetics of activation and deactivation of HERG. Notably, 1 J.lM MK-499 and E-4031 have no effect on HERG when oocytes are voltage clamped to a negative potential and are not pulsed during equilibration with the drug. However, MK-499 does block HERG current if oocytes are repetitively pulsed, or clamped at a voltage positive to the threshold potential for channel activation84. Apparent discrepancies of this result with earlier studies showing significant block of IK,r in isolated myocytes by similar drugs, even in the absence of pulsing may be due to differences in species (HERG versus guinea-pig and mouse IK,r), tissues (oocytes versus myocytes) and/or specific drugs. 46-43-12: In some comparative studies 85, E-4031 in

MK-499

E-4031; lack of K+ channel target selectivity

the micromolar range inhibits several classes of K+selective channels (e.g. transient, sustained, and inwardly rectifying) in addition to a sodium-selective current in dissociated rat taste receptor cells. Other comparative studies have compared £-4031 with sematilide and d-sotaloI 86; (see paragraph 46-43-01).

E-4031; [Mg2 +]i dependence of selectivity

46-43-13: Under normal physiological conditions, E-4031 is a specific blocker of IK,r. However, in the absence of intracellular Mg2 +, E-4031 also partially blocks IK,s (see VLC {K} minK, entry 54). Block of IK,s can be prevented by prior treatment of cells with isoproterenol, suggesting that E-4031 only blocks nonphosphorylated IK,s channels in the absence of intracellular Mg2 +75 . Note: E-4031 (0.01-0.3J.lM) is a potent contractile agent in rat portal vein preparations.

Drug-induced arrhythmias

46-43-14: Block of I K,r has been associated with druginduced cardiac arrhythmias (see paragraph 46-43-02).

Dofetilide (also a 46-43-15: The methanesulfonanilide I Kr blocker Kir2.x family blocker) dofetilide acts as as slow-onset/slow-offset, highaffinity open channel blocker of HERG expressed in oocytes (EC so approx. 12 ± 211M)87. Dofetilide block has been extended to single-channel studies of HERG

_r.......-

e_n_try_4_6_

Table 6. Continued Blocker

Species/homologue, characteristics and references in oocytes 83 . Notably, however, dofetilide has also been shown to potently block inwardly rectifying channels of the Kir2 family (see note 2).

Dofetilide interaction 46-43-16: Chimaeric constructs between hIRKl and ROMK1 (Kir 1.1, dofetilide resistant) have been used sites to show hydrophobic interactions are essential for dofetilide block in hIRK (and possibly HERG channels)88. L691,121 WIN 61773-2 High- and lowaffinity dofetilidebinding sites

II

46-43-17: Competitive binding studies indicate that E-4031, L-691,121 (3,4-dihydro-1'-[2-(benzofurazan-Syl)ethyl]-6-methanesulonamidospiro[(2H)-1benzopyran-2,4'-piperidin]-4-one), and WIN 61773-2 [(R)( + )-4,S-dihydro-4-methyl-1-phenyl-3(2phenylethyl)-( 1H)-2,4- benzodiazepine monohydrochloride] inhibit IK,r channels by interacting at sites distinct from the high affinity [3H]-dofetilide-binding site (see Ligands, 46-47). WIN 61773-2 binding suggests that it is an allosteric modulator of the dofetilide-binding site89. In separate studies, dofetilide has been shown to interact with high- and low-affinity sites in guinea-pig myocytes in a distinct manner, but it is likely that the highaffinity dofetilide-binding site is related to I K ,r 9o .

Dofetilide affecting cardiac pacemaker function

46-43-18: In rabbit sinoatrial node (SAN) cells, dofetilide can separate delayed rectifier current into 'drug-sensitive' current (IK,r) and 'drug-insensitive' current (I K,s)91. The dofetilide-sensitive current activates rapidly and it showed two components of deactivation, the larger of which is very slow, while the dofetilide-insensitive current activates more slowly and deactivated quickly. Dofetilide slows spontaneous activity, suggesting that IK,r contributes to the pacemaker activity of the SAN cell91 . It has been concluded that SAN IK,r plays an essential roles in (i) determining the maximum diastolic potential and (ii) ensuring the firing of the following action potential in SAN cells 92. Other results suggest that IK,r has 'much less effect' on atrioventricular nodal pacemaker activity than on Sl~ pacemaker activity93.

Non-specific blockers (but including I K,l) NE-I0064

46-43-19: The antiarrhythmic agent NE-I0064 (azimilide) has been reported as a 'selective blocker' of the slowly activating component of the delayed rectifier, IK,s (see KvLQT1/minK under VLC {K} minK, entry 54). In ferret cardiac papillary muscle

""---e_n_t_ry_4_6

_

Table 6. Continued Blocker

Species/homologue, characteristics and references preparations, however, NE-I0064 blocks lK,r at an ICso of 0.4 JlM, nearly tenfold greater potency to lK,s (ICso approx. 3 JlM). Note: NE-I0064 also inhibits lea in a use-dependent t fashion 94 .

RP 58866/terikalant

46-43-20: The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, were originally reported to 'selectively block' the inward rectifier K+ current, lkl (see Blockers under lNR K [native), 32-43). However, these drugs also potently block lK,r with ICsos values of 22 and 31 mM, respectively95.

Berberine

46-43-21: The antiarrhythmic drug berberine prolongs action potential duration in cat ventricular myocytes without altering other variables of the action potential and appears to 'preferentially block' lK,r 96 .

Flecainide

46-43-22: The antiarrhythmic class IC drug flecainide also possesses class III effects. 10-30 JlM flecainide inhibits the lK,r but not lK,r in guinea-pig cardiac ventricular myocytes 97. 46-43-23: Along with Kvl.5 98, HERG has been

Terfenadine Desmethylastemizole described as a primary cardiac ventricular target of Erythromycin the non-sedating antihistamine terfenadine with a Ketoconazole Kd(apparent) of 350 nM in oocytes, approx. 10 times more sensitive than Kvl.S (Kd(apparent) of 2.7 J,lM)99. These findings may have relevance to the fact that administration of the antihistamine terfenadine to patients can result in acquired long QT syndrome and ventricular arrhythmias 99 (e.g. Seldane, whose clinical plasma concentration may reach 100nM). In addition to terfenadine, another histamine receptor antagonist (astemizole) has been shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co-medications. Astemizole has been shown to block HERG in oocytes at nanomolar concentrations 100 in addition to its metabolite, desmethylastemizole101 . Other agents commonly assocated with cardiotoxicity (e.g. erythromycin102 and ketoconazole 103 ) also block HERG currents at high potencyl04. Cocaine

46-43-24: Cocaine non-selectively blocks a current identical to 'E-4031 sensitive' current lK,r in isolated guinea-pig ventricular myocytes (IC so rv 4 JlM) in

II

_L.-

e_n_try_4_6_

Table 6. Continued Blocker

Species/homologue, characteristics and references addition to L-type calcium current and the TTxsensitive plateau current at higher concentrations (30-100 )lM)105.

BRL-32872

46-43-25: The novel antiarrhythmic agent BRL-32872 [N-(3,4-dimethoxyphenyl)-N-[3[[2-(3,4dimethoxypheny1)ethyI] propyl]-4-nitrobenzamide hydrochloride] inhibits the IK,r in guinea-pig cardiac preparations (EC so 2.8 )lM)106.

Combretastatin Bl

46-43-26: Combretastatin Bl, a polyhydroxybibenzyl compound extracted from the fruit of Combretum kraussii, (the source of 'hiccup nut' toxin) inhibits HERG-type native K+ channels (ICso 300 )lM)107.

Benzodiazepine derivatives

46-43-27: Characterization of a series of 4,5-dihydro3-[2-(methanesulfonamidophenyl)ethyl]-lH-2,4benzodiazepines as potential antiarrhythmic agents that interact at channels underlying IK,r has appeared108.

Other common blockers

46-43-28: d-eag: Quinine ICso 0.7 ± 0.3 mM (note 1)5; quinidine,0.4±0.15mM; quinine 0.9±0.3mM; quinidine 0.4 ± 0.2 mM (i.e. similar to d-eag).

Notes: 1. ICso indicates concentration of blocker required for half-maximal inhibition of the stated current determined under two-electrode voltageclamp. 2. hIRKI (hKir2.1, see entry 33) has been reported109 as a target for dofetilide (IC so 533nM at 40mV and 20°C) with 'no significant effects' on hKvl.2, hKvl.4, hKvl.5, or hKv2.l. 3. The 'HERG-like' current in F-ll neuroblastoma cells (see Cloning resource, 46-10) shares pharmacological features described for native cardiac IK,rincluding similar sensitivities to E-403l, WAY-123,398, Cs+, Ba2 + and La3 +74. 4. Functionally, a 'Ca2+-inhibitable' property might be expected to specifically amplify excitatory stimuli related to membrane depolarization and [Ca2 +h. 5. In control experiments, Kvl family channels such as Kvl.l and Kvl.2 (entry 48) were not inhibited by 400)lM [Ca2+h. 6. [Ca2+h has been suggested to transduce muscarinic block of M-channels, underlying native M-current (for details see VLC K M-i [native], entry 53). These and other kinetic similarities have been discussed in two serial commentaries 110,111. f".J

II

IL-e_n_ _ry_4_6

_

t

-2

/ (JlA)

-3

0 [Cs+]

(mM)

-4

0 -5

[Ba 2 +]

-5

-6

(mM)

Figure 7. (a) Cs+ inhibition of peak inward HERG current in voltage-clamped oocytes. The figure shows an I- V plot before (.) and after (e) application of 1 mM Cs+ in the bath solution, with inhibition being more effective at hyperpolarized potentials. (b) Ba 2+ inhibition of HERG current, with no apparent voltage-dependent component over the concentration range shown. Bath solution was 20mM KC1, 80mM N-methyl glucamine chloride, 1.8mM CaC12, 1.0mM MgC12, 5mM HEPES (pH 7.4). (Reproduced with permission from Trudeau et al. (1995) Science 269: 92-5.) (From 46-43-06)

in inside-out patches, indicating a 'direct modulation' of Drosophila eag channels by cAMps suggesting binding of cAMP to the channel protein. Comparative notes: 1. Rat eag channel currents are unaffected by 8-BrcAMP or 8-Br-cGMP (2mM) in voltage-clamped oocytes 7. 2. Unlike the vertebrate cyclic nucleotide-gated (CNG) channels, which are relatively voltage insensitive (see ILG CAT cAM~ entry 21 and ILG CAT cGMp, entry 22), activation of eag channels show a 'very steep' voltage dependences,8. 3. A possible role for cAMP-modulation of Drosophila eag-type channels in controlling synaptic efficiency in the central and peripheral nervous systems has been discusseds (see also Selectivity, 46-40 and Voltage sensitivity, 46-42).

Heterologously expressed HERG channel activity is unaffected by cyclic nucleotides 46-44-02: h-erg: The HERG channel contains a segment homologous to a cyclic nucleotide-binding domain near its C-terminus (see Domain conservation, 46-28). Following its heterologous expression of homomultimeric HERG subunits in Xenopus oocytes, application of membranepermeable analogues of cAMP and cGMP to bath solutions show 'no significant effects' on current magnitude or voltage-dependence of channel activation 12 (compare with previous paragraph).

Correction of LQT abnormalities by [K+ 10 modulation of HERG 46-44-03: The profound modulatory effects of extracellular K+ in increasing the amplitude of HERG current12 (see Single channel data, 46-41) implies

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_'--

e_n_t_ry_4_6_

that under conditions of lowered extracellular K+ (i.e. in modest serum hypokalaemia t ) the effects of IK,r blockers would tend to be exaggerated, leading to iexcessive' lengthening of cardiac action potentials, and induction of torsade de pointes. Modest serum hypokalaemia is a commonly observed condition and its incidence has long been associated with ventricular arrythmias l12 and acquired LQTS (see Resen, 1988, under Related sources and reviews, 46-56). Elevation of serum [K+] in hypokalaemic patients receiving medications which block IK,r or in individuals with the chromosome 7-linked LQTS was originally suggested12 as a potential therapeutic intervention for prevention of torsade de pointes. In a later study l13, increase of serum [K+] has been demonstrated to correct abnormalities of repolarization duration, T-wave morphology, QT/RR slope (slope of the relation between Q-T interval and cycle length), and Q-T dispersion in patients with chromosome 7-linked LQTS.

Extracellular divalent cation modulation of rat eag 46-44-04: Extracellular Mg2 + at physiological concentrations 'dramatically slows' activation of r-eag channels expressed in oocytes in a dose- and voltage-dependent manner82 . Similar effects on r-eag activation kinetics by other divalent cations can be observed to an extent that correlates with the ions enthalpy of hydration t. Extracellular H+ ions have been shown to compete with [Mg2 +]0 as the modulatory effects of Mg2 + can be abolished at low pH (i.e. lowering the external p:H also result in a slowing of the activation). A strong dependence of rat eag activation on both [Mg2+]0 modulation and the resting potential have been postulated to constitute a system for fine-tuning iK+ channel availability' in neuronal cells (i.e. where all four r-eag subunits undergo a Mg2 +-dependent conformational transition prior to final channel activation82 .

Ligands Displacement of [3H}-dofetilide with other antiarrythmics 46-47-01: [3H]-Dofetilide binds with high affinity to sites associated with the guinea-pig cardiac IK,r channel (see Table 6; several class ill antiarrhythmic agents, including dofetilide, clofilium, quinidine, sotalol and sematilide, competitively displace [3H]-dofetilide with IC so values that correlate with those for blockade of the IK,r channel (cited in ref. 89) (see also Blockers, 46-43).

Openers 46-48-01: For 'opening' of HERG by extracellular K+ 1 see Channel modulation, 46-44.

Receptor/transducer interactions Mechanisms of arrhythmia suppression by beta-blockade 46-49-01: h-erg: The mechanisms of receptor-coupled (endogenous) control of HERG current in native cardiac cells are presently unclear. Arrhythmias and

1

e_n_t_ry_4_6

_

syncope in LQTS (see Phenotypic expression, 46-14) are induced by sympathetic nerve activity, and because of this, ,B-adrenergic antagonists are frequently prescribed (these agents also suppress the effect of defective KvLQTl channels, see entry 54). Since HERG current does not appear to be affected by cyclic nucleotides (in heterologous cells, see Channel modulation, 46-44) the beneficial effects of ,B-blockade in preventing development of arrhythmia may be due to cAMP-dependent regulation of channel subunits other than HERG, important candidates being voltagegated Na+ channels (see LQT3 in Table 3 under Chromosomal location, 46-18 and Protein phosphorylation under VLG Na, 55-32) and voltage-gated Ca2 + channels (i.e. reducing effects of cAMP-dependent phosphorylation suppressing 'secondary depolarizations' - see Phenotypic expression, 46-14 and Protein phosphorylation under VLG Ca, 42-32). Note however that different LQT2 mutations may affect HERG function in different ways, and there appears to be a role for additional subunits in native HERG function (see Protein interactions, 46-31). These factors make the 'precise' contribution for receptor-linked modulation of HERG difficult to analyse.

Integrin-mediated neurite outgrowth 46-49-02: h-erg: Integrin-mediated neurite outgrowth in neuroblastoma cells appears to depend on the activation of HERG channels in situ - for further details on possible receptor/effector coupling mechanisms, see refs 19,20,114,115 and Developmental regulation, 46-11.

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 46-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction eiJ layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in Introduction etJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouring t analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers), author /reference or nomenclature. Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly reported and related sequences.

II

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entry 46

Eag/Elk/Erg subfamily members Listings are sorted first by subfamily designation then species name (equivalent sequences with different clone names are grouped together in rows). See also notes at foot of table. Type (see field 05)

Original description

Species, DNA source

d-eag

Drosophila eag cDNA sequence

Drosophila head-specific library

m-eag Mouse eag species homologue

Original isolate ORF

Accession Sequence/ discussion

Sequence of cDNA CH20 ORF: 1174aa Mouse brain Mouse brain cDNA library cDNA library ORF: 989aa

gb: M61157

Warmke, Science (1991) 252: 1560-2.

gb: U04294

Warmke, Proc Natl Acad Sci USA (1994) 91: 3438-42. Warmke, Proc Natl Acad Sci USA (1994) 91: 3438-42. Ludwig, EMBOJ (1994) 13: 4451-8.

d-elk

Drosophila elk cDNA sequence

Drosophila elk == ~aghead-specific like K+ library channel

gb: U04246

r-eag

Rat eag cDNA sequence

gb: Z34264

h-erg

Mouse erg species homologue

Rat Reag01/ cerebellum Reag02, cDNA library composite sequence, see Isolation probe, 4612.0RF: 962aa Human erg== ~aghippocampal related gene. cDNA library Isolated by highstringency screen with an m-eag probe:

gb: U04270

Warmke, Proc Natl Acad Sci USA (1994) 91: 3438-42.

Related sources and reviews 46-56-01: Major sources used for compilation of this entrT,7,12; long Q-T syndrome review/molecular mechanisms (1996)116; antiarrhythmic interventions; clinical aspects l17; (1992 review)118; debate relating to resemblance of EAG to native M-current (entry 53)110,111.

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Book references: Rosen, D.M. (1988) Arrhythmogenic potential of class III antiarrhythmic agents: comparison with class I agents. In Control of Cardiac Arrhythmias by Lengthening Repolarization (ed. B.N. Singh), pp.559-76. Futura Publishing, Mount Kisco, New York. Schwartz, P.J., Locatis, E.H., Napolitano, C. and Priori, S.C. (1995) The long QT syndrome. In Cardiac Electrophysiology: From Cell to Bedside (eds D. Zipes and J. Jalife), pp. 788-81. Saunders, Philadelphia. Tseng, C.-N. (1995) in Cardiac Electrophysiology: From Cell to Bedside (eds D. Zipes and J. Jalife), pp.260-8. Saunders, Philadelphia.

Feedback Error-corrections, enhancements and extensions 46-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource T- search criteria). For this entry, send e-mail messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 46-32-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback etJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

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e_n_t _ry_46_1

VLG K Kv-beta Edward C. Conley

Cytoplasmic (Kv,B) subunits co-assembling with pore-forming (Kvo) voltage-gated potassium channel subunits Entry 47

NOMENCLATURES Abstract/general description 47-01-01: The existence of Kv-beta subunit polypeptides (Kv{3, this entry) were first detected in native membranes forming part of co-immunoprecipitated complexes using antibodies raised against various Kv alpha subunit polypeptides. In particular, affinity-purificationt and immunoprecipitation of the a-dendrotoxin (a-DTx) sensitive K+ channel complex from bovine brain initially revealed the presence of 38 kDa (major) and 41-42kDa (minor) {3 subunit polypeptides in association with the 'DTx acceptor complex' including various Kvl subfamily a subunit polypeptides. These early studies provided important direct evidence for native voltage-gated K+ channels consisting of an eight subunit hetero-oligomeric sialoglycoprotein complex ('4a4{3 octamers'). 47-01-02: Availability of cDNA clones encoding both Kva and Kv{3 subunits confirmed the presence of rv38-41 kDa {3 subunit polypeptides in 'tight association' with the pore-forming subunits within expressing cells (see Protein molecular weight (purified), 47-22.). Because Kv{3 subunits do not in themselves form integral, ion-selective pores, they have been termed lauxiliary' or laccessory' proteins associated with pore-forming (a subunit) complexes. Subsequently, heterologous co-expression of Kv{3 with Kva subunits has shown {3 subunits able to profoundly affect channel properties (including modulation of inactivation kinetics, voltage dependence of gating and current amplitudes - see Phenotypic expression, 47-14). Moreover, endogenous Kv{3 subunits (e.g. in cell lines) can affect heterologous expression properties of Kva subunits, and Kv{3-toKva-subunit mRNA ratios are known to influence phenotype in heterologous expression systems (ibid.). Many data are consistent with Kv{3 subunits acting as a 'scaffold' or 'adaptor' to bring about'appropriate' hetero-oligomer formation where cells co-express multiple Kva subtypes. 47-01-03: In vivo, certain Kv{3 subunits may have the capacity to modify neuronal output and firing patterns by regulating the 'phenotypic' expression of A-type t Kv channel activities (ibid., see also VLC K A- T [native], entry 44). Heterologous co-expression experiments have determined that Kv{31a subunits can accelerate rates of inactivation in expressed K+ currents (for details, see Inactivation, 47-37). In the heart, the alteration of several functional properties of hKvl.5 channel subunits by Kv{31b subunits offers a potential mechanism for Kva/{3 associations to generate/modulate regional variations in cardiac repolarizing current.

47-01-04: Hypothetically, Kv{31 subunits may act as sensors for 'oxidative stress' in neurones, by virtue of reversible oxidation/reduction of a 'critical cysteine' in the Kv{31 inactivating ball domain (hypothetically, increases in oxygen radicals t would be predicted to eliminate (3 subunit inactivating functions, and thereby enhance K+ efflux) (for details and an illustration of possible 'redox sensing' mechanisms, see the optimized alignments of Kv{3 N-termini (Domain functions, 47-29) and Channel modulation, 47-44).

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47-01-05: Extensive, comparative Kv,B subunit-specific mRNA hybridization studies (summarized under mRNA distribution, 47-13) have confirmed patterns of differential (heterogeneous) expression, particularly in brain. High densities of Kv,Bl mRNA are detectable in the striatum, the CAl subfield of the hippocampus, and in cerebellar Purkinje cells. High densities of Kv,B2 mRNA can be seen in the cerebral cortex, cerebellum and brainstem. Kv,B3 mRNA has a distinct distribution pattern, but is still predominantly expressed in brain. cRNA probes have characterized several Kv,B variants in various regions of heart and the vasculature. 47-01-06: Several studies have used imlnunocytochemical methods to colocalize K+ channel a and,B subunit polypeptides in brain slices (summarized under Protein distribution, 47-15). Co-localized Kv,B1 and Kv,B2 protein has been shown to be concentrated in neuronal perikarya, dendrites and terminal fields, and in the juxtaparanodal region of myelinated axons. Furthermore, immunoblot and reciprocal co-immunoprecipitation t analyses have predicted that most K+ channel complexes containing Kv,Bl also contain Kv,B2, which is the major Kv,B component in brain; individual Kv channels may contain 'two or more' biochemically and functionally distinct Kv,B subunit polypeptides. 47-01-07: A Kva-Kv,B interaction site motif in KvI subfamily channel N -termini has been described (see Sequence motifs, 47-24). Studies of cytoplasmic Nterminal domains of Kvl a subunits have determined that the Kv,131-binding site overlaps with the region known as the NAB(Kvl) tetramerization domain (for details, see Protein interactions under VLC K Kv1-Shak, 48-31). The NAB domain appears to mediate assembly of both Kva-Kva and Kva-Kv,B complexes. 47-01-08: A number of potential post-translatory modification t sites exist on Kv,B proteins including multiple phosphorylation consensus sites (see Protein phosphorylation, 47-32) and sites for amidation t . As predicted for proteins with cytoplasmic locations, Kv,B subunit sequences do not contain motifs for leader sequencest or N-glycosylation, but structural motifs thought to be involved in NADPHt binding and the hydrogen transfer mechanismt in aldo-keto reductase t superfamily members are conserved. 47-01-09: Kv,B2 subunits have major roles in promoting K+ channel protein surface expression (see Subcellular locations, 47-16). By analogy to an action of a chaperone t protein, Kv,B2 exerts effects on associated Kvl.2 which include promotion of co-translational N-linked glycosylation of the nascent Kvl.2 polypeptide, increased stability of Kv,B2 /Kval.2 complexes, and enhanced Kv1.2 protein turnover. 47-01-10: Stable association of Kv,132 and Kvl.2 a subunits occurs early in K+channel biosynthesis (5 min), most likely at the endoplasmic reticulum. These observations, together with determination of lKvo: lKv,13 subunit stoichiometric assemblies in co-transfected cells have been used to propose a K+channel oj,13 subunit interaction model (as illustrated in Fig. 1 under Subcellular locations, 47-16). 47-01-11: Kv,B subunit-related primary sequences have been identified in flies encoded by the Hyperkinetic (Hk) locus, plants and prokaryotes (see Database listings, 47-53 and Miscellaneous information, 47-55), perhaps

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suggesting that primordial t K+ channels were assembled from Kva:,B subunit combinations. On the basis of primary sequence comparisons, various Kv,B subunit proteins are likely to share structural relationships with members of the NAD(P)H-dependent aldo-keto reductase t superfamilyt (ibid.). Kv,B cDNA sequence variants described thus far show a general pattern of variable N-termini with constant I core' regions; the existence of alternative splicing of Kv,Bl primary transcripts is now well established (see Gene organization, 47-20) and can account for marked sequence variation at N-terminL Subunit genes belonging to different Kv,B subfamilies (e.g. Kv,Bl, Kv,B2 and Kv,B3, see Gene family, 47-05) show much amino acid sequence variability throughout their predicted open reading frames. 47-01-12: No single, systematic nomenclature for Kv,B subunits or genes has been adopted, and this has contributed to some confusion in subunit/gene naming within the early literature. In particular, the late recognition of alternative splicing t of the Kv,Bl primary transcriptt (see Gene organization, 47-20) led to the same name being used for different molecular entities (for co-listing of names in use, see Table 1 under Gene family, 47-05). Overall, studies of Kv,B subunits have suggested further possibilities for extending K+ current diversity, and in consequence, additional mechanisms for modulation of excitability t in native cells.

Category (sortcode) 47-02-01: VLG K Kv-beta, Le. auxiliary (beta) subunits associated with poreforming Kv (alpha) subunits. Note: Although Kv beta subunit proteins (Kv,B, this entry) do not form integral pores their profound significance as 'accessory' or 'auxiliary' subunits for generating functional variation in oligomeric complexes with certain Kv channel a subunit channels (e.g. see Protein interactions, field 31 in entries 48-51) warrants independent description. Different Kva/,B subunit compositions may generate voltagegated potassium channels with properties distinct from the ones expressed by a subunits alone (for review see ref. 2 and other articles listed under Related sources and reviews, 47-56).

Information sorting/retrieval aided by designated gene family nomenclatures 47-02-02: The gene product prefix (used as a unique embedded identifier or VEl) for 'tagging' and retrieving information relevant to the contents of this entry on the CSN website will be of the form VEl: Kvbns where n is a designated number in a [future] systematic nomenclature and (where appropriate) s is a designated ~lice variant letter (e.g. Kv,Bla or where Greek characters are not available, Kvbla). Within this entry, paragraph 'running orders' (sort orders) are largely determined alphanumerically by systematic nomenclatures - Le. denoting species and gene product prefix, sometimes combined with any trivial or clone name(s) where these have been used in the source reference (e.g. hKv,Blb(= clone hKv,B3):). Where properties are likely to apply to all or several subfamily proteins (Le. irrespective of species or isolate) the 'species' term may be omitted.

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Channel designation 47-03-01: Although no systematic designations have been proposed for specification of Kv channels comprising defined Kva/Kv{3 subunit complexes, these would normally make reference to gene family relationships and species of origin. For example, a complex formed between homomeric human Kvl.5a subunits with human Kv,Bl subunits could be designated as 'hKvl.5a:hKv,Bla' or hKvl.5a/,Bla. The known a/{3 subunit stoichiometry of 4a to 4,B subunits is generally assumed, unless the a subunit components are physically associated (tandemly linked at the DNA level) in a defined heteromultimeric complex (see Channel designation under VLG K Kvl-Shak, 48-03). More generally, Kva/,B complexes have been referred to as 'a4,B4 stoichiometric complexes' or '4a4{3 octamers' (see Predicted protein topography, 47-30).

Gene family Gene family assignments (in lieu of a systematic nomenclature) 47-05-01: As described in Subtype classifications (47-06) there was no single nomenclature for Kv,B subunits or their genes at the time of entry compilation, and this has contributed to some confusion in subunit/gene naming within the early literature. In particular, the late recognition of alternative splicing t of the Kv,Bl primary transcriptt (see Gene organization, 47-20) led to the same name being used for different molecular entities and conversely, different names being used for the same gene product. In due course, all nomenclature is likely to converge to gene family-based classifications, but for present purposes, alternative names in use are listed side-by side in Table 1.

Relationship of Kvj3 subunits to the NAD(P)H-dependent oxidoreductase superfamily 47-05-02: Like Kva subunits, ,B subunit-related primary sequences have been identified in flies encoded by the Hyperkinetic (Hk) locus, plants and prokaryotes (see Database listings, 47-58 and Miscellaneous information, 47-55). The representation of Kv,B-like subunits over such a wide 'evolutionary gap' has led to the suggestion that primordial t K+ channels were assembled from Kva:,B subunit combinations2 . Furthermore, sequence and secondary structure alignments have indicated a distant gene family relationship between genes encoding various Kv,B subunit proteins and members of the NAD(P)H-dependent aldo-keto reductase t superfamilyt, see Miscellaneous information, 47-55.

Subtype classifications Classifications/nomenclatures for entry

Kv~j

genes/subunits used in this

47-06-01: Kv,B gene/subunit names used in this entry are largely as they have appeared in the source references based on the initial nomenclature

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Table 1. Gene family nomenclature and Inon-systematic' names in use for {3 subunits associated with vertebrate voltage-dependent K+ channels. cDNA Iclone' names (isolates) in common use are indicated in bold. For background to modified nomenclatures used as underlined prefixes to entry paragraphs, see Subtype classifications, 47-06. (From 47-05-01) Paragraph Human Rat Mouse Other designation isoforms isoforms isoforms species Subfamily Kv{31 Kv,81a Names in use and refs (see notes 1 and 2) Kv,81b Names in use and refs Kv,81c Names in use and refs

hKv{31 transcripts hKv,81a: hKv,81 3

hKv,81b: hKv,83 5 hKv,83 6 hKv,83 7 hKv,81c: hKv,81.3 8

rKv{31 transcripts rKv,81a: rKv,81 4

mKv{31 transcripts

rKv,81b: rKv,83 7

Other

fKv,83: Ferret Kv,81b 7

Subfamily Kv{32 Names in use and refs

hKv{32

rKv{32 rKv,82: rKv,82 RCK,82 9

mKv{32

Other bKv,82: Bovine Kv,82 9

Subfamily Kv{33 Names in use and refs

hKv{33

rKv{33 rKv,83: rKvj33 10,11 (see note 7)

mKv{33

Other

Notes: 1. For reasons outlined in Subtype classifications, 47-06, the underlined prefix names are used to establish a 'running order' of entry paragraphs, with any 'clone' or 'isolate' names appended (e.g. hKv,81b (== clonehKv,8a). 2. rKv{31a, rKv{31b and hKv{32 subunits share >82 % identity in the Cterminal 329 aa and show 'low identity' in the N-terminal 79 aa, indicating the likely position of splicing - see the alignment under Encoding, 47-19. 3. Numerical subscripts have sometimes been used to designate Kv{3 subunit types in the literature, but designations in this entry have been largely cited as non-subscripted forms (in line with ref. 4 ). 4. All Kv{3 subunit designations are tentative (for updates on Isystematic' nomenclature of Kv beta subunits, see the IUPHAR pages on the CSN). 5. For convenience, references to the Drosophila Hyperkinetic subunit/gene are prefixed by dHK{3. 6. For sequence accession numbers, see Database listings, 47-53. 7. Rat Kv{33 (as designated in refs.l0,11, but see note 4) is a 403 amino acid residue protein with a 680/0 amino acid sequence homology to Kv{3 1.1.

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established in ref. 4 (with occasional use of initial letters to designate species). Note, however, that two cDNAs t encoding voltage-gated K+ channel {3 subunits isolated from human heart, originally designated hKv {33 (in ref. s) and hKv,81.3 (in ref.8) represent alternative splice variants t arising from a single gene (i.e. encoding all human Kv,81 variants; as confirmed independently in ref. 12 ). Before this was established, England et a1. 8 suggested a simplified nomenclature for Kv{3 subunits encompassing all the then known gene and vertebrate species variants. To retain consistency with other entries, however, this nomenclature has not been adopted exactly as proposed. Designation of splice variants of the Kv{31 gene as Kv{3I.I, Kv{3I.2 and Kv{3I.3 (as proposed8 ) is inconsistent with designation of splice variants in other K+ channel systelnatic nomenclatures, which are normally designated by serial letters -- e.g. Kv3.2a, Kv3.2b, Kv3.2c and Kv3.2d (i.e. all products of the single Kv3.2 gene - see VLG K Kv3-Shaw, entry 50) or Kirl.la, Kirl.lb, Kirl.lc, Kirl.ld and Kirl.le (i.e. all products of the single Kirl.l gene - see INR K [subunits), entry 33). Adoption of Kv{3I.I, Kv{3I.2 and Kv{3I.3 (as proposed8 ) might also incorrectly imply these gene products arise from separate genes within a gene subfamily (as correctly implied for the established KVQ gene subfamily nomenclature series Kvl.l, Kvl.2, Kvl.3, etc. (see VLC;' K Kv1-Shak, entry 48). For the present purposes therefore, designation of alternative splice variants of the Kv{31 gene will follow the series Kv,81a, Kv,81b and Kv,81c. This nomenclature retains consistency with other entries, and helps indicate their possession of a common C-terminal exon (see Gene organization, 4720). The designations Kv{3la, Kv{3lb and Kv{3lc appear as underlined prefixes coupled to original clone names and have determined the 'running order' of entry paragraphs. All alternative names in use are listed and compared in Table I under Gene famil'y, 47-05. For updates on nomenclatures applicable to Kv{3 subunits and other K+ channel subunits (including newly isolated clones appearing after going to press) see the CSN website (www.le.ac.uk/csn/).

Trivial names 47-07-01: Because Kv{3 subunits do not in themselves form integral, ionselective pores, they have been termed lauxiliary' or laccessory' proteins associated with pore-forming (Q subunit) complexes. Selected crossreferences for {3 subunits associated with voltage-gated channels other than the Kv family are listed under Miscellaneous information, 47-55.

EXPRESSION

Cell-type expression index 47-08-01: See Isolation probe, 47-12, mRN~ distribution, 47-13, Subcellular locations, 47-16, Protein distribution, 47-15 and Protein molecular weight (purified) 47-22.

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Channel density 47-09-01: For notes on the capacity of the Kv,B2 subunit co-expression to alter cell surface expression characteristics of K+ channel complexes (and in consequence channel densities), see Phenotypic expression, 47-14.

Cloning resource 47-10-01: For sources of mRNA used to construct eDNA libraries for retrieval of presently known Kv,B subunit isolates, see Database listings, 47-53. Low molecular mass fractions of rat brain poly(A)+ mRNA have been shown capable of slowing13, accelerating or modifying the surface expression14 of outward K+ currents mediated by A-type channels (following co-injection with KVQ subunit mRNA in oocytes). The (unidentified) factors encoded by these fractions may include ,B subunit activities (for significance, see Phenotypic expression, 47-14 and other fields).

Developmental regulation 47-11-01: No specific examples of Kv,B gene or protein regulation in a developmental context were described at the time of compilation. From the phenotypic roles already established for Kv,B subunit variants, however, (see Phenotypic expression, 47-14) several known developmental 'cues' (e.g. phosphomodulation t or altered redox states t ) could be expected to exert profound changes on cellular excitability or Kv channel expression determinants.

Isolation probe Microsequencing procedures used to isolate Kv{3 subunit prototypes 47-12-01: bKv,81a: Affinity purificationt and immunoprecipitation of the adendrotoxin (a-DTx)-sensitive K+ channel complex from bovine brain1S- 18 revealed the presence of both 38 kDa (major) and 41-42 kDa (minor) ,B subunit polypeptides in association with the DTx acceptor complex including various Kvl subfamily Q subunit polypeptides (see Protein interactions, 47-31 and for background, see Blockers, 47-43). Direct microsequencing t of 'separated' bovine ,B subunits initially proved technically difficult, and it was first necessary to purify proteolytic fragments using trypsin t , V8t and Asp Nt proteases 9 . Microsequencing of 10 PAGEtpurified peptides yielded nine new amino acid sequences which did not match any known KVQ subunit in database searches 9 . The partial sequences were used to design oligonucleotide peR primer pairs to retrieve eDNA fragments, and later a full-length eDNA sequence from a Agtl0 bovine brain eDNA library (367 aa, see Database listings, 47-53). Using cloned eDNA encoding the bovine DTx acceptor ,B subunit 9 as a probe, cDNAs encoding two homologous polypeptides, initially designated Kv,Bl and Kv,B2 were isolated from rat brain cortex cDNA libraries by conventional low-stringency hybridization4 . Note: The inability to align one peptide sequence derived from microsequencing of ,B subunits associated with QDTx-sensitive K+ channels 9 appeared consistent with the presence of a

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(minor) alternative {3 subunit isoform with a divergent N-terminus and 78% sequence identity to the (major) bovine Kv{32.

Early co-immunoprecipitation experiments suggesting the presence of (3 subunits 47-12-02: The first studies using toxin cross-linking and immunoaffinity procedures identifying multiple Kva/{3 subunit interactions in native K+ channel subunit assemblies (i.e. as part of large hetero-oligomeric t sialoglycoproteint complexes) are outlined in Table 5 under Protein molecular weight (purified), 47-22. Similarly, immunoprecipitation of K+ channel complexes from radioiodinated rat brain membranes using antibodies specific for the rKv2.1/drkl K+ channel a subunit revealed the presence of a 38 kDa {3 subunit polypeptide in 'tight association' with the 'delayed rectifier' complex19. (Note, however that the molecular species is likely to be distinct from Kv{31 or Kv,81, since the latter do not appear to co-precipitate with Kv2.1.)

Isolation of likely splice variant forms of Kv(31 47-12-03: hKv{31b (== clone h Kv,83): A peR-generated eDNA fragment corresponding to nt 435-1089 of rKv{32 was used to screen a A eDNA library derived from cardiomyopathic human heart left ventricular mRNA pools5. A 'partial' clone exhibiting predicted homology (but little identity) to rKv{31a and rKv{32 was used to retrieve a further clone from a eDNA library derived from healthy human cardiac left ventricular eDNA. This clone had a 3.2 kb insert including a large internal ORF predicting a 408 aa coding region for clone hKv{33 (hKv{31b, lor clarification, see Subtype classi-

fications, 47-06).

mRNA distribution General notes on Kv(3 mRNA distributions (see also this field under VLG K Kv1-Shak, 47-13) 47-13-01: mRNA distribution studies which take into account the likely alternative exon t usage from Kv,8 genes were incomplete at the time of compilation. Reported patterns may therefore be different according to which region(s) of Kv{3 sequences were used as a probe, and, in particular, some exon probe sequences may be shared between splice variants. Further investigation of Kv,8 gene organization and transcript mapping t is likely to define probes unique to each 'isoform'. Furthermore, although mRNA distribution patterns may indicate intracellular sites of channel protein biosynthesis (generally the endoplasmic reticulum t in 'expressing' cell types), they do not necessarily report the 'final' distribution(s) of subunits into mature assembled channel complexes, and post-translational transport of protein to distal sites are well-documented. This factor is of some significance for (at least) Kv{32 subunits, whose expression exerts marked changes in cellular localizations of Kva/{3 channel complexes (see Phenotypic expression, 47-14). With these caveats, the following paragraphs

summarize results of Kv{3 mRNA distribution studies, defining the probe in each case.

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Comparative Kv{31, Kv{32 mRNA versus protein expression patterns 47-13-02: rKv(31; rKv(32: ISH: A summary of expression patterns described for Kv(31 versus Kv(32 subunits in adult rat brain from the extensive study by Rhodes et al. (1996)20 is given in Table 2. This work directly compared subunit-specific mRNA hybridization to immunolocalization patterns especially in neocortex/hippocampus, striatum/basal forebrain, thalamus/ hypothalamus, midbrain and cerebellum/brainstem. In general, (3 subunit genes appeared heterogeneously expressed, with high densities of rKv(31 mRNA in the striatum, CAl subfield of the hippocampus, and cerebellar Purkinje cells. High densities of rKv(32 mRNA were observed in the cerebral cortex, cerebellum and brainstem (for details and original autoradiographs, see ref. 20).

Kv{31a mRNA is predominantly expressed in the rat nervous system 47-13-03: Kv(31a/other splice variants: ISH: Using antisense oligonucleotide probes complementary to the 3' untranslated sequence of Kv(31 (nt 15911542 and nt 1696-1647 in ref. 4 ) in situ hybridization of rat brain sections shows 'notably high' expression of Kv(31 mRNA in (i) the CA1/CA2 fields of the hippocampus; (ii) the caudate putamen. 'Moderate to high' expression is observed in (i) dentate gyrus; (ii) neocortical layers (pyramidal cells); (iii) cerebellum (Purkinje and granule cells); (iv) the CA3 fields of the hippocampus and in (v) thalamic nuclei4 . Comparative note: Sensitive RTPCRt analyses also do not detect Kv(31a mRNA expression in human cardiac ventricle or atriums. Note: Kv,83 (as designated in refs. 10,11, see Table 1 under Gene family, 47-05) is primarily expressed in brain but with a distinct distribution to those of Kv(31.1 and Kv(32.

Kva/Kv{3 mRNA co-distribution patterns (see also Protein distribution, 47-15) 47-13-04: ISH: Comparisons of KVQ and Kv(3 mRNA co-expression patterns may indirectly suggest in vivo co-assembly of their subunits (but see caveats in paragraph 47-13-01). In this regard, it is interesting that Kvl.l mRNA (e.g. RCK1) mRNA is expressed in Purkinje cells, but not in corpus striatum21 , while Kvl.4 mRNA (e.g. RCK4) is highly expressed in corpus striatum, but not in Purkinje cells 21 . Comparison with Kv,B1 mRNA expression (ref. 4, paragraph 47-13-03) may therefore suggest selective assembly of Kvl.l/Kv(31-type K+ channels in Purkinje cells as opposed to Kvl.4/Kv(31-type K+ channels in corpus striatum4 . Irrespective of these specific interpretations, it is clear that differential Kv(3 subunit gene expression can significantly contribute to the structural and functional diversity of Kv channels in the mammalian nervous system (see other fields). Independent in situ hybridization studies using cRNA probes for Kv(31 and Kv(32 20 also reveal heterogeneous expression patterns in adult rat brain, with high densities of Kv(31 mRNA in the striatum, CAl subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv(32 mRNA in the cerebral cortex, cerebellum and brainstem20 (see also Protein distribution, 47-15).

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Table 2. Distribution of Kvf31 and Kvf32 mRNA and protein in rat brain (From 47-13-02) Kvf32 Kvf3l mRNA mRNA Subfield/lamina/sublamina Region

Kvf32 Kvf3l Immunoreactivity Immunoreactivity

++ ++ +++ +++ +++ + + ++ + + ++

+ +++ +++ ++ ++ + + ++ + + ++

+ ++ ++ +++ ++ + + ++ +++

+ +++ +++ +++ ++ + + ++ +++

Infragranular Granule cell Inner third Middle third Outer third

++ +++ + + +

+++ + + ++ + + +

+ ++ + ++ +

+ ++ + +++ ++

S.oriens S. pyramidale S. radiatum S. moleculare

++ + + ++ ++ +

++ +++++ ++ +

++ ++ ++ +

++ + + ++ +++ +

+++++ +++++ +++++ ++

++ ++ ++ ++

++ ++ ++ + + ++

++ ++ ++ + + ++

Cortex I II ill IV V VI

Hippocampus Dentate gyrus

CAl

Striatum Caudate Accumbens Olfactory tubercle Globus pallidus

I! ~

........

Basal forebrain

(b

Medial septal n. Lateral septal n. Diagonal band vert. Diagonal band hor. Nucleus basalis

+++ + +++ +++ +++

+++ + +++ +++ +++

+++ + +++ +++ +++

+++ + +++ +++ +++

Basolateral n.

+++

+ + ++

++

++

Anterior n. Lateral n. Laterodorsal n. VL VPM VPL Lateral geniculate n. Medial geniculate n.

+++ ++ ++ +++ +++ +++ ++ ++ ++

+ + ++ ++ ++ +++ ++ +++ +++ ++ ++

++ +++ ++ +++ +++ +++ +++ ++ ++

++ +++ ++ ++ ++ ++ ++ ++ ++

Medial n. Lateral n.

+ ++

++ ++

++ ++

+ +

Sup. colliculus Inf. colliculus Substantia nigra Pars compacta Pars reticulata Redn. nITI

+++ + + ++

++ +++

+++ ++

++ ++

++ + + + ++ +++ +++

++ + + + ++ +++ +++

++ + + ++ +++++ +++ + + ++

++ + + ++ +++++ +++ + + ++

Amygdala Thalamus

Hypothalamus Habenula

Midbrain

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Table 2. Continued

Region

Subfield/lamina/sublamina

Kv,B1 mRNA

Kv,B2 mRNA

Kv,B1 Kv,B2 Immunoreactivity Immunoreactivity

mnV nVI nVII nVIII

+++ +++ +++ ++

+++ +++ +++ ++

+ + ++ + + ++ + + ++ ++

+ + ++ + + ++ + + ++ ++

mnIX mnX mnXI mnXll

+++ +++ +++ +++

+++ +++ +++ +++

+ + + +

+ + + +

Purkinje cells Granule cells Interneurons Deep nuclei

+ + ++ ++ + +++

++ ++ ++ +++

+++ + + +++

Pons

Medulla

+ + + +

++ ++ ++ ++

+ + + +

++ ++ ++ ++

Cerebellum

+++ + ++ +++

Reproduced with permission from Rhodes et al. (1996) TNeurosci 16: 4846-60.

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Kvf31b subunit mRNA distribution in human heart 47-13-05: hKv{31b (== clone hKv{33): Northern t blotting analysis 5 using probes

specific for the unique N-terminal segment of hKv{31b shows its mRNA is (i) f".Jtwofold more abundant in human cardiac ventricle than in atrium and (ii) expressed in both 'healthy' and cardiomyopathic human heart tissue. Probes directed against conserved C-terminal segments (ibid.) are f".Jfourfold more abundant in left ventricle than in left atrium, suggesting further {3 subunit mRNAs are present in heart 5 . RT-PCRt analyses with Kv{31bspecific primer pairs detected mRNA expression in human brain cortex cDNA5 . Note: This study also reported Kv{32-specific cDNAs derived from heart tissues. Kv{31b == Kv{33: The Kv{31b transcript (designated Kv{33 in ref. 7) is detectable in multIple tissues, but is most abundant in aorta and left ventricle of the heart 7.

Comparative mRNA analyses using RNAase protection assays 47-13-06: A quantitative expression analysis of Kv{3 subunit mRNAs plus 16

different Kva channel mRNAs in rat sympathetic ganglia has been made using an RNAase protectiont assay22. Eleven a subunit genes and two {3 subunit genes were expressed in sympathetic ganglia; evidence for differential expression (between the superior cervical, coeliac and superior mesenteric ganglia) was obtained for Kv{31, Kval.2, Kval.4 and Kva2.2 22 . Notes: 1. Kv{31 transcripts (and those of Kval.2, Kval.4 and Kva2.2) were 'more abundant' in the pre-vertebral ganglia. 2. None of the distributions obtained in this study22 'matched' those of the M-current or the D2current, which are both prominent in electrophysiological studies of sympathetic neurones (see VLC K M-i, entry 53).

Phenotypic expression 47-14-01: General note: Functional changes 'conferred' by Kv{3 subunits when

co-expressed with Shaker subfamily (Kvla) subunits may have important implications for the 'reproduction' of native channel properties from 'cloned' channels in heterologous cell expression systems. For further discussion of this topic (including the importance of 'appropriate' heterooligomer formation, see also the following fields under under VLC K Kv1Shak (entry 48): Channel designation (48-03), Cell-type expression index (48-08), Developmental regulation (48-11), Phenotypic expression (48-14) and Protein interactions (48-31).

Regulation of native Kv channel properties by Kvf3 subunit expression 47-14-02: Kv{31a: In general, Kv{31 subunits can modify neuronal output and

firing patterns by regulating the 'phenotypic' expression of A-type t Kv channel activities4 : For example, co-expression of rKv{31a subunits with either the rKvl.l a subunit or rKvl.4 a subunit accelerates the rate of inactivation of the expressed K+ currents4 (compared to the rate of inactivation arising from these a subunit polypeptides expressed alone - for details, see Inactivation, 47-37). Association of rKv{31a with a subunits thus confers rapid 'A-type" inactivation on non-inactivating Kvl.l channels ('delayed

II

_L--

e_n_t_ry_4_7_

rectifiers', see also following paragraphs). This effect is probably mediated by an inactivating lball domain' within the variable Kv,Bla N-terminus (for further details, see Channel modulation, 47-44).

Possible functional role of Kv{3 subunits as sensors for toxidative stress' in neurones 47-14-03: Reversible oxidation/reduction of a critical cysteine in the Kv,Bl inactivating ball domain (see above) has been proposed as a potential sensor for oxidative stress in specific neurones 4 . According to this hypothesis, abnormal increases in oxygen radicals t (as in ischaemia t) would be predicted to eliminate ,B subunit inactivating functions, and thereby enhance K+ efflux. For putative mechanisms, see Channel modulation, 47-44.

tConversion' of hKvl.5 delayed rectifier channels to tpartially inactivating' channels 47-14-04: hKv,Bla ( == clone hKv,B3): The hKv,Blb subunit derived from human

heartS alters several functional properties of hKvl.5 channel subunits (which are also normally expressed in heart tissue) when heterologously coexpressed in Xenopus oocytes (see VL(; K Kvl-Shak, entry 48). hKv,Blb cDNA expression 'converts' hKvl.5 froln a delayed rectifier to a channel with rapid, but 'partial' inactivation (for details, see Inactivation, 47-37). hKvl.5/clone Kv,Blb channels also activate at more hyperpolarized voltages and show 'dramatically slower' deactivation (ibid.). Hypothetically, ldif_ ferential association' of channels such as hKv1.5 with ,B subunits in cardiac atrium and ventricle could explain rt~gional differences in repolarizing currents. Commonly encountered difficulties in 'matching' native channel currents to those obtained following heterologous expression of 'cloned' channels are outlined under Channel designation of VLC K Kvl-Shak, 4804. See also following paragraphs.

Kv{3 to KvO'. subunit mRNA ratios influence phenotype in heterologous expression systems (example) 47-14-05: hKv,Bla ( == clone hKv,B3): When cRNAs encoding hKvl.5 and hKv,Bl b subunits are mixed and co-injected into Xenopus oocytes the observed effects (see previous paragraph) depend on the ratio of the two mRNAs: higher Kv,B:KvQ subunit mRNA ratios inhibi1~ hKvl.5 current, whereas lower Kv,B:KvQ ratios may not influence hKvl.5 current (Kv,B alone does not express current)s. In practice, hKv,B mRNA is 'titrated-in' with fixed KVQ to maximize any observable phenotypic effects (see Inactivation, 47-37).

Endogenous Kv{3 subunits in cell lines can affect heterologous expression properties 47-14-06: hKvl.5 Q subunit expression properties differ when heterologously expressed in HEK-293 and mouse L-cells (see below). These differences can be accounted for by the presence of an endogenous Kv,B2.1 subunit coassembling with the transfected hKvl.5 protein in L-cells23, in contrast to HEK-293 cells where no Kv,B protein or mRNA is detectable. In the absence of Kv,B2.l (in HEK-293s), midpoints for activation and inactivation for hKvl.5 were -0.2 ± 2.0 and -9.6 ± 1.8 mY, respectively. Subsequently,

II

1'--_e_n_t_ry_4_7

---'_

the 'L-cell phenotype' (including the kinetics and voltage dependence of activation and slow inactivation) could be 'completely reconstituted' in HEK-293 cells following heterologous expression of Kv,82.1 23 • These observations further underlined the importance for characterization of endogenous ion channel components in heterologous expression systems prior to extensive biophysical characterizations or comparisons.

Role of Kvf32 subunits in K+ channel protein surface expression, stability and turnover 47-14-07: Kv{32: Within the Xenopus oocyte expression system, rKv,82 does not alter macroscopic t K+ channel current properties when co-expressed with rKvl.l or rKvl.44 . Notably, the rKv{32 subunit lacks an N-terminal inactivation 'ball domain' motif (compare previous paragraphs). Heterologous co-expression of Kvl.2 and Kv,82 has, however, been shown to (i) increase aKvl.2 protein turnover and (ii) promote the transport of Kvl.2 to the cell surface (for further details, see Subcellular locations, 47-16). For additional phenotypic features of Kv,82, e.g. modulation of the inactivation properties of Kvl.4 a subunit complexes, see Fig. 4 under Inactivation, 47-37.

Phenotypic functions of Drosophila Hyperkinetic 47-14-08: dHk,8: The Hyperkinetic (Hk) gene encodes a Drosophila homologue of mammalian Kv,8 subunits24 . Mutations in the Hk gene alter firing patterns and A-current properties in native and cell preparations, 'similar to but "milder" than' Sh mutations25 and epistasis t of Sh and Hk has been observed in double mutants. Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant reveals defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. Co-expression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence and secondary structure alignments of Hk with mammalian Kv,8 sequences show they represent an additional branch of the aldo-keto reductase t superfamily (see Gene family, 47-05 and Miscellaneous information, 47-55).

In vivo effects of mutations in the Drosophila Hyperkinetic (Hk) gene (examples) 47-14-09: dHk,8: Drosophila Hk (Hyperkinetic) mutants were initially shown24 to induce a leg-shaking phenotype under anaesthesia (which showed similarities to mutational phenotypes at the Shaker locus, albeit with less 'severity'). Hk mutations were also associated with rhythmic bursts of spontaneous activity in motor neurones of thoracic ganglion in adult flies 25,26. Current-clamp t of cultured 'giant' neurones derived from Hk mutants can distinguish two types of spontaneous sustained firing (occurring over >5 min) which do not occur in wild-type t neurones. In the first type, action potentials display little undershoot t and pre-potential t , while the second is characterized by strong undershoot and pronounced pre-potentiaI27. Voltageclamp t studies in Hk giant neurones displaying abnormal spontaneous firing are associated with (i) approx. 50% reduced I A compared to wild-type neurones, with all-or-none action potentials under current injection t i (ii) slower recovery

11II

_1...-

en_t_ry_4_7_1

Table 3. In vivo functional effects of Hk beta subunit mutants in the Drosophila larval muscle preparation. (From 47-14-10)

Wild-type Hk (various alleles)

Wild-type Hk (various alleles)

IA (nA/nF)a

IK (nA/nF)a

tp

7

(ms)b

(ms)b

5.3±0.3 3.2±0.3

3.6±0.3 3.2±0.2

9.3±0.2 13.6±0.4

10.4±0.3 14.9±0.2

Vmli2 (mV)b

Vmsl ope (mV/e-fold)b

Vh 1/ 2 (mV)b

Vhslope (mV/e-fold)b

-13.5 -11.0

8.0 6.5

-39 -33

4.5 4.5

Values were derived following step-depolarization of larval muscle fibres (-80 to OmV) at 11°C (n == 9-15, mean ± SEM). b t p, time to peak; 7, inactivation time constant of IA; Vml/2, voltage for halfconductance of IA; Vmslope , limiting slope for activation curve; Vh 1/ 2 , voltage for half-inactivation of IA; Vhslope, limiting slope for inactivation curve. Data a

of I A from inactivation and (iii) a relative insensitivity to Q-dendrotoxin27. These findings are consistent with predictions24 that Sh-Hk heteromeric channels would activate earlier in the action potential (with greater magnitude) and hence would be more efficient at repolarization.

Effects of Hk mutations on A-current modulation in Drosophila larval muscle (examples) 47-14-10: dHk,B: The role of the Hyperkinetic gene product in the in vivo modulation of Drosophila larval muscle fA is supported by various Hk mutants displaying (i) reduced current amplitudes and (ii) substantially slower kinetics of channel activation, inactivation and recovery compared to wild-types28 (summarized in Table 3). Notably, no significant defects in Drosophila I K are detectable (in contrast to the marked effects on [A); furthermore, effects with Hk null alleles t are 'no more extreme' than with other Hk alleles, indicating a modulatory role for the ,B subunit in channel gating and conductance28 .

Effects of Drosophila Hyperkinetic following heterologous co-expression with Shaker (examples) 47-14-11: Co-expression of wild-type Drosophila Hyperkinetic (Hk) ,B subunits with Drosophila Shaker (Sh) Q subunits in voltage-clamped Xenopus oocytes24,29 show (i) ""twofold increases in current amplitude in Sh plus Hk co-expressors compared to when the Q subunit is expressed alone; (ii) acceleration of activation kinetics; (iii) acceleration of inactivation kinetics and (iv) an approx. 10mV shift of the voltage dependence of activation and inactivation in the hyperpolarizing direction. When co-expressed with Sh Q subunits lacking a fast inactivation domain, the effects on current amplitude and activation kinetics persist29, consistent with predicted functions of Hk in

vivo (see previous paragraphs).

1'--_e_n_t_ry_4_7

_

Protein distribution Co-localization of K+ channel a and f3 subunit polypeptides in rat brain 47-15-01: A summary of reported co-localizations patterns for o./{3 K+ channel

subunits and other notable {3 subunit protein distributions appears in Table 4. Note that a number of antibodies used in these studies have been reported as having both species and isoform cross-reactivity. This property is largely dependent upon which region of the {3 subunit polypeptide is used to design the peptide epitope t, with C-terminal-derived antibodies having notable cross-reactivity amongst presently known isoforms (see sequences under Encoding, 47-19 and Domain conservation, 47-18). Cross-reactivity has both advantages and disadvantages within comparative distribution studies (for a discussion, see ref. 30).

Subcellular locations Hypotheses accounting for observed a/f3 subunit distributions in neurones 47-16-01: Immunohistochemical staining and co-localization patterns of Kvo. and Kv{3 subunits (see Protein distribution, 47-15 and Protein interactions, 47-31) predict mechanisms that can 'direct' the selective interaction of K+ channel 0. and {3 subunit polypeptides (discussed in ref. 30).

These data are consistent with properties of K+ channels in specific subcellular domains regulated by the formation of heteromultimeric K+channel complexes containing limited combinations of 0. and {3 subunits. At the time of compilation, little direct information regarding specific mechanisms of subcellular 'targeting' of {3 subunits in native membranes was available (e.g. to somatic, dendritic, axonal or synaptic membranes). Evidence for o.4{34 stoichiometric complex assembly as an early event in channel biosynthesis within transfected cells has been used to propose a model of K+channel o./{3 subunit interaction (see next paragraph and Fig. 1).

Promotion of cell surface expression and stability of Kvl.2 by co-expression with Kvf32 47-16-02: Stable association of the Kv{32 and Kv1.2o. subunits occurs early in biosynthesis, presumably at the endoplasmic reticulum31 . In COS cells transfected with expression constructs encoding Kvl.2 alone, the majority of the expressed protein appears 'trapped' within the cell (as determined by immunocytochemistry). Although Kv{32 has no effect on the kinetics of 0. subunit inactivation (ref. 4, see Inactivation, 47-37) heterologous coexpression of Kv1.2 and Kv{32 promotes the transport of Kv1.2 to the cell surface. These o.j{3 interactions in COS cells exhibit a predicted stoichiometry of 1: 132, in agreement with studies in native cells (see Protein molecular weight (purified), 47-22). By analogy to an action of a chaperone t protein, Kv{32 exerts effects on associated Kv1.2 which include promotion of co-translational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv{32jKvo.1.2 complexes, and increased efficiency of cell surface expression of Kv1.2 (as above). Furthermore, Kv1.2 protein turnover occurs

_'---

e_n_t_ry_4_7_

Table 4. Summary of Kv{3 subunit co-distributions detected in brain (see also VLC K Kv1-Shak entry 48) (From 47-15-01) Kv{3 Properties and cross-references Kv,81a Most K+ channel complexes containing Kv{31 also contain Kv{32 Kv,81b 47-15-02: Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies has revealed that the Kv{31 and Kv{32 polypeptides frequently co-localize20. Immunoblot and reciprocal co-immunoprecipitationt analyses predict that most K+ channel complexes containing Kv{31 also contain Kv{32. Co-localized Kv{31 and Kv{32 protein has been shown to be concentrated in neuronal perikarya, dendrites and terminal fields, and in the juxtaparanodal region of myelinated axons. These data further suggested that individual Kv channels may contain 'two or more' biochemically and functionally distinct Kv{3 subunit polypeptides20. Table 2 (under mRNA distribution, 47-13) compares protein versus mRNA expression patterns described by Rhodes et al. (1996)20 in adult rat brain. Kv,82 47-15-03: Immunohistochemical staining30 using antibodies directed against the C-terminal18 aa residues of the bovine Kv{32 subunit (associated with the dendrotoxin-sensitive K+ channel complex - see Protein molecular weight (purified), 47-22 and Database listings, 4753) shows 'widely distributed' immunoreactivity in adult rat brain30 . The antibody was cross-reactive (see paragraph 47-15-01), detecting two major bands of 50 kDa (non-specific) and 38 kDa with a lessprominent band of 41 kDa in rat brain membrane preparations (ibid. 30 ). The cellular distribution of {3 subunit immunoreactivity corresponds closely with that for Kvl.2 (and to a lesser extent Kvl.4) but not with Kv2.1, consistent with other findings from the same study (see Protein interactions in this entry, 47-31 and under VLC K Kv-Shak, 48-31). Further detailed descriptions relating differential distributions of anti-Kv{32 immunoreactivities (e.g in neocortex, hippocampus, piriform cortex, striatum, cerebellum, cranial nerve nuclei and most major white matter pathways) are illustrated in ref. 30. 47-15-04: Using immunoblot and reciprocal co-immuno-precipitationt analyses an extensive independent studro concluded (i) that Kv,82 is the major {3 subunit present in rat brain membranes and (ii) Kv{32 is a component of 'almost all' K+ channel complexes containing Kv1Q subunits (see also mRNA distribution, 47-13).

Notes: 1. Determination of K+ channel Q subunit polypeptide associations with Kv{3 subunits have been performed using ~reciprocal co-immunoprecipitation' methodologies. In these assays, crude membrane preparations are subjected to immunoprecipitation using antibodies specific for Q or {3 subunit polypeptides; resultant immmunoprecipitation products are then analysed by immunoblotting, using either the Q or {3 subunit-specific antibodies. 2. In-situ protein localization studies for Kv{3 subunits (e.g. ref. 30) contain detailed descriptions relating {3 subunit expression to specific brain regions. To help consolidate all such data into a single reference source and make it available on the WWW, please forward citation details to the e-mail address shown under Feedback, 47-57.

(D

=

51

f""t'

~

Kv1.2a protein

~

""'" 4. Early KV1.2a:Kv~2 interaction promoting surface expression possibly via assisting correct folding or assembly

3

k--'

Cytoplasm

~

ER lumen

1

1. Exit of nascent polypeptide from ribosome complex

, 3. Post-translational glycosylation (oligosaccharide chain addition) Asn-207

IKeYI II

~

Direction of movement for newly-synthesized Kv1.2 polypeptide

'r

Consensus site for N-linked 9'ycosy'ation on Kv1.2 (a unique site between domains 51 and 52, see Sequence motifs, 47-24).

Figure 1. Model for association of Kv{32 subunits with partially synthesized Kv1.2a subunits (steps 1 to 4). The co-translational association (indicated at step 4) has been proposed to affect the efficiency of co-translational oligosaccharide addition (step 3) due to effects on Kv1.2 folding either before or during ER translocation (step 2) of the Sl and S2 transmembrane segments. (From 47-16-02)

_ 1 . . . . . - - - -_

_.

entry47

I ,

at a higher rate in Kvl.2/Kv,B2 co-transfectants, and these cells display increased binding of (l 25 I]-DTx (see Protein interactions under VLC K KvShak, 48-31) by increasing Bmax without affecting K0 31 . Additional features

of a/,B subunit interactions and a proposed model for these early biosynthetic events (occurring ~5 min of nascent protein synthesis) are outlined in Fig. 1, this field.

Transcript size 47-17-01: rKv,Bl/hKv,Blb (= clone hKv/3a): No extensive study of transcriptional control/mapping of Kv,B genes was published at the time of entry compilation, although the existence of alternative splicing of Kv,Bl primary transcripts is now well established (see Cene organization, 47-20). Using a Kv,Bl riboprobe t (nt 669-694) in Northern t blots of total cell RNA extracted from rat brain, two transcripts of approx. size 3.6 and 3.9 knt are observed4 . These signals are absent in mRNA pools from heart, skeletal muscle or kidney. The size of the hKv,Blb(=clonehKv,Ba) transcript in various human heart mRNA pools has been estimated as ",4 knt 5 .

SEQUENCE ANALYSES

Chromosomal location 47-18-01: hKv,Bl: The human Kv,Bl.l gene ('KCNAIB' - see note 1 below) has been mapped to human chromosome 3q26.1 by fluorescence in situ hybridization t (FISH) confirmed by PCR screening of the CEPH YACt library and a chromosome 3 position predicted by somatic cell hybrid mapping33 . hKv,Bb (= clone hKv,Ba): In separate studies, the gene encoding clone hKv,B3 was also localized to human chromosome 3 using a human/ rodent cell hybrid mapping panel and Southern blot analyses 5 . hKv,B2: The human Kv beta 1.2 gene ('KCNA2B' - see note 1 below) has been localized by FISH to human chromosome Ip36.3 consistent with a chromosome 1 location as indicated by somatic cell hybrid mapping33 . Notes: 1. These gene names and human chromosomal locus names appeared in ref. 33 and are subject to review (compare with Cene mapping locus designation under VLC K Kv1-Shak, 48-54 for established locus names for the Kv1a subunit genes, which are an unrelated gene family). 2. In Drosophila, Hk is located at polytene bands 9B7-8 (Schlimgen, A.K., cited in ref. 24 ).

Encoding Variable N-termini linked to a constant aida-keto reductase core 47-19-01: Kv,B subunit cDNAs (as described thus far) encode alternative Nterminal regions coupled to a conserved ('constant') region homologous to the aldo-keto reductases (the laldo-keto core', see also Domain conservation, 47-28). An illustration of this arrangement, together with an alignment of rKv,Bla, hKv,Blb and rKv,B2 amino acid sequences (modified from ref. 5) is shown in Fig. 2. Kv,Blb = Kv,B3: A cDN.A (designated Kv,B3 in ref. 7) has a

II

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_

L..--

(a)

....__....",, Alternative

-..-

Aldo-keto reductase-homologous 'core' region "

:~; • • •~ • •II-• • •H • • •_II• •I

t-

N-termini

....__.....r;'

;';';'

(b)

•

MHLYKPACAD 1PSPKLGLPK SSESALKCRW HLAVTKTQPQ AACKPVRPSG MQV S1ACTEHNL- -RNGEDRLLS KQSS-APNVV N-ARAKFRTV MYPESTTGS

hKv~3 rKv~l rKv~2

*.* 0 51 AAEQKYVEKF LRVHG1SLQE TTRAETGMAY RNLGKSGLRV SCLGLGTWVT 44 -11ARSLGT- TPQ-H---K- S-AKQ---K- ---------- ---------10 P-RLSLRQTG SPGM1Y-TRY GSPKRQLQF- ---------- ---------101 FGGQ1SDEVA ERLMT1AYES GVNLFDTAEV YAAGKAEV1L GS11KKKGWR 94 ---------- ---------- ---------- ---------- ---------60 -----T--M- -H---L--DN -1-------- --------V- -N--------

o.

••

151 RSSLV1TTKL YWGGKAETER GLSRKH11EG LKGSLQRLQL EYVDVVFANR 144 ---------- ---------- ---------- ---------- ---------110 ---------1 F--------- ---------- --A--E---- ----------

•

201 194 160

PDSNTPMEE1 VRAMTHV1NQ GMAMYWGTSR WSAME1MEAY SVARQFNM1P ---------- ---------- ---------- ---------- -----------P------T ---------- ---------- --S------- -------L--

251 244 210

PVCEQAEYHL FQREKVEVQL PELYHK1GVG AMTWSPLACG 11SGKYGNGV ---------- ---------- ---------- ---------- ----------1-------M ---------- ---F------ ---------- -V----DS-1

301 294 260

PESSRASLKC YQWLKER1VS EEGRKQQNKL KDLSP1AERL GCTLPQLAVA ---------- ---------- ---------- ---------- ----------PY------G -----DK-L- ----R--A-- -E-QA----- --------1-

•

..

--

351 WCLRNEGVSS VLLGSSTPEQ L1ENLGA1QV LPKMTSHVVN E1DN1LRNKP 344 ---------- ---------- ---------- ---------- ---------310 ---------- ----A-NA-- -M--1----- ---LS-S1-H ---S--G---

•

401 YSKKDYRS 394 -------360 --------

Figure 2. (a) General Kv{3 subunit arrangement encoding alternative Nterminal regions {white boxes} coupled to a conserved tconstant'} region {black boxes} homologous to the aldo-keto reductases {the aldo-keto reductase core} (Based on figure from ref.12.) (b) Alignment of rKv{31a, rKv{32 and hKv{31b {clone hKv{33} amino acid sequences, illustrating several features described in the text. Key: -, identical amino acids; *, consensus sites for phosphorylation by PKC common to rKv{31a and rKv{32 but not hKv{31b {clone hKv{33}; ., consensus sites for phosphorylation by PKC in clone hKv{33, some of which are conserved in other {3 subunits, with Ser13 and Thr71 being unique to clone hKv{33; 0, consensus site for phosphorylation by cAMP-dependent protein kinase {Ser153 in clone hKv{33} conserved across all Kv{3 subunits; D, location of a potential splice site predicted by the nucleic acid sequence {notably, this coincides with a point of divergence between the {3 subunit N-termini}. (Alignment modified from England et al. {1995} Proc Natl Acad Sci USA 92: 6309-13.) {From 47-19-01}

II

_ entry 47

"-----------

408 amino acid open reading frame, and possesses a unique 79 amino acid Nterminal leader (but is otherwise identical with rat Kv,B1a over the 329 Cterminal amino aCids 7). Kv,B3 (as designated in refs 10,11, see Table 1 under Gene Family, 47-05) also has a long N-terminal structure and induces inactivation in N-terminal deleted Kv1.4 (but not in other members of the Kv1 channel family).

Gene organization Alternative splice variants 47-20-01: 'Direct' sequence comparisons between genomic and cDNA sequences encoding Kv,B subunits were not published at the time of compilation. From cDNA sequence variants described thus far, there appears to be a general ,B subunit pattern of variable N-termini with constant 'core' regions (see also Domain conservation, 47-28 and Domain functions, 47-29). Analysis of nucleic acid sequences, suggests5 the point of amino acid sequence divergence between rKv,B1a and clone hKv,B3 is an alternative splice junction (illustrated in Fig. 2, under Encoding, 47-19). This is supported by the majority of the 'C-terminal 4/Sths' being identical between rKv,B1a and hKv,B3 (ibid.). In c.omparison, the first 72 N-terminal amino acids of rKv,B1a do not align with the Kv,B2 N-terminus; in this case, however, differences in the 'C-terminal 4/Sths' of the rKv,B1 and rKv,B2 sequences are 'interspersed' (albeit they represent largely conservative substitutions)4.

Homologous isoforms Species homologues of Kvf3 subunit proteins show notably high identities

47-21-01: rKv,B1/bKv,B2: The rat Kv,82 amino acid sequence4 shares approx. 990/0 amino acid identity with the previously obtained sequence for the bovine Kv,82 subunit9 (Le. they differ by only 4 residues out of 367). A 100% identity has been noted between the C-terminal 329 aa segment between the rat Kv,B1 sequence and the human clone Kv,83 (derived by alternative splicing from the Kv,81 gene in humans). These identities indicate that the respective proteins have 'equivalent' or at least similar physiological roles in the different species. General note: Subunit genes belonging to different Kv,B subfamilies (e.g. Kv,B1, Kv,B2 and Kv,B3, see Gene family, 47-05) show much antino acid sequence variability throughout that predicted by their open reading frames. For further interspecies comparisons, see also the sequences shown under Database listings, 47-53.

Protein molecular weight (purified) Kva/f3 subunit assemblies revealing the native constitution of voltage-gated K+ channel 47-22-01: A summary of protein Mr estimates for recombinant Kv,B subunits and native protein complexes containing Kv,B subunits appears in Table 5. Minor variations in gel mobilities/molecular weights for Kv,B subunits have

II

II...-_e_n_try_4_7

_

Table 5. Protein molecular weight estimates for recombinant Kv{3 subunits and native protein complexes containing Kv{3 subunits (From 47-22-01) Kv{3 Mr (purified), notes and cross-references designation/ complex 47-22-02: Antibodies raised against a C-terminal 18 aa peptide epitopet of the {3 subunit originally associated with the bovine dendrotoxin-sensitive K+ channel complex (bKv{32, see below and Database listings, 47-53) have been used to characterize the related {3 subunit polypeptides in rat brain (ref. 30, see Protein distribution, 47-15). These cross-reactive antibodies (ibid.) can detect a 'major' 38kDa polypeptide and a 'minor' 41 kDa polypeptide in rat brain membranes, possibly corresponding to the predicted sizes of the rKv{32 and rKv{31a respectively (compare this table, below and Protein molecular weight (calc.), 47-23). (See also notes 1-3). 47-22-03: Historically, use of 12sI-labelled a-DTx (see Early studies first defining Blockers, 47-43 and Ligands, 47-47) in immunoprecipitation native K+ experiments34,35 with rat cerebrocortical synaptosomes were important in establishing (i) that the toxin receptors were channels as large heterointegral membrane proteins, while subsequent cross-linking studies36,37 led to the first identification of K+ channel a oligomeric subunits with Mr estimates falling between 65 kDa (Nsialoglycoproteins deglycosylated, see below) and 78 kDa (glycosylated), depending on the electrophoretic conditions used 18 . Anomalous 47-22-04: Note: Channel proteins cross-linked to toxin molecules may exhibit lanomalous' migration in SDSmigration of a subunits but PAGEt analyses· digestion with sialidase t and peptide not {3 subunits N-glycosidase FT abolishes anomalous migration but bands may still be 'broad and diffuse' indicating subunit heterogeneity. Beta subunit bands do not exhibit anomalous migration behaviour since they are not glycosylated (see Sequence motifs, 47-24). Immuno47-22-05: Following a-DTx affinity purification from bovine precipitates of brain (see above), fast-activating, voltage-sensitive K+ intact K+ channels can be purified into distinct oligomer populations channel with (i) a major, 'large form' population (containing Mr complexes 39 kDa (3 subunits) being in the molecular weight range 370(a-DTx 420 kDa (after correction for detergent binding) with a minor acceptor 'small form' population (lacking 39 kDa (3 subunits) in the range 240-265 kDa 16 . Note: These estimates were for the populations) protein moiety of the hetero-oligomeric sialoglycoprotein complexes, and in order to correct for attached carbohydrate, the degree of glycosylation was assumed to be that for the Na+ channel (f'./30 0/0 w/v hexose; u = 0.61 ml/g). Beta subunits within DTx-sensitive complexes

47-22-06: On the basis of these ranges, {3 subunit-containing forms are typically cited as the '400 kDa complex', as

II

_L....-

en_t_ry_4_7_1

Table 5. Continued

Kv{3 designation/ complex

Mr (purified), notes and cross-references

determined from sedimentation analysest using the buoyant density method (giving 'large form' Svedberg unitt values of 11.2S and 9.9S in H20 and 2H20 respectively16). See consistency with Q;4{34 stoichiometric complexes under Predicted protein topography, 47-30). 47-22-07: Using gel filtrationt analysis, a Stoke's radiust of 8.6 nm has been determined for the large form of the oligomeric receptor 9,16. This estimate agrees with studies carried out on solubilized Q;-DTx acceptors. Kv,81a

Note: rKv{31a ORF: 401 aa

47-22-08: Kv{31a: An Mr value of 41 kDa has been determined for rat brain Kv{31a (Nakahira et. a1., cited in ref. 30 ). rKv{31a transiently expressed in COS-l cells has a 'mobility similar to, but slightly larger than' 41 kDa (ibid.). (See also note 3.)

47-22-09: hKv{31b (== clone hKv(33): Specific estimates of Note: hKvj31b purified Mr were not found during compilation, but may be expected to be close to those for Kvj31a (compare ORFs in ORF: 408aa first column and conserved regions between Kvj31a/Kvj31b under Domain conservation, 47-28).

Kv,81b

Kv,82

Note: bKvj32 ORF: 367aa

47-22-10: ~2: Minor variations in reported Mr also exist for Kvj32, most commonly estimated at 38 kDa30. (See also notes 2 and 3.)

Notes: 1. On the basis of similar gel mobilities under denaturingt (reducingt) or nondenaturing t conditions, the 31 kDa and 41 kDa subunits do not apparently contain extensive intra- or interchain disulphide bridges (cited in ref. 30). 2. In independent studies19, a 38 kDa polypeptide co-immunoprecipitating with Kv2.1/drkl subunits probably represent a 9istinct 13 subunit to the Kvj31a and Kv{32 proteins described above (for Kv2.1/d.rk1, see VLC K Kv2-Shab, entry 49). 3. Non-specific bands at rv50 kDa (sometimes observed in co-immunoprecipitations of Kv 13 subunits3o ) are likely to represent heavy chains of rabbit IgG reacting with anti-rabbit secondary antibodies. 4. Purification (detergent/buffer) and storage conditions enhancing stability of Q;-DTx receptor complexes are detailed in ref. 16 and references therein. 5. Protein kinase A appears to phosphorylate a 40 kDa protein which coimmunoprecipitates with the type n channel (hKvl.3) from membranes of Jurkat T cells38 (see Table 16 in Protein phosphorylation under VLC K Kv1Shak, 48-32 and also the description of Kvj3 subunit expression in activated T lymphocytes39 (abstract). 6. Compare protein mol. wt of (i) mammalian Kv{3 subunits (above) with that of the mammalian maxi (BKCa) channel (31 kDa (for refs, see ILC K Ca, entry 27, and Miscellaneous information, field 55, this entry) and (ii) an Arabidopsis thaliana 13 subunit (38.4 kDa) (see ref.40).

II

lL...--e_n_ _ry_4_7 t

_

been reported between studies, but these may be attributable to differences in electrophoretic conditions used. Molecular weight differences of Kv0/{3 complexes are probably due to differential glycosylation patterns and 0 subunit compositions. Table S also summarizes studies of a-dendrotoxin receptor complexes t, where the Kvo subunit components correspond to several Shaker-related Kv1 subfamily proteins tightly but non-covalently associated with smaller Kv{3 subunit variants. These studies provided important direct evidence for native voltage-gated K+ channels consisting of large eight-subunit hetero-oligomeric complexes ('404{3 octamers', see also Predicted protein topography, 47-30).

Protein molecular weight (calc.) 47-23-01: Kv{31a: Predicted from cDNA sequence (ORF 401 aa) rv44.7kDa 4. hKv{31b (~e hKv{33): Predicted rv4S.0kDa (for the full ORF of 408 aa); note that if cleavage by amidation t were to occur (see Sequence motifs, 4724 and Resource C - Consensus sites and motifs, entry 62) the predicted molecular mass for clone Kv{33 would be rv36 kDa 5 . Kv{32: Predicted from cDNA sequence (ORF 367 aa) rv41 kDa (40983 Da)4,9. - -

Sequence motifs A KVQ-Kv{3 interaction site motif in Kvl subfamily channel N-termini 47-24-01: The Kv1.S N-terminal region (90 aa residues, positions 112-201) has been determined sufficient for interactions of Kv1.So and Kv{31 subunits (this entry, see also Protein interactions under VLC K Kv1-Shak, 48-31). One study41 has further delineated an interaction site motif (FYE/ QLGE/DEAM/L, residues 193-201 in Kvo1.S) which has been (i) found only in N-termini of the Shaker-related Kv1 subfamily channel proteins and (ii) shown necessary for Kv{31-mediated rapid inactivation of Kv1.S currents. These results also indicated that hetero-oligomerization between 0 and Kv{31 subunits is restricted to Shaker-related potassium channel 0 subunits. (See also Protein interactions, 47-31 and next paragraph.)

The NAB domain mediates assembly of both KVQ-KVQ and KVQ-Kv{3 complexes 47-24-02: In an independent study, mapping of the Kv,L31-binding site to regions in cytoplasmic N-terminal domains of Kv10 subunits have determined that it overlaps with the region known as the NAB(Kvl) tetramerization domain1 (for details, see Protein interactions under VLC K Kv1Shak, 48-31). By means of channel chimera/deletion experiments, it has been shown that the NAB(Kv1) domain is essential for reproducing Kv{31mediated inactivation1. (See also Protein interactions, 47-31 and paragraph 47-24-01). 47-24-03: On the basis of primary sequence comparisons (see Fig. 2 under Encoding, 47-19) a number of potential post-translatory modification t sites exist on Kv{3 proteins including multiple phosphorylation consensus sites

II

_'--

e_nt_ry_4_7_1

(see Protein phosphorylation, 47-32) and for amidationt. The motif associated with protein amidation is conserved in all presently known Kv{3 subunits (indicated by a short horizontal line in the alignment shown in Fig. 2). For predicted consequence of subunit cleavage by amidation, see Protein molecular weight (calc.), 47-24. For background refs on peptide amidation, see Resource G - Consensus sites and motifs, entry 62.

Lack of leader and N-glycosylation motifs 47-24-04: As predicted for proteins with cytoplasmic locations, Kv{3 subunit sequences do not contain motifs for leader sequencest or N-glycosylationt (consistent with the deduced lack of added carbohydrate chains on Kv{3 subunits within experiments 9 described in Table 5). For conservation of structural motifs associated with Kv{3 sequences thought to be involved in NADPHf binding and the hydrogen transfer mechanism t in aldo-keto reductase t superfamily members, see also Channel modulation, 47-44 and Miscellaneous information, 47-55.

STRUCTURE AND FUNCTIONS

Amino acid composition 47-26-01: Hydropathyt analyses of Kv{3 subunits (e.g. in refs. 4,9) do not indicate the presence of hydrophobic stretches of sufficient length to act as transmembrane domains (i.e. at least 19 residues with an average hydropathy index t of >1.6 by the criteria of Kyte and Doolittle42 ). Kv{3 subunits appear to be peripheral membrane proteins with a 'dominant hydrophilic character' based on analyses by the method of Klein et. a1. 43 .

Domain arrangement 47-27-01: By the criteria of Garnier et a1. 44 the Kv,82 subunit appears to include four major a-helical t domains spanning residues 66-103, 126-140, 193-236 and 270-298 9 (for sequence, see Fig. 2 under Encoding, 47-19). Comparative note: This a-helical pattern has also been predicted for cloned ,8 subunits of voltage-gated calcium channels from skeletal muscle (see VLG Ca, entry 42).

Domain conservation Kv{31, Kv{32 and Kv{33 show high sequence conservation in their C-terminal portions 47-28-01: hKv,81b (== clone hKv{33): As illustrated in Fig. 2 (under Encoding, 47-19) homology between the presently known Kv,8 subunits is greater in their C-terminal regions, with 'significant differences' occurring in the Nterminal region. As pointed out in ref. 5, the C-terminal 329 aa of rKv{31a and hKv,81b (== clonehKv,83) are 1000/0 identical and share rv85% identity to rKv{32. Notably, however, the N-terminal 79 aa of rKv,81a and hKv,81b share only rv25 % identity, with this region also being 'difficult to align'

II

l'---e_n_t_ry_4_7

_

with the rKvj32 sequence5 (see Domain functions, 47-29). The point of divergence between Kvj3la and Kvj3lb (clone hKvj33 (see Fig. 2) has been suggested as a candidate alternative splice junction5 (see Subtype classifications, 47-06 and Gene organization, 47-20).

Molecular basis of selective interactions between Kva and Kv{3 subunits 47-28-02: Kvj31 and Kvj32 exhibit indistinguishable Kvla subunit selectivity in complexes containing Kval.l, Kval.2, Kval.3, Kval.5 or Kval.6 32. Reciprocal co-immunoprecipitationt studies have further indicated that selective interaction between 'compatible' Kva and Kvj3 subunits are mediated through conserved domains (analogous to the 'TI' or 'NAB' domain in Kva subunit N-termini - for details, see Protein intereactions under VLG K Kv1-Shak, 48-31). Notably, the interaction(s) of Kvj3 subunits with Kvl (Shaker-related) a subunits do not require the 13 subunit Nterminal domains (Le. mutants Kvj3l~N70, lacking 70 aa residues, and Kvj32~N22, lacking 22 aa residues)32. Note: These authors used these observations to argue that a previously observed failure of Kvj31 N-terminal mutants to modulate inactivation kinetics of Kvl family members could not be simply due to a 'lack of subunit interaction' (see also Protein interactions, 47-31).

Structural relationships between Kv{3 subunits and aldo-keto reductase superfamily members

47-28-03: Kvj3 subunits were originally described4 as showing 'no significant

homologies' to other proteins. Since their original description, however, sequence similarities to members of the NAD(P)H-dependent aldo-keto reductase t superfamilyt have been pointed out24,25 (see Miscellaneous information, 47-55). These analyses indicate that the 'most conserved' residues are those aligning to key secondary structure determinants (see Fig. 6, ibid.). Notably, many of the structural motifs known to be involved in NADPH binding and the hydrogen transfer mechanism in aldo-keto reductase t superfamily members are also conserved in mammalian Kvj3 and Drosophila Hk subunits 24 .

Conservation of primary sequences between Drosophila hyperkinetic and mammalian Kv{3 47-28-04: In general, the physiological effects seen when Hk is co-expressed

with Sh are distinct from those reported for either mammalian Kvj3la/b or Kvj32 subunits.

Domain functions The rKv{31a subunit N-terminal tball' domain confers inactivation properties upon rKvl.l 47-29-01: The inactivating 'ball' domain in the N-terminus of Kvj31 promotes

rapid closure of open Kv channels which cannot otherwise inactivate rapidly4 (see Phenotypic expression, 47-14). Strikingly, the oxidation or reduction of a ~critical cysteine' in the inactivation ball domain of Kvj3la reversibly

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_4_7_

switches the channel inactivation mode from 'fast' to 'slow' and vice versa (for further details, see Channel modulation, 47-44, Fig. 3, this field, and ref. 4).

Comparison of rKv{31a and hKv{31b subunit N-termini 47-29-02: hKv,Bla; hKv,Blb (== clone hKvj33): hKv,Blb induces an inactivating property upon hKvl.5 delayed rectifier currents, but to a lesser extent than hKv,Bla on rKvl.l (see previous paragraph). Although the level of identity between rKv,Bla and hKv,Blb N-termini is low (ca. 250/0, see Domain conservation, 47-28) predictions based on the algorithms of Chou and Fasman suggest that rKv,Bla and hKv,Blb N-termini have 'nearly identical' secondary structures (cited in ref. 5). Furthermore, optimized N-terminal alignments (different to those shown in Fig. 2 under Encoding, 47-19) which introduce gaps and highlight conservative substitutions between rKv,Bla and hKv,Blb termini have helped identify residues that appear important for conferment of inactivating properties (as denoted and summarized in Fig. 3, this field).

Predicted protein topography Differences in mass between Kva and Kva/b complexes tbest fit' a a4{34 stoichiometry 47-30-01: Summations of 'accurate' protein molecular weight differences between a-DTx acceptor complex populations purified from bovine brain consisting of (i) major 'large' Mr range complexes of 370-420 kDa (containing 39 kDa ,B subunits) and (ii) minor 'small' range complexes of 240-265 kDa (lacking 39 kDa (3 subunits) are most consistent with native K+ channel oligomers containing four Kvn and four Kv,8 subunits (generally designated as 'Q4,B4 stoichiometric complexes' or '4a4,8 octamers'46). Note: Alternative combinations (such as Q4,B3, Q4,B2 or Q4,Bl) are incompatible with this data (see also Protein molecular weight (purified), 47-22). For the collective evidence supporting tetrameric assemblies of KVQ subunits, see the VLC K Kv series, entries 48 to 51 inclusive.

Protein interactions Significance of co-expressed Kv{3 subunits in accounting for tnative' K+ channel currents 47-31-01: The full 'variety' of protein subunit interactions which generate native cell K+ current diversity have only recently begun to be explored (see brief discussion within Channel designation of the entry VLC K Kv1Shak, 48-03). 'Correlation' or 'matching' of endogenous cell currents with heterologously expressed K+ channel Q subunit channel currents is not straightforward, since the latter show only a limited range of diversity (Atype r versus delayed rectifier t currents, often with similar pharmacological properties). The demonstration of functional heterotetrameric t Kv channel assemblies (ibid.) and the existence/association properties of Kv,8 subunits in vivo (this entry) can both profoundly alter functional properties and therefore must be taken into account in comparative studies. Most

1'--_e_n_t_ry_4_7

_

biochemical data are consistent with the {3 subunit being 'firmly associated' at intracellular sites with a subunits (as initially proposed9 ). There is no evidence for disulphide linkages between Kva/{3 subunits, but the Itight' non-covalent interactions in oligomeric subunit complexes are not disrupted by high salt concentrations or the vigorous procedures involved in co-immunoprecipitations using anti-a subunit antibodies (see below).

Direct evidence for the hetero-oligomeric constitution of Kv channel complexes in vivo 47-31-02: Characterization of seven monoclonal antibodies raised against adendrotoxin-sensitive K+ channels (purified from bovine cerebral cortex) first showed Kv{3 subunits to be distinct proteins (and not proteolytic fragments of the larger subunit)47. 'Authentic' subunit compositions of neuronal K+ channels purified from bovine brain have been analysed using monoclonal t antibodies reactive with Kva subunits (e.g. mAb 5 selective for Kv 1.2)46 or polyclonal t antibodies specific for fusion proteins containing C-terminal regions of several mammalian Kv proteins (ibid.). Western blottingt of K+ channel complexes from several brain regions using adendrotoxin (a-DTx, see Blockers, 47-43), show precipitation of several different Kva subunits in variable amounts according to brain region. Results from this type of investigation are summarized in Table 6, which also cites early descriptions of Kv{3 subunits co-purifying within native Kva/{3 complexes.

Kvla subfamily-selective interactions with Kvj3 subunits 47-31-09: Both the Kv{31 and Kv{32 have been shown to display 'robust and selective' interaction with five members of the Shaker-related (Kvl) a subunit subfamily when co-expressed in mammalian (COS) cells32 (Kvl.l, Kvl.2, Kvl.3, Kvl.5, and Kvl.6 - see VLC K Kvl-Shak, entry 48). Conversely, interaction of Kv,81 and Kv,82 with members of the Kv2 (Shabrelated) and Kv3 (Shaw-related) a subunit subfamilies could not be detected by immunoblot analysis. Note: Although a member of the Kv4, Shal-related subfamily, Kv4.2 was determined to interact with Kv{3 subunits in this study32. Notably, however, this interaction showed characteristics distinct from that typical between Kv{3-Kval family members: Co-immunoprecipitation t could not be disrupted by SDS at <0.60/0 (compared to Kvl.2:Kv{31 interactions, which can be partially disrupted by the addition of SDS to 1"V0.20/0, and completely disrupted by SDS at 0.40/0 )32. These findings in heterologous t cells complement independent observations in situ, where only Kvl a subunits (and not Kv2, Kv3, Kv4) have been co-localized in rat brain slices30 (see also the Kva-Kv{3 interaction site motif and NAB tetramerization domain under Sequence motifs, 47-24).

Protein phosphorylation Multiple protein kinase motifs are found in Kvj3 subunit sequences 47-32-01: hKv{31a; hKv{31b (== clone hKv(33): Several consensus phosphorylation motifs are apparent in Kv{3 subunit sequences, and in vitro phosphorylation of {3 (and a) subunit components has been demonstrated in

II

II

Ica) Residue number rKv~la N-terminus Ser/Cys motif (aa 4-24) Cys-7

11

1 ~CZi

SIACTE

HN~

~

21 31 gRNGEDRLLS KQSS:IAPNW N~

41

KFRl:

51 V~IIARSLGT

+ .+ •••• + ••• +

.s.

61 71 KLRV1!GISLQ ~~E -

-

--

-

-

TGMAX -

---

*

(b) rKv~la N-terminus Cys-7 (& +ve clustering)

71 ~KQTGMKX

*

Residue number 1 11 21 31 41 51 hKv~lb N-terminus ,MHLYKPACAD. IPSPKbGLPlS .§.SESALECRW HLAV1:KTQPQ ~CEPVBP G~QKYVEK Ser/Cys motif (aa 25-55) - •• +•••••••• + ••••••• + •• + ••••••• + Cys-8

61 E:TPOJ!HISLK ~

(c) MQVgIA£TEHNL

KSRNGEDRLLSKQS

+ +

*

+

+

rKv1.4 N-terminus MEVAMVSAESSG£NSHMPYGYAAQARARERERALHSRAA Cys-13 (& +ve clustering) * + + + + + rKv3.4 N-terminus Cys-6 (& +ve clustering)

Drosophila Shaker (+ve cluster but no Ser/Cys motif)

MISSV£VSSY

RGKKSGNKPPSKTC

+ ++

*

+

+

MAAVAGLYGLGEDRQHRKKQ

+ +++

Figure 3. (a) Optimized alignment of rKv{31a and hKv{31b N-terminal regions. Following heterologous co-expression, Kvl.l-rKv{31a and hKvl.5-hKv{31b channels displcy inactivating properties that are dependent upon the presence of the {3 subunit (see text). The figure denotes some common features between the N-terminal regions of these proteins despite the relative divergence of sequence compared to the C-terminal regions (see Domain conservation, 47-28). Identical residues in the optimized alignments are double underlined, while conservative substitutions are indicated by a single underline. A Ser/Cys motif followed by a series of positively charged amino acids considered to be the inactivating domains in rKv{31a (aa 4-24, see text) is indicated by the symbols. and +. This motif is also present in Kvl.4a subunits (see below), which produce inactivating currents following heterologous expression 4 (see also Domain functions under VLC K Kvl-Shak, 48-29). A Ser/Cys motif resembling the rKv{31a is denoted in the hKv{31b sequence (aa 2555, from ref. 5). A cysteine residue that has been shown to render N-type inactivation properties susceptible to

9 t""t"

~ ~ ~

oxidation is conserved between hKv{31a and hKv{31b (Cys7 and Cys8 respectively, as indicated by asterisk symbols, see below). (Modified from England et al. (1995) Proc Natl Acad Sci USA 92: 6309-13.) (b) Optimized alignment of rKv{31a, rKv1.4 (RCK4), rKV3.4 (Raw3) and Drosophila Shaker N-terminal regions. The alignment illustrates broadly conserved features between a Kv{31 subunit N-terminus and those of Q subunit channels comprising the inactivating ball domain. Annotations are as for panel (a). This broad structural conservation and the presence of the cysteine residue (asterisked) can help interpret similarities in redox sensitivity of both inactivating KVQ subtype channels and KVQ/Kv{3 channel complexes (for details, see Channel modulation, 47-44). Note: The bracketed SGN symbol denotes the three amino acids (SGN) that replace the tripeptide GED at this position in peptide Kv{31 - N wt to generate the peptide Kv{31 - NSGN (see also Fig. 5). (Original alignment and analysis from Rettig et al. (1995) Nature 369: 289-94.) (c) Model for KVQ/ {31 subunit association showing proposed role of inactivating ball domains. The figure shows a hypothetical representation of only two KVQ and two Kv{3 subunits within an Q4{34 (octameric) voltage-gated K+ channel complex (see Protein molecular weight (purified), 47-22 and Predicted protein topography, 47-30). This simplified model is consistent with Kv{31 subunit N-termini providing a ball domain that can occlude the inner mouth (ball receptor) of open Kv channels (analogous to N-termini of KVQ channels, assuming that a single ball domain is supported by site-directed mutagenesis studies examining redox susceptibility - the symbol SH denotes the sulphydryl group of the cysteine which renders the inactivating ball domain sensitive to oxidation; for details, see Channel modulation, 47-44). (Reproduced with permission from Rettig et al. (1995) Nature 369: 289-94.) (From 47-29-02)

II

(b

=:s t"'t

~ ~ ~

_____________________ en_t_ry_4_7_1

Table 6. Summary of protein interactions reported for Kv{3 subunits within native protein complexes (From 47-31-02) Subunit designations

Description and cross-references

Early studies

For studies first defining native K+ channels as large hetero-oligomeric sialoglycoproteins, see Protein molecular weight (purified), 47-22. 47-31-03: Initial N-terminal microsequencingt of the 0 subunit of purified bovine o-DTx acceptors 18 (see Protein molecular weight (purified), 47-22) identified an 0 subunit subtype 'almost identical' to the rat Kv1.2 channel clones RCK5, BK2 and RBK2.

o4{34 octamers

See ref. 16 under Predicted protein topography, 47-30.

IS elective' association of Kvo and Kv{3 subunits in brain membranes by co-immunoprecipitation experiments

47-31-04: Immunoprecipitation of the DTx-acceptor complex (see Isolation probe, 47-12) using antisera specific for the Kvl.2o subunit indicates that Kv1.1, Kv1.4 and Kv1.6 are also components of this complex, consistent with detection of heteromultimer forms all containing Kv1.2 (for details, see Protein interactions under VLC K Kv1-Shak, 48-31). 47-31-05: Different Kvo-specific antibodies generally precipitate different proportions of the channels detectable with [125I]-o-DTx in different brain regions (anti-Kv1.2> 1.1 » 1.6> 1.4) consistent with a widespread distribution of these oligomeric subtypes48 . 47-31-06: Hetero-oligomeric combinations show Ilack of additivity' upon precipitation with a mixture of antibodies to Kv1.1 and Kv1.2. Furthermore, 'cross-blotting' of multimers precipitated by mAb 5 show they contain all four Kv proteins48 • 47-31-07: In independent 'reciprocal co-immunoprecipitation' experiments (see note 1 in Table 4) the {3 subunit polypeptides associated with the DTx-sensitive K+ channel complexes have been shown to be associated with rKvl.2 and rKvl.4, but not rKv2.1 o subunits in rat brain membranes30 (see also Isolation probe, 47-12; Subcellular locations, 47-16; Protein molecular weight (purified), 47-22 and Database listings, 47-53).

Other putative interactions

II

47-31-08: Probable Kv{3 subunit locations on the peripheral 'inner leaflet' of membranes (see Subcellular locations, 47-16) may suggest a role in cytoskeletal interactions, as proposed for the {3 subunit of L-type calcium channels (see VLC Ca, entry 42).

l_e_n_try_4_7

--'_

vitro following addition of protein kinase A (PKA). As denoted in the legend to Fig. 2 (under Encoding, 47-19) alignment of the presently known Kv,B subunit sequences identifies (i) a PKA consensus site conserved across all subunits (Ser146 in Kv,Bla, Serl12 in Kv,B2 and Ser153 in Kv,Blb) and (ii) several potential protein kinase C (PKC) sites conserved in the C-termini with an equal numbers of PKC sites in the N-terminus of rKv,Bla and hKv,Blb. Multiple consensus sites for phosphorylation by casein kinase II also exist in Kv,B subunit sequences (for loci, see refs. 4,9).

Action of 'endogenous' phosphorylating activities on Kva/{3 subunit complexes 47-32-02: Findings consistent with 'extensive phosphorylation in vivo' prior to the purification of the bovine Kv,B2 subunit have been described Y• Moreover, in the absence of added kinases, a low level of 32 P-Iabelling of Kva and Kv,B subunits is also observed, at sites with apparently different specificity to protein kinase A. Functional note: As hypothesized and discussed in ref. 4, modulation of Kv,B subunit activity by kinases may lead to switching of inactivation modes in native A-type Kv channels analogous to demonstrated redox modulatory effects (see Channel modulation, 47-44). Such 'long-term' functional changes may therefore include alteration of firing frequencies by either increasing or attenuation of cellular excitabilityt

(see also Phenotypic expression, 47-14).

ELECTROPHYSIOLOGY

Activation Accelerated activation/inactivation times for Kva/{3 complexes compared to Kva complexes 47-33-01: The difficulties involved in 'predicting' specified Kva subunit 'contributions' to voltage-gated K+ currents observed in native cells are outlined elsewhere (e.g. Channel designation under VLC K Kv1-Shak, 4803). In particular, heterologously expressed Kva subunits may exhibit slower activation times in comparison with (likely) native 'counterparts'. Examples of the 'accelerating' effect on rKvl.4a channel activation following co-expression with rKv,Bla are included under Inactivation, 4737. Note: The rKvl.4a/rKv,Bla combination was initially selected for these studies because of their notable co-distribution patterns in brain (see mRNA distribution, 47-13, Protein distribution, 47-15 and Protein

interactions, 47-31).

Effects on activation threshold - 'hyperpolarizing shift' (examples) 47-33-02: hKv,Blc == hKv,Bl.3 (as designated in ref.8; for inconsistencies in nomenclatures, see Table 1 under Cene family, 47-05): When hKv,Bl.3 is co-expressed in Xenopus oocytes with hKvl.5, it (i) induces a timedependent inactivation during membrane voltage steps to positive potentials; (ii) induces a 13 mV hyperpolarizing shift in the activation curve and (iii) slows deactivation (7 == 13 ± 0.5 ms versus 35 ± 1.7 ms at -40 mV)8

II

_"--

en_t_ry_4_7__1

(see also Current-voltage relation, 47-35). rKv,B2: Co-expression of Kv,B2 with Kvl.S causes a voltage shift in the activation threshold of Kvl.S of about -lOmV, which has been taken to indicate a putative physiological role for this interaction49. In separate experiments, Kv,B2 (i) induces a similar, but quantitatively smaller effect on Kv1.1 channels; (ii) accelerates the activation time course of Kvl.S, but (iii) had no marked effects on channel deactivation49.

Current type 47-34-01: Together with Kvo subunit composition within heteromultimeric complexes, the presence or absence of Kv,B subunits can profoundly alter the 'type' of current observed in heterologous and native cell expression systems (see other fields, e.g. Phenotypic expression, 47-14). As denoted by their description as 'accessory' or 'auxiliary' subunits, Kv,B subunits are not capable of forming ion channels when expressed alone.

Current-voltage relation A unique functional role for the hKvj31.3 (Kvj31c) splice variant 47-35-01: hKv,Blc == hKv,Bl.3 (as designated in ref. 8 ; for inconsistencies in nomenclatures, see Table 1 under Gene family, 47-05): When hKv,Bl.3 is co-expressed in Xenopus oocytes with hKvl.S (i) it converts the Kvl.S (outwardly rectifying) current-voltage relationship to one showing an apparent inward rectification8 , while (ii) the channels begin to open at more negative potentials (see Inactivation, 47-37).

Inactivation Altered inactivation properties of neuronal Kvl channels by Kvj31a subunits (examples) 47-37-01: rKv,Bla: Association of rKv,Bla with Kvo subunits confers rapid A-type inactivation on non-inactivating rKvl.l (delayed rectifier) channels following their heterologous co-expression in Xenopus oocytes. This effect is mediated by properties of an inactivating ball domain in the Kv,8la Nterminus 4 : Modulation of Kv,8la activity appears to be a function of the redox state of a cysteine residue near the Kv,8la N-terminus (for further details, see Channel modulation, 47-44).

Functional changes conferred on hKvl.5 by co-expressed hKvj31b 47-37-02: hKv,8lb (== clone hKv,83): As illustrated in Fig. 4, microinjection of Xenopus oocytes hKv,8lb mRNA with mRNA encoding the human cardiac delayed rectifier 0 subunit, hKvl.S (see VLG K Kv1-Shak, entry 48) induces (i) an inactivating component at pulse potentials> 0 mY; (ii) an 18 mV hyperpolarizing shift in the activation curve and (iii) slowed deactivation (7 == 8.0ms vs. 3S.4ms at -SOmV)5 (for further details, see legend to Fig. 4). Notes: 1. The hyperpolarizing shift described above would give rise to significant fractions of open channels at membrane potentials where hKvl.S alone would not normally be open (e.g. in the range -40 to -30mV).

II

l_e_n_t_ry_4_7

_

(a)

hKv 1.5

fr r - - - - -

-~--~/

~

40 msec (d)

(c)

C 1.0

~

Kvl.S + hKvP3~~-a

C 1.0

V I12=-24 mV

~

='

=' u

to)

+

+

~

]

.~ ~

~

0.5

~

0.5

.~

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E o

E o z

z

0.0 -80

-40

0

40

Membrane potential (m V)

o. 0

~

- - - - r - - , - - -__- - - .

r-j

0.0

0.5

1.0

1.5

2.0

Recovery Time (sec)

Figure 4. Functional expression ofhKv(31b : hKv1.5 channels. hKv(31b (= clone hKv(33): Voltage-dependent hKv1.5 current in oocytes in the absence (a) and in the presence (b) of hKv(31b shows a pronounced inactivating component induced by the (3 subunit. This effect is only observed at pulse potentials >OmV (currents shown were produced at a holding potential of -80mV with test pulse from -90 to +50mV in 10mV increments, followed by a return to -40mV). (c) The normalized current-voltage relation, calculated from deactivating tail currents at -40 m V and indicated by arrows in panels (a) and (b), illustrates the approx. 20mV hyperpolarizing shift in the activation curve observed in the presence (e) versus the absence (0) of hKvb1b. Differences in hKvl.5 recovery time following inactivation are also affected by the presence of hKv(31b: Following induction of inactivation (500ms pre-pulse to +50mV) currents were elicited by a step back to -BOmV for various time periods (d) followed by a test pulse to +50mV to test the degree of inactivation (measurement of peak normalized current flowing during the text pulse). As indicated in panel (d), recovery time courses were best fitted with an exponential function with a time constant of 51 ms. (Reproduced with permission from England et al. (1995) Proc Natl Acad Sci USA 92: 6309-13.) (From 47-37-02)

II

_

.

entry 47 "--------------

2. rKvl.l/hKv,Bla channels do not display any hyperpolarizing shift in the activation curve as seen with hKvl.S/hKv,Blb (ref. 4 and previous paragraph). 3. hKvl.S currents do not become fully inactivated by the presence of hKv,Blb (Fig. 4b) even after a 500 ms inactivating pre-pulse (Fig. 4d). Although non-stoichiometric expression ratio of a:,B subunits could be responsible for 'partial' inactivation (see paragraph on a:,B mRNA .ratios under Phenotypic expression, 47-14-05) there is presently no direct evidence to support this 5 . 4. Similar 'conversion' of a 'delayed rectifier type' to a 'rapidly inactivating type' channel has been described following co-expression of clone RBKVI.S (==rabbit Kv1.5, see VLG K Kv1-Shak, entry 48) with Kv,BI 5o. 5. Further functional characterization of Kvj31 subunits co-expressed with various K channel a subunits showed49 that (i) Kvj31 could induce inactivation in members of the Kvl subfamily with the exception of Kvl.6, while (ii) it could not induce inactivation of Kv2.I, Kv3.4-Ll2-28 and Kv4.1 channels49.

Kv{31b (clone Kv(33) alters Kvl.4 channel states which follow --activation 47-37-03: Kvj31b (== clone Kvj33): ferret Kval.4: When co-expressed in oocytes with a Kvl.4a subunit (clone FK1, see VLG K KV1-Shak, entry 48), clone Kvj33 has been shown51 to (i) accelerate both the fast and the slow component of Kvl.4 inactivation; (ii) alter the relative contributions of the two components of inactivation by increasing the contribution of a slow component to the inactivation process; (iii) slow recovery from inactivation for Kv1.4 (but not for a Kvl.4 deletion mutant lacking N-type inactivation) and (iv) slow deactivation kinetics. Steady-state activation (and the time course of Kvl.4 current activation) were not strongly influenced by co-expression of clone Kv,B3 51 .

Modulation of hKval.4 and Kval.5 current kinetics by clone hKv{33 in oocytes 47-37-04: Kvj31b (== clone hKvj33): hKvj33 subunits (cloned from human atrial mRNA, as designated in ref. 6 - see Table 1 under Gene family, 4705) also accelerate the inactivation of co-injected hKvl.4 currents and induce fast inactivation of non-inactivating co-injected hKvl.5 currents in oocytes 6 . Clone hKvj33 has no apparent effect on hKval.I, hKval.2 or hKva2.1 currents in this system. Note: Ra~ Kv,83 (as designated in refs 10,11, see Table 1 under Gene family, 47-05) also has a long N-terminal structure and induces inactivation in N-terminal deleted Kvl.4 (but not in other members of the Kvl channel family)ll. 47-37-05: Kvj3lb == Kvj33: Co-expression of Kvj3lb (designated Kvj33 in ref. 7) with K+ channel a subunits can increase the rate of inactivation from 4- to 7-fold in a Kvl.4 or Shaker B channels, with other kinetic parameters being unaffected 7. In contrast to Kvj3la (this field), Kvj3lb has no apparent effect on Kvl.l channel kinetics in oocytes 7 •

N-terminal peptide segments of Kv{31 alone cannot induce fast inactivation properties 47-37-06: Certain N-terminal peptides of the Kvj31 subunit alone are unable to mimic the increases in the rate of inactivation of whole-cell mKvl.1

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_

1~_e_n_t_ry_4_7

currents (as observed with the entire Kv,81 subunit)52 (compare with (i) the ability of N-terminal pepides from fast-inactivating Kv channel a subunits to induce N-type inactivation in Inactivation under VLC K Kv1-Shak, 48-37 and (ii) the minimal interaction domain motif described under Sequence motifs, 47-24).

Kv(32 modulates the inactivation properties of Kvl.4a subunits 47-37-07: Kv,82: Although the Kv,82 subunit has been shown to increase

protein turnover and promote the transport of Kva to the cell-surface (see Subcellular locations, 47-16), it can also 'modulate' the inactivation properties (i.e. increase the inactivation rate) of Kval.4 when co-expressed in Xenopus oocytes 12. Notably, Kv,82 does not induce inactivation when coexpressed with certain non-inactivating (N-terminal truncated) Kvl.4 channel constructs or non-inactivating Kvl.l channels. Moreover both Kv,81 and Kv,82 are able to increase the amplitude of Kvl.4 currents expressed in Xenopus oocytes. Note: In a different study49, Kv,82 did not induce inactivation when co-expressed Kvla subunits; by means of Kv,81/ Kv,82 N-terminal chimaeras, however, it was shown that Kv,82 subunits do co-assemble with these Kva subunits. Notably, Kv,81/Kv,82 and Kv,83/Kv,82 chimaeras were able to induce fast inactivation of several Kvl channels, also indicating that Kv,82 associates with these a subunits49.

PHARMACOLOGY

Blockers High-affinity toxin-binding studies instrumental in the discovery of Kv(3 subunits 47-43-01: The use of a-dendrotoxin (a-DTx) and its homologue 6-dendrotoxin (8-DTx, exhibiting a subtly different specificity, see Dolly et al., 1994, under Related sources and reviews, 47-56) has been of central importance in

defining Kva/,8 subunit associations within native K+ channel complexes using immunoaffinity-binding methods (for description, see Isolation probe, 47-12). Historically, such approaches led to the first identification of K+ channel a subunits and were important for establishing K+ channels from large, hetero-oligomeric sialoglycoproteins t in native tissues (for references, see Protein molecular weight (purified), 47-22). In general, a-DTx efficiently inhibits fast-activating, voltage-dependent, aminopyridine-sensitive K+ currents that exhibit slow inactivation53,54. Related, but rapidly inactivating K+ channel variants in neuronal preparations have been described as 'less susceptible' or 'insensitive' to a_DTx36,55; see also Dolly et al., 1987, under Related sources and reviews, 47-56. Notes: 1. The presence of K+ channel,8 subunits may alter sensitivity to block by pyridines (see ref. 13). 2. {3Bungarotoxin binds with high affinity to a subpopulation of a-DTx sites56 . 3. As determined by site-directed mutagenesis t, a-DTx itself binds to residues close to the extracellular mouth of intact channel complexes, without any reported affinity for the constituent ,8 subunits themselves (see Protein interactions, 47-31 and refs in the VLC K Kv series, entries 47 to 52; also

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_

...

e_n_t_ry---,-4_7_

compare to toxin binding properties of fJ subunits of calcium-activated K+ channels, under ILG K Ca, entry 27).

Utility of neurotoxin probes in channel expression/distribution and structure/function studies 47-43-02: In general, binding of the dendrotoxins and {3-bungarotoxin in brain

slice preparations reveal 'distinct, yet overlapping' patterns of distribution (e.g. as in rat, refs 57,58, see also Dolly et al., 1987, under Related sources and reviews, 47-56, and Protein distribution, 47-15). The ability to readily (i) modify rrimary structures of toxins for which the high-resolution crystal structures are known and (ii) purify large quantities of active recombinant toxins from bacterial hosts may help refine 'subtype-specific' immunoaffinity probes for native K+ channel complexes (see review; ref. 59).

Channel modulation tOxidoreductive' modulation of Kv{31 N-terminal tball' domain/ inactivation properties 47-44-01: Alignment of the N-terminal domain of Kv{31 subunits with those of Kvo subunits that undergo rapid N-terminal (N-type) inactivation demonstrates some broad structural conservation (illustrated in Fig. 3 under Domain functions, 47-29). The inactivating ball domain in the N-terminus of Kv{31 promotes rapid closure of open Kv channels which cannot otherwise inactivate rapidly (ref. 4 , see Phenotypic expression, 47-14 and Inactivation, 47-37). The redox state (Le. oxidation or reduction) of a Icritical cysteine' in the inactivation ball domain of Kv{31a reversibly switches the channel inactivation mode from 'fast' to 'slow' and vice versa4 : When serine is substituted for cysteine at position 7 (Kv{31C7S, see Fig. 3 under Domain functions, 4729) the sensitivity of (3 balls to oxidation is eliminated. This result is similar to that observed when 0 ball cysteines (ibid.) are replaced by serine60 and suggests that such substitutions 'destabilize' the interaction between the ball and its receptor bordering the channel pore. Figure 5 summarizes additional experiments which contributed to the proposal of inactivating {3 ball domains which are sensitive to redox modulation. Notes: 1. The term '{3 ball' was introduced to distinguish these domains from 'a ball' domains of Kvo subunit channels that undergo fast inactivation. 2. Inactivation induced by Kv{33 (as designated in refs 10,11, see Table 1 under Gene family, 47-05) is also regulated by the intracellular redox potential11 . 47-44-02: Comparative note: For further potential modulations of of{3 channel

complexes by oxidoreductive biochemical pathways, see Miscellaneous information, 47-55. For other examples of ion channel modulation in relation to cellular redox state, see refs 45,61 and Channel modulation under ELG CAT GLU NMDA, 08-44, ILG CI ABC-CF, 23-44, ILG K Ca, 27-44 and INR K ATP-i, 30-4462 .

Cytochrome P-450 inhibitors reduce native Kv currents in pulmonary arterial myocytes 47-44-03: Comparative note: Cytochrome P-450 (P-450) is an NADPH-

requiring and O2 -dependent mono-oxygenase system expressed in lung

II

Il..--e_n_t_ry_4_7

-----'_

(a)

(c)

(b)

red

RCK4

RCK4~1-110

RCK4~1-IIO

(e)

(d)

(f)

+ KvPI

200 ms ... ox -red

RCK4~1-110

RCK4~1-110

RCK4~1-110

+ KvPIC7S

+ Kv~1~1-34

+ peptide

Figure 5. 'Switching' of inactivation mode in Kvl.4 (RCK4) channels by oxidation or reduction of Kv{31 N-terminal ball domains. For background to experiments see paragraph 47-44-01. (a) For outward currents elicited by depolarizing pulses (to +50mV from an h.p. of -100mV in inside-out oocyte patches) N-type inactivation properties are lost following exposure of wild-type Kvl.4 channels to oxidative treatments (ox., by addition of 0.1 % H2 0 2 to the bath). N-type inactivation is restored following reducing treatments (red., by addition of 5 mM glutathione to the bath). (b) N-type inactivation and redox sensitivity are lost following removal of the Kvl.4 ball domain, as exemplified by the Kvl.4 deletion mutant RCK4~1-110, lacking the first 34 N-terminal amino acids 4 ,63. (c) Co-expression of RCK4~1-110 and Kv{31 subunits restores a rapidly inactivating current under reducing conditions (7[ == 4.2 ± 0.8ms). (d) Traces obtained when wild-type Kvl.4 is co-expressed with Kvf31 subunits are indistinguishable from those in (c). (e) Kv{3 subunits lacking their first N-terminal 34 amino acids (Kv{31~1-34) cannot confer fast-inactivating properties when coexpressed with RCK4~1-110. (f) 'Partial' restoration of inactivation to RCK4~1-110 channels is observed when a peptide comprising aa 1-24 of the Kv{31 N-terminus is applied to the bath (Kv{31-N wt ; 100 jjM). When the N-terminal peptide containing a CED ~ SCN substitution with increased net positive charge is similarly applied (Kvf31-N SGN ; 100 jjM; for residues see Fig. 3) a fast inactivating current results which is comparable to that seen with intact Kv{31 subunits. Note: The 'residual' slow current decay in the absence of N-type inactivation (e.g. under oxidizing conditions in panels a and c) is attributable to C-type inactivation involving the channel pore structure (see refs 64 ,65 and Inactivation under VLC K KvlShak, 48-37). (Reproduced with permission from Rettig et a1. (1995) Nature 369: 289-94.) (From 47-44-01)

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e_n_t _ry_4_7

-----J

which may act as an '02 sensor' in hypoxic pulmonary vasoconstriction. In pulmonary arterial myocytes, the P-450 inhibitors clotrimazole, miconazole, and 1-aminobenzotriazole (l-ABT) 'significantly and reversibly inhibit' native steady-state voltage-gated K+ currents and depolarize the cells bathed in either Ca2+-containing (1.8 mM) or Ca2+-free bath solution66 • These data may support a role for the P-450 system in linking the regulation of pulmonary vascular tone to the alteration of cellular redox status via its influence on Kv currents and sensitivity to O2 tension and NADPH66 . Notes: 1. Pre-treatment of pulmonary arterial smooth muscle with 4-aminopyridine (4-AP, 10 mM) but !lot tetraethylammonium prevents the subsequent inhibitory effect of P-450 inhibitors on Kv currents. 2. The effects of the P-450 inhibitors resemble those induced by hypoxia, reduced glutathione, and 4-aminopyridine.

Ligands Radioligands for co-precipitation of Kv{3 subunit acceptor complexes 47-47-01: Availability of radioiodinatedt derivatives of a-DTx, 8-DTx and {3bungarotoxin has allowed high-affinity acceptor complexes to be identified, purified and characterized in mammalian brain (for further details, see Isolation probe, 47-12 and Blockers, 47-43).

Receptor/transducer interactions 47-49-02: Although no studies have been reported regarding the functional regulation of Kv,8 subunit components coupled to specified receptor/second messenger systems, the known patterns of protein phosphorylation (see field 32, Protein phosphorylation) and possibly redox modulation (field 44, Channel modulation) might predict their existence. Hypothetically, receptor-coupled Kv{3 subunit modulation may influence in vivo phenotypes explicable in terms of those demonstrated for recombinant Kv{3 subunits in heterologous expression systems (e.g. see Phenotypic expression, 47-14, Subcellular locations, 47-16 and Inactivation, 47-37).

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 47-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction etJ layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in Introduction etJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily

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"'--.e_n_t_ry_4_7

_

accessed by one or two rounds of neighbouring t analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBlt ). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers) author /reference or nomenclature. Following direct accession, however, neighbouring t analysis is strongly recommended to identify newly reported and related sequences. Kv,B designation (this entry)

Species, original isolate name

ORF

cDNA source

Sequence/ discussion

rKv,81a

Rat Shaker ,81 subunit4

ORF: 401 aa rat gb: X70662 brain cortex cDNA library

Rettig, Nature (1994) 369: 289-94.

=

rKv,81

gb: not found McCormack, FEBS lett (1995) 370: 32-6.

Human brain cDNA Kv,81 clone3

hKv,81a

Accession

hKv,81b hKv,83

=

clone

Human, ,83 subunit clones

ORF: 408 aa cardiacgb: L39833 ventricular cDNA

England, Proc Nati Acad Sci USA (1995) 92: 6309-13.

hKv,81b hKv,83

=

clone

Human, ,83 subunit clone 7

ORF: 408 aa cardiac atrial cDNA

gb: U16953

Majumder, FEBS Lett (1995) 361: 1316.

gb: U17968

Morales, TBiol Chern (1995) 270: 6272-7.

ORF: 408 aa gb: U17966 (Musteia putorius)

Morales, TBioi Chern (1995) 270: 6272-7.

rKv,81b = clone Rat cDNA 7 rKv,83 partial seq.

399 nt segment

gb: U17967

Morales, TBioi Chern (1995) 270: 6272-7.

hKv,81c

ORF: 419 aa

gb: L47665

England, TBioi Chern (1995) 270: 28531-4.

ORF: 367 aa bovine Bas prirnigenius (aurochs) brain cDNA

gb: X70661 (as gb) cIte as X70662 in ref. shown at right

Scott, Proc Natl Acad Sci USA (1994) 91: 1637-41.

hKv,81b = clone Human, cDNA 7 400 nt segment hKv,83 partial seq. fKv,81b fKv,83

=

=

clone

Ferret heart, ,83 subunit 7

Kv,81.3 Human cardiac ventricular cDNA8

bKv,82

Bovine Shaker ,82; 'major' component of a-dendrotoxinsensitive K+ channels 9

hKv,82

Human brain cDNA Kv,82 clone3

Yi

gb: not found McCormack, FEBS Lett (1995) 370: 326.

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_L..... Kv{3 designation (this entry)

e_n_t_ry_4_7_

Species, original isolate name

rKv,82 or RCK,82 Rat Shaker ,82 subunit 9

ORF

Accession

cDNA source ORF: 367 aa rat gb: X76724 brain cortex cDNA library

Sequence/ discussion Scott, Froc Natl Acad Sci USA (1994) 91: 1637-41.

rKv,83

Rat brain Induces partial gb: not found Heinemann, Kv,83 10,11. Shares inactivation in Biophys T(1995) 68% identity channels of the Kvl 68: A361. with rKv,81 subfamily Heinemann, FEBS Lett (1995) 377: 383-9.

dHk,8

Drosophila, ORF: 546 aa adult gb: U23545 hyperkinetic (Hk)head cDNA 24 gene locus

Chouinard, Froc Natl Acad Sci USA (1995) 92: 6763-7.

Note: As part of the,8 subunit homology searches described in ref. 45 (see Miscellaneous information, 47-55), several partial cDNA clones were identified from rice and Arabidopsis (gb: D24756 j gb:Z30863 j gb: Z18389 j gb: D24673, of unknown function). These clones showed >40-60% identity with the Shaker ,8 subunits over their entire length of up to 96 aa.

Gene mapping locus designation 47-54-01: For chromosomal locus names that have been used (KCNA1B and KCNA2B) but are under review, see the notes under the field Chromosomal location, 47-18 (see also online resources such as OMIM for confirmation of

locus designations).

Miscellaneous information Cross-references to studies of accessory ({3) subunits associated with other channels 47-55-01: Accessory proteins or {3 subunits have been shown to modulate voltage dependence of gating, current amplitudes and inactivation kinetics of voltage-gated Ca2 + and Na+ channels (see refs 67- 77 and also relevant fields of VLC Ca, entry 42 and VLC Na, entry 55). Structural and functional features of a non-pore forming, 'accessory' {3 subunit of 31 kDa associated with pore-forming (maxi) K ca channel a subunits (e.g. refs. 78-82) are described in Protein molecular weight and Predicted protein topography under ILC K Ca, 27-22 and 27-30 respectively. An Arabidopsis thaliana cDNA encoding a 38.4 kDa polypeptide with homology to the mammalian Kv{3 subunits has been described40 . Kv{3 subunits possess unrelated primary sequences to all of the 'accessory' subunits indicated above, but do exhibit general structural similarity in that they are predominantly basic t (pI ~ 9.5) and hydrophilic, reflecting their common location at the cytoplasmic face of the membrane 83 . (see also Related sources and reviews, 47-56).

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l..--e_n_t_ry_4_7

A4 B 8 C 8

il

~1

L

H 41 I 75

S1 V T PNSSAQQ 1SIINIPFAE NLGKS RVSCL NLGKS RVSCL

J 1 K 2

SQARPATVLGAMEMlRRMDV----TSSSASVRA-----FLQ~TE1~If~GQS~1LIPLGLGLG

D 10 E 5 F 13 G 7

I

P3

f34

U3

loop A

~ A 63

~

B 70 C 70 D 72 E 64

74 66 108 142 1 K 63

F G H I J

-----W -----W

----------------------------------------

RSI-----CKVK1ATlAAp----MFGKTLKlADIRFQIET

------l I

U4

A B C D E F G H

125 DESGNVVPSDTN1L 132 DEHGKLLFETVD1C 132 DENGK1LFDTVDLC 134 DENGRV1YHKSNLC 127 NADGT1CYDSTHYK 136 EVEDLLPFDV---KG 117 -----------RWL 166 ------------ME I 200 ------------MEE1V J 24 ------------LEEOM K 117 ------------1ESDLQ

U6 A B C D E F G H

193 200 S 200 202 193 NE 199 172 223

A B C D E F

232 240 240 244 232 234 202 290 324 141 243

I

~ I

KS1 KS1 KS QA TKA1 RS1

~

I

I

E~!li

L

I EEF L NPF FSF I L L PSVG------FAN PNTI------FAN SNT ------1FYSIRVDATT ------FY~FIDHGT -------

Us

~

I

r-

HLQVEIKPG--LKY CRQLE KPG--LKY RRQLE KPG--LKY RRQLE KPG--LKY I SRQ1D SVAS----V SVKKLQN SVAT----1 TEPMLKTL1DETG----V WSSME1MEAYSVARNFNL1 F -QR WSAME1MEAYSVARQFNM1 CE F-QR SYSPELTQKAAA1LKEER-VPLF1H PNYNMF-NR SWEVAEDCTLCKKNGW1MlTVY MYNA1-TR

II~

.a

loop B

1QYCQSK----I1VVTIY SP-DRPWAKPEDI----------------ILIDP---------LDYCKSK----D11LVSYCT SSRDKTWVDQKS ---------------- LDDP---------DFCKSK----D1VL SHREEPWVDPNS ---------------- L DP---------EVSASSM---TSF1 TCRNPLWVNVSS P--------------L---------SS-DRAWRDPDE ---------------- L P---------1AHCQAR----IEVT EFCKEN---- 11VT RKGASRGPN---------------------E D---------FHDEH---- 1RTES RRS--------------------------~L Q---------EVNLPELFHK1 GAMT CG1VSGKYDSG~PYSRASLKGYNWLKDK1IS RRQQA---KL I 257 EVQLPELYHK1 GAMT CG11SGKYGNG ESSRASLKCYQWLKER1VS RKQQN---KL J 80 W1ENGLLDTLG-E1 GC1VF QGLLTSRYLNG1 GARANQGGS--LKASAQ LL------GR1 GGLLTGRYKYQDKDGKNPESRFFGNPFSQLYMDRYWKEEHFNG1A K 174 QVETELFPCLRH-F RFYlF

Us

G H I

J K

UH2 ~

EISQDMTTL Q SEDMKAL Q SEEMKA1 S KEEMKD1 TFSPEEMKQL EQDHHK1 W D ADQVDA1 1GA1QVLPKLSSS11H GA1QVLPKMTSHVVN ALVEEGPLEPAVVDA

loop C

---j ----------:....---------1 LSIRVCALLS---------CTSHKDlPFHEEF DG FRYNNAKY---------FDDHPNHPFTDE DG RYLTLD1---------FAGPP~PFSDEY EA RFVEMLM---------WSDHP PFHDEY NA RY1VPMLTVDGKRVPRDAGHP PFNDPY SQ1SQSRL1SGPTK---PQLADLWDDQ1 259 SGLERGRLWDGD-----------------PDTHEEM 350 E1DS1LGNKP--------------YSKKDlRS I 384 E1Ql1LRNKP--------------YSKKQlRS 1-1

A B C D E F G H

289 297 297 300 289 291

J 183

K 311 FDQAwlLVAHECP---------------NlFR

Figure 6. Amino acid alignment of selected putative NADPH-dependent aldo-keto reductase superfamily members including Kv{3 subunits. Alignments are related to known secondary structure elements of aldose reductase (top line A) as annotated.

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e_n_t_ry_4_7_

Key to Fig. 6 alignment: Sequence Protein (selected putative in NADPH-dependent aldo-keto alignment reductase superfamily members)

Species

Database reference (sp: Swissprot; gb: Genbank)

Aldose reductase 3-a-hydroxysteroid reductase Trans-1,2-dihydrobenzene-1,2diol dehydrogenase 3-oxo-5-~-steroid 4 dehydrogenase Aldehyde dehydrogenase NAD(P)H-dependent 6'deoxychalcone synthase 2,5-diketo-D-gluconic acid reductase Shaker ~2 subunit Shaker ~1 subunit igrA gene product (potential, partial sequence) Aflatoxin B1 aldehyde reductase

Human Rat Human

sp: P15121 sp: P23457 sp: Q04828

Rat

sp: 31210

Human Soybean

sp: P14550 sp: P26690

Corynebacterium Bovine Rat Pseudomonas

sp:P15339 gb: X70661 gb: X70662 gb: M37389

Rat

gb: X74673

A

B C D

E F G H I

T K

Annotations: ~1 to ~8: Segments forming ~ strands in aldose reductase. al to a8: Segments forming a helices in aldose reductase. Loops A to C: Loop regions in aldose reductase. Loops aHl and QH2: Two additional C-terminal helices. Numberings indicate portions of original amino acid sequences that were used in the alignment. Predominant amino acid identities are indicated as white lettering on black (see also Database listings, 47-53). Note: Other superfamily proteins with noted homologies24 to Kv~ and Hk proteins include bovine prostaglandin F synthase and lens crystallin from frog. Similar homology alignments (using a number of different algorithms) have resulted in significant matches with over 60 putative superfamily members45 . (Reproduced with permission from ref.45.) (From 47-55-02)

Structural relationships within the aldo-keto reductase superfamily 47-55-02: Structural analyses of Drosophila Hyperkinetic sequences (see Chouinard et al., 1993, under Related sources and reviews, 47-56 and ref.24) and amino acid homology searches for Kvf3 subunit homologues across many phyla45 have identified structural similarities of Kv~ subunits to members of the NAD(P)H-dependent aldo-keto reductase t superfamily t. This gene family encompasses a 'structurally similar but functionally diverse' group of cytosolic enzymes that all use NADPHt as a cofactor. Figure 6 shows an alignment of selected putative NADPH-dependent aldo-keto reductase superfamily members in relation to the known secondary structure of aldose reductase 84 . Similar homology alignments to Fig. 6 (using a number of different algorithms) have resulted in significant matches with over 60 putative superfamily

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-----..J_

members45 . Although percentage amino acid identities between Kv{3 and aldoketo reductase superfamily members are relatively low (typically 15-30%) this partly reflects the wide species coverage of sequences in alignments (identical residues or conservative substitutions occur within several key structural determinants - see Fig. 6). Notably, many of the residues known to be involved in NADPH binding and the hydrogen transfer mechanism are conserved in both mammalian Kv{3 and Drosophila Hk subunits24 . While it is premature to draw 'functional' conclusions on the basis of alignment data alone, it has been tentatively suggested that (3 subunits might be functional oxidoreductase enzymes. If this is the case, it might imply that (i) K+ channels are modulated through the redox state of NADP(H) or NAD(H) in vivo and/or that (ii) the {3 subunits themselves might be regulated by K+ channel activity45. For further discussion of these hypotheses, see refs24,25; see also Channel modulation, 47-44 and the annotated legend to Fig. 6.

Related sources and reviews 47-56-01: Reviews on structural and functional aspects of Kv{3 subunits2,59; minor part of cardiac potassium channel molecular physiology review85; see also the commentary on modification of channel inactivation properties86; auxiliary subunits of other voltage-gated ion channels83; commentary on calcium channel {3 subunits87 (see also VLC Ca, entry 42 and VLC Na, entry 55).

Book references: Chouinard, S.W., Schlimgen, A.K. and Ganetzky, B. (1993) Abstract. In Neurobiology of Drosophila, p.96. Cold Spring Harbor Laboratory Press, Plainview, NY. Dolly, J.D., Stansfeld, C.E., Breeze, A.L., Pelchen-Matthews, A., March, S.T. and Brown, D.A. (1987) In Neurotoxins and their Pharmacological Implications (ed. P. Jenner), pp. 114-16. Raven Press, New York. Dolly, J.D., Munitz, Z.M., Parcej, D.N., Hall, A.C., Scott, V.E.S., Awan, K.A. and Owen, D.G. (1994) In Neutrotoxins and Neurobiology (eds K.F. Tipton and F. Dajas), pp. 103-22. Ellis Horwood, Chichester.

Feedback Error-corrections, enhancements and extensions 47-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource T- Search criteria). For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 47-24-03). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback etJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

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_L....REFERENCES Yu, Neuron (1996) 16: 441-53. Pongs, Semin Neurosci (1995) 7: 137-46. 3 McCormack, FEBS Lett (1995) 370: 32-6. 4 Rettig, Nature (1994) 369: 289-94. 5 England, Proc Natl Acad Sci USA (1995) 92: 6309-13. 6 Majumder, FEBS Lett (1995) 361: 13-16. 7 Morales, TBiol Chem (1995) 270: 6272-7. 8 England, TBiol Chem (1995) 270: 28531-4. 9 Scott, Proc Natl Acad Sci USA (1994) 91: 1637-41. 10 Heinemann, Biophys T (1995) 68: A361. 11 Heinemann, FEBS Lett (1995) 377: 38~3-9. 12 McCormack, FEBS Lett (1995) 370: 32-6. 13 Rudy, Neuron (1988) 1: 649-58. 14 Chabala, T Gen Physiol (1993) 102: 713-28. 15 Parcej, Biochem T(1989) 264: 623-4. 16 Parcej, Biochemistry (1992) 31: 11084·-8. 17 Muniz, Biochemistry (1992) 31: 12297-303. 18 Scott, T Biol Chem (1990) 265: 20094-·7. 19 Trimmer, Proc Natl Acad Sci USA (1991) 88: 10764-8. 20 Rhodes, TNeurosci (1996) 16: 4846-60. 21 Kues, Eur T Neurosci (1992) 4: 1296-308. 22 Dixon, Eur TNeurosci (1996) 8: 183-91. 23 Uebele, T Biol Chem (1996) 271: 2406--12. 24 Chouinard, Proe Natl Aead Sci USA (1995) 92: 6763-7. 25 Stern, TNeurogenet (1992) 8: 157-72. 26 Stern, T Neurogenet (1989) 5: 215-28. 27 Yao, Soc Neurosci Abstr (1995) Abstract 121.11. 28 Wang, Soc Neurosci Abstr (1995) Abstract 121.10. 29 Wilson, Soc Neurosci Abstr (1995) Abstract 121.8. 30 Rhodes, TNeurosci (1995) 15: 5360-71. 31 Shi, Neuron (1996) 16: 843-52. 32 Nakahira, T Biol Chem (1996) 271: 7084-9. 33 Schultz, Genomics (1996) 31: 389-91. 34 Black, Biochem T (1986) 237: 397-404. 35 Black, Biochemistry (1988) 27: 6814-20. 36 Dolly, T Physiol (Paris) (1984) 79: 280-303. 37 Mehraban, FEBS Lett (1984) 174: 116-22. 38 Cai, T Biol Chem (1993) 268: 23720-7. 39 Prystowsky, FASEB T(1996) 10: 2504. 40 Tang, Plant Physiol (1995) 109: 327-30. 41 Sewing, Neuron (1996) 16: 455-63. 42 Kyte, TMol Bioi (1992) 157: 105-32. 43 Klein, Biochim Biophys Acta (1985) 815: 468-76. 44 Garnier, TMol Biol (1978) 120: 97-120. 45 McCormack, Cell (1994) 79: 1133-5. 46 Scott, Biochemistry (1994) 33: 1617-23. 1

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Muniz, Biochemistry (1992) 31: 12297-303. Schuhmann, FEBS Lett (1994) 341: 208-12. Heinemann, TPhysiol (1996) 493: 625-33. Sasaki, FEBS Lett (1995) 372: 20-4 (correction in Vol. 379: 202). Castellino, Am T Physiol (1995) 38: H385-91. Stephens, FEBS Lett (1996) 378: 250-2. Stansfeld, Neurosci Lett (1986) 64: 299-304. Stansfeld, Neuroscience (1987) 23: 893-902. Halliwell, Proc Natl Acad Sci USA (1986) 83: 493-7. Breeze, Eur T Biochem (1989) 178: 771-8. Awan, Neuroscience (1991) 40: 29-39. Pelchen-Matthews, Brain Res (1988) 441: 127-38. Dolly, Biochem Soc Trans (1994) 22: 473-8. Ruppersberg, Nature (1991) 352: 711-14. Lee, FEBS Lett (1992) 311: 81-4. Islam, FEBS Lett (1993) 319: 128-32. Hoshi, Science (1990) 250: 533-8. Lopez-Barneo, Recept Channels (1993) 1: 61-71. Hoshi, Neuron (1991) 7: 547-56. Yuan, Am T Physiol (1995) 37: C259-70. Dewaard, T Physiol (1995) 485: 619-34. Dewaard, Neuron (1994) 13: 495-503. Isom, T BioI Chern (1995) 270: 3306-12. Patton, T BioI Chern (1994) 269: 17649-55. Isom, Science (1992) 256: 839-42. Messner, T BioI Chern (1985) 260: 10597-604. McHugh-Sutkowski, T BioI Chern (1990) 265: 12393-9. Makita, Genornics (1994) 23: 628-34. Makita, T BioI Chern (1994) 269: 7571-8. Yang, Neuron (1993) 11: 915-22. Bennett, FEBS Lett (1993) 326: 21-4. Garcia-Calvo, T BioI Chern (1994) 269: 676-82. Knaus, T BioI Chern (1994) 269: 3921-4. Knaus, T BioI Chern (1994) 269: 17274-8. McManus, Neuron (1995) 14: 645-50. Tseng-Crank, Proc Natl Acad Sci USA (1996) 93: 9200-5. Isom, Neuron (1994) 12: 1183-94. Wilson, Science (1992) 257: 81-4. Deal, Physiol Rev (1996) 76: 49-67. Aldrich, Curr BioI (1994) 4: 839-40. Bean, Nature (1994) 368: 15-16.

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Vertebrate K+ channels related to Drosophila Shaker (Kva: subunits encoded by gene subfamily Kvl) Edward C. Conley

Entry 48

Note on coverage: Features of Kvo. subunits forming K+ -selective, voltage-gated channels in heterologous cell expression systems are listed within this and the following three entries according to their gene family relationships (VLG K Kv1Shak, entry 48; VLG K Kv2-Shab, entry 49; VLG K Kv1-Shaw, entry 50 and VLG K Kv1-Shal, entry 51). The roles of Kv(3 subunits in extending the structural and functional diversity of voltage-gated K+ channels by association with specified Kvo. subunits are described under VLG K Kv-beta, entry 47. General properties applicable to voltage-gated K+ channels in native cells (largely where the subunit composition has not been determined) are summarized under VLG K A-T native ('transient outward' K+ channels, entry 44) and VLG K DR native ('delayed rectifiers', entry 45). Inwardly rectifying, K+ -selective channels formed from subunits encoded by the Kir gene family are described under the entries beginning INR K (entries 29 to 33 inclusive). Because of their distinct functional or structural properties, separate entries have also been assigned to voltage-gated K+ channels in the eag family (VLG K eag/elk/erg, entry 46), calcium-activated K+ channels (ILG K Ca, entry 27), sodium-activated K+ channels (ILG K Na, entry 28), channels underlying native cell M-current (\lLG K M-i native, entry 53), and 'minimal' K proteins (VLG [K) minK, entry 54). In all entries, there is a bias of coverage to vertebrate K+ channel types - see special note under Category (sortcode), field 02.

NOMENCI-JATURES

Abstract/general description 48-01-01: Vertebrate K+ channel subunits whose primary amino acid sequences are most closely related to those of Drosophila Shaker are grouped in the voltage-gated K+ channel subfamily 1 (Kvl, this entry). In mammals, the Shaker-related gene subfamily consists of seven characterized isoforms (Kvl.l to Kvl.7); initial reports of further Shaker-related isoforms have appeared (see Gene family" 48-05). Shaker and Shaker-related channels probably represent the most intensively studied group of K+ channels. 48-01-02: Most Kv channels display marked heterogeneous expression patterns, many of which appear subject to developmental control (for specific examples, see Developmental regulation, 48-11). Spatial- and temporal-selective induction of Kv channel activities in development (developmental heterogeneity) presumably reflects functional specialization within particular 'developmental compartments' or terminally differentiated cell types (for discussion, see TUN [connexins), entry 35). Localization (segregation) of different Kv proteins to specific subcellular domains may also significantly affect native channel properties, particularly where they are co-localized with modulatory proteins. Much indirect evidence exists for developmental co-regulation of genes encoding multiple channel types channel and channel-modulatory proteins (i.e. 'co-ordinated' multigene expression).

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48-01-03: Kvl subfamily channel developmental ontogeny has been studied extensively in Xenopus, taking advantage of well-characterized morphological differentiation of identified neurone subsets (and the opportunity for comparing channel subtypes versus native currents this offers). For example, within the embryonic nervous system, transcripts encoding the Kvl.l homologues XShal and (at a lower level) XSha2 (a Kvl.2 homologue) are detectable. Furthermore, the onset of neurogenesis in Xenopus (stage 13) is associated with the induction of Kvl.2 gene expression (isolate XSha2) (see Developmental regulation, 48-11). Well-characterized development models involving Kvl subfamily channels also exist for non-excitable cells, for example Kvl.l and Kvl.3 in lymphoid thymocyte precursors (ibid.). 48-01-04: Several novel aspects of gene expression control have been described for Kvl subfamily members. These include (i) multiple hormonal regulation of Kvl.S gene transcription (and protein induction) in a tissue-specific manner (e.g. TRH or dexamethasone); (ii) sensitivity to 'cold stress' responses; regulation by cAMP and K+-induced depolarization (the former mediated by a cAMP-response element t (CRE) in the S/-non-coding region of the rKvl.S gene interacting with CRE-binding protein (CREB); (iii) regulation by chronic morphine administration; (iv) cell-specific negative regulation of rKvl.S by a gene silencer t element and (v) heterologous positive regulation of the native hKvl.S gene by a locus control region or 'gene activation element' (for details of each of these, see Developmental regulation, 48-11.) 48-01-05: A number of approaches have been used to deduce 'function' of a given K+ channel subunit in vivo, and in most cases this is not a simple task (see Phenotypic expression, 48-14). 'Clues' to function may be derived from, for example, naturally occurring mutant Kvl channels in populations, perhaps associated with an inherited disease (as for mutant Kvl.l/episodic ataxia, ibid.); non-random subcellular distributions or subunit co-assemblies (ibid.); specific functions suggested by a limited Kv1 subtype expression profile in a specified cell type (e.g. Kv1.3 in T cells, microglia and osteoclasts, ibid.); transgenic overexpression (i.e. 'forcing' expression of a Kv channel in a particular cell type, as in the case of Kv1.5 in pancreatic j3 cells, ibid.) or gene knockout t approaches. Much debate has centred upon the difficulties of 'predicting' specified Kv subunit 'contributions' to voltage-gated K+ currents observed in native cells (for discussion, see Channel designation, 48-03 and in VLC K Kv-beta, entry 47). Despite these difficulties, many authors have pointed out 'notable similarities' between native and heterologously expressed channel properties (see Table 4 under Phenotypic expression, 48-14). 48-01-06: Many cell types express voltage-gated K+ channel subpopulations that are characterized by different functional and pharmacological properties Generally, Kvl subunit genes show distinct but overlapping expression patterns within brain and other tissues. However, the expression pattern of some Kvl subunit mRNAs in native tissues appear ubiquitous. Tabular summaries of reported tissue distributions for Kvl subfamily mRNAs by techniques such as in situ hybridization, Northern hybridization, RNAase protection assay and RT-PCR appears in Table 3 under mRNA distribution, 48-13.

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48-01-07: In general, Kv channel subunits in native cell types are expressed at 'low densities', with more than one subtype co-expressed per cell- factors that have made 'direct' purification methods for specific Kv channel proteins technically formidable without toxin immunoaffinity methods (see next paragraph, Protein molecular weight (purified), 48-22 and Blockers, 48-43). Notably, however, certain membrane-associated putative guanylate kinases such as PSD-95, SAP97, chapsyn-110 and hdlg have been shown to promote differential Kv channel clustering by associating their PDZ domains with Cterminal residues in Kvl.l, Kvl.2, Kvl.3 and Kvl.4. Chapsyn-IIO associates tightly with the post-synaptic density in brain and mediates the clustering of both Kv channels and NMDA receptors (entry 08) in heterologous cells (see Channel density, 48-09). 48-01-08: Native Kv channel assemblies immunoprecipitated with highaffinity toxins such as a-dendrotoxin (o-DTx) are octameric (a4,84) sialoprotein complexes (for details, see VLG K Kv-beta, entry 47). Subtypes of a-DTx acceptor complex in bovine brain using a monoclonal antibody selective for Kv1.2 (mAb 5) and polyclonal antibodies for other Kvo subunits indicate that Kv1.1, Kv1.4 and Kv1.6 can be components of the acceptor, while 'virtually all' DTx receptors contain the Kvl.2 0 subunit (see Protein molecular weight (purified), 48-22). Anti-Kv antibodies have been important tools for understanding events in early channel biosynthesis and assembly, defining specific subunit interactions (e.g. within heteromultimers) and in measuring channel Mr values (ibid.). 48-01-09: Mechanisms contributing to the origin of the Kv multigene family include many different types of genetic duplication, recombinational exchange and transfer mechanism that have operated during evolution. In particular, localized, tandem gene duplication (followed by sequence divergence) is associated with the generation of clusters of functionally related genes, and this appears typical for Kv channel gene arrangements (Fig. 1 and text under Chromosomallocat:ion, 48-18). Large-scale duplications and/or rearrangements (e.g. affecting whole chromosomes or substantial segments of them) can also generate contiguous segments of linked genes that have a corresponding (paralogous t) region of related, linked genes elsewhere in the genome. This 'clustered' distribution of several groups of Kv channel genes in the mouse (ibid.) has confirmed and extended the existence of such paralogous regions. Genomic studies such as these have allowed proposal of evolutionary time-scales for ]{v gene duplication and divergence from primordial types (Fig. 1). 48-01-10: In Drosophila, K+ channel subtype heterogeneity is determined by alternative splicingt of the limited set of K+ channel genes; in the vertebrate Shaker-related gene subfamily (all single-copy genes), most protein-coding regions are intronless (the exception being Kvl.?, which has a single intron interrupting the structural gene in the region encoding the Sl-S2 extracellular loop - see Gene organization, 48-20). A summary of intron sequences found in Kvl subfamily (within non-coding regions) is listed in Table 9. A potential role in control of channel mRNA stability conferred by ATTTA and ATTTG motifs in the 3' untranslated region of the Kvl.4 gene (KCNA4) has been described (ibid.).

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48-01-11: Prior to the availability of high-resolution K+ channel molecular structure information494, modelling approaches reliant on interpretation of structure-function t data (e.g. 'iterative' testing of structural predictions by means of site-directed mutagenesis t followed by functional analysis of mutant proteins) had been a dominant approach. These models are subject to continual refinement, and the numbers of protein motifs (field 24) involved with designated 'functions' are increasing, often following detection of motif homologies between protein families. The 'delineation' of named domains and their description within the Kv1 family are described under fields such as Domain conservation, 48-28, Predicted protein topography, 48-30 and Protein interactions, 48-31. Structural elements for homophilic interaction between Kv1 subunits have been extensively characterized (ibid.). 48-01-12: Phosphorylation of KVQ subunits (and/or their accessory protein components) can alter characteristics such as channel current amplitude, voltage-dependence of gating and the kinetics of activation (see Protein phosphorylation, 48-32). Phosphorylation of K+ channels is thus an important general mechanism for modulating calcium entry, action potential firing patterns (threshold, frequency, height, width) and 'effector' responses coupling membrane excitability to secretion, muscular contraction or gene transcription (see Phenotypic expression, 48-14). Activation of neurotransmitter receptors commonly alter excitability and synaptic efficacy by generating intracellular second messengers, with several second messengers acting through protein kinase and phosphatase proteins that alter properties of K+ channels. Specific examples of Kv1 subfamily phosphomodulation are described in Table 16 under Protein phosphorylation, 48-32. 48-01-13: The molecular mechanisms of channel activation and inactivation following displacement of voltage-sensing domains has been most extensively studied in Drosophila Shaker channels (see Activation, 48-33, Inactivation, 48-37 and Voltage sensitivity, 48-42). For example, there is an extensive literature on the origins of gating current t (i.e. movements of electronic charge across the membrane field during voltage activation), Ntype inactivation (field 37) and C-type inactivation (ibid.). These topics are further discussed within structured tables under these fields. 48-01-14: In physiological ionic gradients, Kv channels show high selectivity for K+ over Na+ ions. A large number of studies (Table 21 under Selectivity, 48-40) have determined that ion selectivity functions in voltage-dependent K+ channels are predominantly associated with a segment of 21 contiguous residues known as the P-region (i.e. the pore region between domains S5 and S6). The P-region, along with the S6 segment and the S4-S5 linker appear to contain most of the pore determinants. The P-region is the only recognizably conserved segment in all K+ -selective channels, irrespective of subfamily or species (ibid.). Kv1 subfamily channels have unitary conductances in the range 8-18pS (see Table 22 under Single-channel data, 48-41). 48-01-15: A large number of studies have reported properties of peptide toxin, ionic and pharmacological blockers of Kv 1 subfamily channels in heterologous expression systems (for overvie~ see Blockers, 48-43). High-throughput screening methods have begun to yield compounds with claimed high

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selectivity and potency against Kvl channel subfamily members. In addition, sophisticated 'toxin docking' approaches (based on assignment of 'pairwise' amino acid interactions between blocker and target) have helped define spatial arrangements of Kv channel residues (ibid.). Scanning mutagenesis t (see Glossary) can identify sets of inhibitor residues critical for making energetic contacts with the channel, using thermodynamic mutant cycle analysis. Issues of selectivity, relative potency and mechanisms of block (listed by blocker and by target) are discussed within structured tables under Blockers (field 43). 48-01-16: A number of physiological and pharmacological factors/agents that have been cited as modulating the electrophysiological characteristics of KvI subfamily channels, including arachidonate, Ca2 + ions, K+ ions and redox state are discussed under Channel modulation, 48-44. The large size of the Kv subunit family (and the considerable number of potential couplings to receptor/second messenger systems) predict a considerable number of receptor/transducer/effector combinations operating within native cells. Despite the large potential number of couplings, only relatively few have been demonstrated directly (see Table 28 under Receptor/transducer interactions, 48-49). 48-01-17: Homologues of voltage-gated K+ channel genes are now known to exist in an extraordinarily wide evolutionary range of organisms from Escherichia through Streptomyces, ciliate protists (e.g. Paramecium), Arabidopsis, jellyfish, worms, squid and (as most extensively characterized) flies and vertebrates. The Escherichia coli sequence predicts a protein 417 residues long with 'extensive similarity' to eukaryotic proteins in amino acid sequence, six apparent transmembrane regions with a P-region motif between S5 and S6. Given that there is lack of direct evidence that the Escherichia K+ channel gene was 'imported' from genomes of higher organisms, its existence supports the view that the Shaker K+ channel subfamily was 'functionally established' prior to the first major radiation of

metazoans.

Category (sortcode) 48-02-01: VLG K Kvl-Shak, Le. a subunits of vertebrate voltage-gated potassium channels encoded by Kv gene subfamily 1 whose primary (amino acid) sequences show highest identity to those of Drosophila Shaker (see Gene family, 48-05). Homomultimeric t channels formed from Kvla subunits are normally named in accordance with the cDNA name (see Channel designation, 48-03). Conventional names of Kv gene (chromosomal) loci are listed under Gene mapping locus designation, 48-54.

Special note: Bias of coverage to vertebrate K+ channels 48-02-02: The fundamental and continuing importance of studies in Drosophila for delineation of K+ channel gene family and structurefunction relationships cannot be underestimated, and several comparative features are listed in the entry. For reasons of space, however, this entry excludes many of the known properties specifically derived for Drosophila

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and other non-vertebrate Shaker-subfamily channels (but see references in Related sources and reviews, 48-56).

Problems of 'single entry' coverage for large Kv channel families 48-02-03: 'Grouping' of channels using the criterion of primary sequence relatedness (for the purposes of print publication, as in the Kvl-Kv4 subfamilies, entries 48 to 51) has both advantages and disadvantages: Advantages include the ability to compare homomeric channel properties applicable in heterologous t expression systems and 'common' subfamily features. Disadvantages include the large number of apparent 'variables' between studies and the lack of a simple method to index properties applicable to heteromultimeric subunit combinations in native and heterologous cells; these difficulties are not unique to K+ channel subunit families.

Information sorting/retrieval aided by designated gene family nomenclatures 48-02-04: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: Kvl.n where n is a designated !!umber in the systematic nomenclature (e.g. Kvl.4). Within this entry, paragraph 'running orders' (sort orders) are largely determined alphanumerically by systematic nomenclatures - i.e. denoting species and gene product prefix combined with any trivial or clone name{s) where these have been used in the source reference (e.g. rKvl.4/RCK4:). Where properties are likely to apply to all or several subfamily proteins (i.e. irrespective of species or isolate) the 'species' term may be omitted. Entry updates covering novel isoforms will index information using these conventions established from systematic nomenclatures based on gene family relationships.

Channel designation 48-03-01: Vertebrate voltage-dependent K+ channel genes/cDNAs/channels related to Drosophila Shaker are assigned to Shaker-related subfamily 1 (this entry), presently designated as Kvl.l to Kvl.7 by the published nomenclature 1,2. Species 'equivalents' (see next paragraph) are indicated in a Kv name by a species prefix (e.g. human Kvl.l, rat Kvl.l, mouse Kvl.l) or conventionally by their initial letter (e.g. hKvl.l, rKvl.l, mKvl.l). For descriptions of vertebrate K+ channel subunit genes segregating into homology groups defined by Drosophila Shab, see VLC K Kv2-Shab, entry 49; for Drosophila Shaw-related Kv genes, see VLC K Kv3-Shaw, entry 50 and for Drosophila Shal-related Kv genes, see VLC K Kv4-Shal, entry 51. Strictly, Kv genes t (i.e. chromosomal sequences) should be referred to by their gene locus designation (see Cene mapping locus designation, 48-54) although in practice 'Kv numbers' are used to conveniently 'specify' genes, mRNAs, cDNAs, cRNAs and protein subunit variants arising from or associated with expression of a given Kv gene product or locus. Note: Two additional mammalian cDNAs for which Drosophila homologues have not (presently) been identified, are tentatively classified {in the absence of

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functional expression) as cDNA IK8 as K(v)5.1 and cDNA K13 as K(v)6.1 (for a brief account, see VLC K Kvx, entry 52). This nomenclature conflicts with a designation used for the Aplysia clone aKv5.1 (for a brief description of clone aKv5.1, see Abstract/general description under VLC K Kvx, 52-01).

Intended use of the term tisoform' within this entry

48-03-02: Properties of specific isoforms t with the same tKv number' are

intended to designate genes/protein subunits that are 'so closely related' at the primary sequence level that they might be judged 'equivalent' in different vertebrate species. The presumption of physiological 'equivalence' is open to debate, since channel function and modulation in situ (i.e. within the 'same' tissue of different species) may critically depend on the coexpression of other proteins which may have arisen since speciation (i.e. evolutionary separation) occurred. These complex issues, including the evolutionary antecedents of vertebrate K+ channels, assemblies of 'functional motifs' in primary sequences and the conservation of the Shaker, Shal, Shab and Shaw subfamilies as 'essential sets' of excitable t channels have been reviewed3,4 (see also Cene family, 48-05).

Parallel designation of tsystematic' and tnon-systematic' (isolate/ clone) names 48-03-03: In addition to the systematic nomenclatures based on sequence homology groupings (Table 1), non-systematic (original 'isolate' or 'clone')

names may persist in use for descriptive or 'discriminatory' purposes. Independently isolated (and named) subunit designations are listed in Table 1 under Gene family, 48-05. Subtype- or 'isoform-specific' information is indicated in this entry using an underlined prefix (e.g. RCKl:) within appropriate fields. In an attempt to preserve a 'logical' running order, properties are mostly (but not exclusively) listed in 'ascending' Kv number (coupled to the clone/isolate name, e.g. Kvl.l/RCKl:).

Other specific designations in occasional use 48-03-04: (i) In cases where it is important to distinguish Kv gene loci from Kv

gene products conventional gene locus designations can be used (see Gene mapping locus designation, 48-54). (ii) Specific designations of native and heterologously expressed K+ channel subunit complexes may have to take account of known subunit associations/stoichiometries that occur in cells (e.g. see Channel designation under VLG: K Kv-beta, 47-03 and paragraph 47-04-05). (iii) In the special case of tandem linkage of cDNAs encoding different Kv subunits (within a single open reading frame t construct) additional nomenclature may be needed to specify subunit order/position and predicted composition (i.e. A-B-A-B versus A-A-B-B etc.). Furthermore, phenotypic t expression of site-directed mutations t in one (or more) of the tandemly linked subunits may show some subunit 'position-dependence'. It should also be noted that tandem linkage of K+ channel subunits may not guarantee stoichiometric t relationships of the expressed channels5 (described under Protein interactions, 48-31).

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tMatching' native channel currents with those of heterologously expressed Kv subunit channels 48-03-05: Several factors underline the difficulty of 'matching' properties of native cell voltage-gated K+ channels to the 'contribution' of individual Kv subunits: Heterologous t expression of single Kv subunits (i.e. in the specified absence of endogenous channel subunits capable of co-assembling with the heterologous component) is generally assumed to occur via formation of homomultimerict (i.e. homotetramerict) channels6 . Relatively few 'native' K+ channel currents can be 'accounted for' in this way (for

possible exceptions, see paragraph in Phenotypic expression, 48-14). Functional heteromultimerict channels can be assembled from subunit components belonging to the same Kv subfamily (e.g. between members of the Kvl subfamily) but not between members of different subfamilies within the same tetrameric complex (for details and mechanisms, see Protein interactions, 48-31). Despite this, 'closely related' channels from the same subfamily, even when expressed in the same cell, need not coassemble and as a result may be differentially localized within the cell (for an example, see Subcellular locations, 48-16). Furthermore, the presence of certain Kv,B subunits can significantly influence the inactivation, modulation and expression properties of voltage-gated K+ channels formed from Q subunits alone (for details, see VLC K Kv-beta, entry 47). Heterologous expression systems may not 'reproduce' significant modulatory conditions found in native cells (e.g. those dependent on co-expressed receptor/ transducer protein activities, cytosolic modulators of channel function or other 'critical' post-translatory modifications t). Finally, there are numerous examples of native/heterologous current 'matching' which have not anticipated 'contributions' from novel channel subunits that were 'uncloned' at the time of analysis.

Current designation 48-04-01: For currents conducted by homomultimeric t channel assemblies in heterologous cells, this could follow the shorthand form I Kvn .n , where n.n is the Kv subfamily number of the gene encoding the monomer, although this type of designation is rarely used.

Gene family An tessential set' of K+ channel subunit genes conserved from flies to humans 48-05-01: The Kv channel Q subunit gene family constitutes the most extensive and diverse group within the voltage-gated channel superfamilyt (see VLC key facts, entry 41). Drosophila Shaker, Shal, Shab and Shawrelated sequences 7- 9 define four subfamilies of voltage-gated K+ channels which are conserved in vertebrates 3,10 (subfamily Kv1, this entry, and subfamilies Kv2, Kv3 and Kv4, entries 49 to 51 inclusive). Conservation of this subfamily structure over such a wide 'evolutionary gap' suggests a common relationship to ancestral gene(s) in existence prior to the divergence of mammals and insects (see refs. 3 ,4 and paragraphs describing

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the putative K+ channel gene kch in the bacterium Escherichia coli, under Miscellaneous information, 48-55). The similarities between multiple genes in the Sh superfamily indicates that ancestral gene(s) encoding K+ channels underwent extensive duplication and subsequent variation (selection) in the chordate lineage (generating a large number of closely related genes). The availability of lcomplete' genomic sequences for several prokaryote organisms (together with increased knowledge on gene homologue relationships in lower and higher eukaryotes in coming years) may clarify these 'adaptive' functions of Kv and other gene products to their various physiological roles in differentiated cell types. Note: To indicate their relatedness to the Sh superfamily t of genes, some authors have used the designation ShI to designate mammalian homologues of Shaker. Selected references describing the original isolation and characterization of the Drosophila Shaker K+ channel gene products can be found under Related sources and reviews, 48-56. For a clarification of gene family relationships of the KATl and AKTl channels from Arabidopsis thaliana (which share some structural features with the Kv subfamilies) see Domain conservation under VLG K eag/elk/erg, 46-28).

The vertebrate Kvl gene subfamily: channel subtype diversity 48-05-02: Whereas in Drosophila K+ channel subtype heterogeneity is determined by alternative splicingt of the limited set of K+ channel genes, vertebrates primarily use multiple distinct genes to encode channel subtypes (although several alternative splicing events are known within the Kv gene family, affecting both non-coding and coding regions (for known examples, see Gene organization, field 20 of entries; particularly in VLG K Kv3-Shaw, 50-20). A listing of vertebrate Shaker-related genes/cDNAs encoding voltage-gated K+ channel subunits, with 'systematic' and 'isolate' (clone) names is given in Table 1.

A gene family tree for Kvl subfamily members 48-05-03: Figure 1 shows a Shaker subfamily phylogenetic tree including some non-mammalian channel sequences, based on the 1994 analysis of Chandy and Gutman (1994, see Related sources and reviews, 48-56) and an earlier analysis by the same authors 55 . The later analysis groups Xenopus Xshal with Kvl.l and Xsha2 with Kvl.2.

Basic criteria for Kv subfamily assignments 48-05-04: Generally, the central hydrophobic core of vertebrate Kv channels can be readily identified as homologous t to that of a channel encoded by one of the Drosophila genes, although there is less conservation of sequence in the C-terminal and N-terminal cytoplasmic regions. As a general guide, members within the same subfamily are ''"''700/0 identical' at the amino acid sequence level, whereas members among different subfamilies show '4050%' sequence identity8. Note that tpercentage homologies' or identities within a group of channel sequences may be influenced by the number of channel sequences included in the analysis, the regions of proteins chosen for alignment (i.e. total versus hydrophobic tcore' sequences), species of origin, and the sequence alignment algorithm employed (see Resource D

II

I

entry48

_

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- Diagnostic tests, entry 59). Conserved subfamily lcompatibility' determinants in the N-terminal region of Kv channel have been subjected to extensive structure/function analyses (for details, see Protein interactions, 48-31).

Subtype classifications Kv channel classifications/nomenclatures are subject to modification 48-06-01: Voltage-gated K+ channel genes reported after July 1991 usually have followed the 'Kv nomenclature' as an essential part of their description1 . Existing classifications of Kv channels may require 'ongoing refinement' (e.g. to accommodate new isolates which do not show sufficient amino acid homologyt for classification as members of the Shaker/Shab/Shaw/Shal subfamilies). This is exemplified by the putative K+ channel cDNA isolates K13 and IK8 (briefly described under VLC K Kvx, entry 52) which appear to be representative of novel K+ channel gene subfamilies. The 'relatedness' of presently known Kv1 subfamily isolates is indicated under Cene family, 48-05.

Earlier 'classifications' of voltage-gated K+ channels are inadequate 48-06-02: As further described in VLC K A-T [native], entry 44 and VLC K DR [native], entry 45, it is likely that the use of the terms 'A-type' and 'delayed

rectifier' will assume less significance for voltage-gated K+ channel classification: These divisions cannot account for the known subunit heterogeneity expressed in native cells and defined hetero-oligomeric arrangements of Kva subunits (channel-forming, this entry and entries 49 to 52 inclusive) complexed with Kv,8 subunits (see VLC K Kv-beta, entry 47) may be able to account for many observed inactivation properties at the level of specified native cell types (ibid.).

Trivial names 48-07-01: The Shaker-related voltage-gated K+ channel subfamily. Kv1a

subunits; pore-forming Kv1 subfamily channels. Other 'trivial names' of Kv channel subunits/genes/cDNAs are indicated in Table 1 under Gene family, 48-05.

EXPRESSION In order to compare 'similarities and differences' information has been collated in tabular format for several fields for each of the subtypes of K+ channels mostly by 'ascending' Kv number. Note, however, that many 'independently reported' results for Kv isoforms t show only minor or no differences, thus 'full listing' of data 'by isolate name' is superfluous. Where apparent discrepancies exist, or where specific information might be useful, data are listed under both the Kv nomenclature 1 and the original gene/subunit ('clone') name. Source references for 'sequence-related'

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Table 1. Cene family nomenclature and original names in use for vertebrate voltage-dependent K+ channel cDNAs/subunits related to Drosophila Shaker, subfamily 1. cDNA 'clone' names (isolates) in common use are indicated in bold. See also VLC (key facts, entry 41) and Subtype classifications, 48-06. (From 48-05-02) Kv designation

Human isoforms

Rat isoforms

Mouse isoforms

Other species

Kvl.l Names in use and refs (notes 4 and 5)

hKvl.1 HuK(I)11-13

rKvl.1

mKvl.1 MBKl 18 MKl 16,19

Other Hamster Kvl.1 16 Rabbit Kvl.1 2o Xenopus Xsha1 21

Kvl.2 Names in use and refs

hKvl.2 HuK(rIV)11-13

rKvl.2 BK2 15 RCKS 22 NGK1 23 RK2 17 RAK24 RH1 25

mKvl.2 MK2 19 MKS 16

Other Bovine Kvl.2 (BGKS)26 Rabbit Kvl.2 2o Xenopus Xsha221 Canine Kvl.2 (CSMKl)27

Kvl.3 Names in use and refs

hKvl.3 HuK(III)11,12 hPCN3 28 HLK3 29

rKvl.3 RCK3 22 RGKS 31 Isolate KV3 (see

mKvl.3 MK3 16,19,33

Other Rabbit Kvl.3 2o; see also Rabbit kidney I glibenclamide-sensitive' clone rabK (vl.3)34

mKvl.4 MK4 16

Other Bovine Kvl.2 (BAK4)39 Canine Kvl.2 (dKvl.2)27 Rabbit Kvl.4 2o (see note 4) Ferret Kvl.4 (FKl)4o

(see note 7)

RBKl 14 BKl 15 RKl 16,17

HGKS 30

Kvl.4 Names in use and refs

RCKl 10

hKvl.4 HuK(II)11-13 hPCN228,35 HK1 36

note 2)32 rKvl.4 RCK4 22 RHK1 37 RK4 16,38 RKS 16

(tl

l:j t""t-

~ ~

00

Kvl.5 Names in use and refs

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hKvl.5 HuK(VI)11,12 HK241 hPCN1 28 HCK1 42 fHK 43

rKvl.5 Isolate KVl (see note 2)32 RK3 38 RMK2 44

mKvl.5 mKvl.5 45

Kvl.6 Names in use and refs

hKvl.6 HBK248 HuK(V)11,12

rKvl.6 RCK2 48 Isolate KV2 (see note 2)32

mKvl.6 MK2 16 MK6 49

Kvl.7 Names in use and refs

hKvl.7 Kvl.7 50,51 (abstracts) (diabetic (3 cells)

rKvl.7 RK6 16

mKvl.7 Other MK6 16 Hamster Kvl.7 16 52 MK4 mK(v)1.7 (see note 8)

K(v)1.8 (see note 6)

rK(v)1.8

rK(v)1.8

mK(v)1.8 Other mK(v)1.8 (see note 9)

Other Hamster Kvl.5 16 Bovine BAK5 46 Rabbit Kvl.5 (RBKVl.5)47

Other Hamster Kvl.6 16

Notes: 1. If the isolate (clone) name is used to identify a data source in this entry, the Kv nomenclature 1 also appears. Amino acid sequence alignments for these proteins are shown under Encoding, 48-19. 2. To ensure there is no confusion between Kv subfamily numbers and certain clone (isolate) names (KVl, KV2 and KV3)1,2, all occurrences of the latter (capitalized) are preceded by the word 'isolate'. 3. An 'RCK2' clone named in the original RCKI paper10 has now been re-named RCKlai RCK2 is defined as the rat brain 'equivalent' of the human brain K+ channel HBK2 (nomenclature Kvl.6). 4. A series of partial Shaker-related sequences have been amplified by RT-PCR t from rabbit kidney cortex and the renal epithelial cell line LLC-PK 1 20 (see Channel density, 48-09). A further partial Shaker-like cDNA (KC6, similar to Kvl.2/RBK2) from rabbit kidney cDNA was later reported53, bringing the total number of detectable Kv cDNAs in kidney to six. One of these isoforms (KC22) appeared to be a novel K-channel gene that is highly expressed in the rabbit kidney and in primary cell cultures of rabbit distal tubule and likely to be involved in epithelial K+ transport53 (see also clone 'rabK(v1.3)' described under Phenotypic expression, 48-14).

~

::s

t"1"

~ ~

00

n

5. Only supplementary or contrasting information for channel/equivalents' are listed in entries - these data are quoted under isolate names to enable identification and comparison of source data (see INFORMATION RETRIEVAL section). 6. Genes encoding K+channels which are expected to be voltage-gated (but which have not been experimentally confirmed) are generally designated by a K(v) prefix. 7. The tabulation is not exhaustive, and subunit genes not indicated or incorrectly designated can be communicated to the entry e-mail feedback file (see Feedback, 48-57). 8. Cited as mouse K(v)1.7, K. Kalman et al. unpublished data, in Chandy and Gutman, 1994, under Related sources and reviews, 48-56. 9. Cited as mouse K(v)1.8, B. Tempel (ibid.); a partial cDNA has been isolated from mouse cochlea by RT-PCR; chromosomal mapping (see field 48-18) and genomic sequencing of the mKvl.8 region is cited as in preparation within ref. 54).

(1)

::s

t"1"

~ ~

00

l_e_n_t_ry_48

_

mKv1.1 (MK1) rKv1.1 (RBK1) hKv1.1 (HUK[I)) ~---xKv1.1 (XSHA1) mKv1.2(MK2) rKv1.2(RGK5) hKv1.2(HUK[IV)) dKv1.2 bKv1.2(BGK5) ~---xKv1.2(XSha2) mKv1.3(MK3) rKv1.3(RGK5) hKv1.3(HPCN3) rKv1.5(KV1 ) hKv1.5(HPCN1 ) mKv1.6(MK6) rKv1.6(RCK2) hKv1.6(HBK2) ' - - - - - - - - mKv1.7 mKv1.4 rKv1.4(RCK4) hKv1.4(HPCN2) bKv1 .4(BAK4) ~-----APLK ~--------Shaker

Figure 1. Shaker subfamily phylogenetic tree. (Reproduced with permission from Chandy and Cutman (1994) In Handbook of Receptors and Channels (ed. R.A. North). CRC Press, Boca Raton.) (From 48-05-03)

features can be found under Database listings/primary sequence discussion, 48-53. In the case of missing or incorrect data, see the Feedback field, 48-57.

Cell-type expression index Cell-type expression patterns implied using molecular probes specific for Kv1 family members are described under Cloning resource, 48-10; Isolation probe, 48-12; mRNA distribution, 48-13 and Protein distribution, 48-15. Kv channels are ubiquitously expressed in excitable cells 48-08-01: K+ -selective channels are generally considered to be expressed in all vertebrate cells, although marked differential expression is observed at the cell type level (e.g. in terms of channel subtypes, co-assembly properties, relative densities and 'timing' of developmental onset). These variables in part account for the remarkable functional diversity of voltage-gated K+ channels in native vertebrate cells, including their major roles in regulation of membrane potential in excitable t cells such as nerve and muscle (see also VLC K A-'J: entry 44 and VLC K DR, entry 45). Within single cell types, there is direct evidence for large numbers of co-expressed Kv gene family members (e.g. in the PC12 phaeochromocytoma cell line model, where nine separate gene family products have been detected56; notably, this study could not use the full complement of presently known probes for Kv channels).

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Diversity of cardiac Kv channel expression 48-08-02: In the heart, voltage-gated K+ channels incorporating Kv subunits are likely to contribute to modulation of action potential frequency and duration (see also the INR K series, entries 29 to 33 inclusive, VLC K eag/ elk/erg, entry 46 and VLC K Kv-beta, entry 47). Molecular cloning t studies have shown multiple (>7) Kv subunit genes to be expressed in rat or human heart from the Kvl, Kv2 or Kv4 subfamilies (not including the KvLQTl- or erg-related families) (see mRNA distribution, 48-13 and under VLC K Kv2Shab, 49-13 and VLC K Kv4-Shal, 51-13).

Voltage-gated K+ channel expression is not restricted to excitable cells 48-08-03: Notably, a number of voltage-gated K+ channels are known to be expressed in non-excitablet cells (i.e. those cells which do not pass action potentials, such as lymphocytes) although no studies have reported Kv channel subunit expression in native differentiated erythrocytes. Voltagesensitive K+ channels are also generally pot observed in patch-clamp studies of native epithelial cells. Note, however, sensitive RT-PCRt techniques have revealed several Shaker subtype-specific mRNAs2o,53 in rabbit kidney cortex and the renal epithelial cell line LLC-PKl, implying such channels do exist in these tissues at very low densities (see also mRNA distribution, 48-13).

Heterologous cell expression of Kv channels 48-08-04: Functional studies of ion channels or other signalling proteins encoded by cDNAs or genes require that the proteins be correctly assembled in a transmembrane confi~ration. This is usually accomplished by the injection of messenger RNAt, cRNAt, or by stable transfection t of DNA into heterologous cells which do not normally express the protein(s). For stable expression, simple 'driving' of expression by strong tissue-specific promoterst and enhancerst does not guarantee optimal functional expression: 'Correct' folding and membrane insertion of channels may also require co-expression of accessory subunits or chaperonet functions in the heterologous system (e.g. see effects of Kvf32 in Phenotypic expression under VLC K Kv-beta, 47-14). Stably expressed channel activity may also be susceptible to endogenous kinase/phosphatase regulation, which in turn may be influenced by growth factor signalling, developlnental factors or cell cycle position. While these technical problems remain, heterologous cell expression methods (used in combination with voltage-clampt or patchclampt analysis of currents) have been especially powerful in the identification of structure-function relationships in ion channels. Other 'cell-free' approaches designed to overcome potentially 'confounding' influences of endogenous ion channels, neurotransmitter receptors and receptor-channel subunits have been described for Shaker channels57.

Limitations of Kv channel analysis by heterologous cell expression 48-08-05: The subunits of the Kv series probably underlie the properties of a 'significant proportion' of voltage-gated K+channels expressed in native tissues (see mRNA expression, 48-13, but see also VLC K eag/elk/erg, entry 46 and VLC {K} minK, entry 54). The 'spatial distribution' of channel

II

entry48

_

I" - - - - - - - - - -

subunit-specific mRNAs presumably reflects some 'functional specialization' in particular cell types, tissue regions or systems: Distribution patterns of 'kinetically similar' delayed rectifier K+ channels are distinct in brain58 with the implication that 'delayed rectifiers' and other channels possess functional differences that may be impossible to infer from heterologous expression studies in isolation (see also (i) note on 'matching native current properties' under Channel designation, 48-03 and (ii) the significant roles of accessory subunits in native channel complexes under VLG K Kv-beta, entry 47). Methodological note: Application of single-cell RT-PCRt eDNA amplification methods combined with patch-clamp methods (e.g. ref. 59, as applied to AMPA receptor-channels, see ELG CAT GLU AMPA/KAIN, entry 07) may help determine subunit 'contributions' to native cell currents. Products of at least nine distinct Shaker subfamily genes have been shown to express in single cells56, see also 60 . Alternatively, or in addition, use of clonal cell lines where known channel isoforms are expressed (e.g. hKvl.3 ~ type n channel of Jurkat T lymphocytes61 ) are also important models for channel genotype-phenotype studies.

Channel density Channel clustering at the plasma membrane by PDZ domain-Kv C-terminal interactions 8-09-01: Kv1 subfamily: The membrane-associated putative guanylate kinases PSD-95, SAP97, chapsyn-ll0 and hdlg have been shown to promote differential Kv channel clustering by associating their PDZ domains with Cterminal residues in Kvl.l, Kvl.2, Kvl.3 and Kvl.4 (for details and refs, see Protein interactions, 48-31 and Fig. 7). PSD-95 induces plaque-like clusters of K+ channels at the cell surface, while SAP97 co-expression results in the formation of large, round intracellular aggregates into which both SAP97 and the K+ channel proteins are co-localized. The 'efficiency' of surface clustering by PSD-95 has been shown to vary with different Kvl subunits: whereas 'striking' Kvl.4 clustering occurs in >600/0 of co-transfected cells, Kvl.l and Kvl.2 form clusters with PSD-95 at low efficiency (approx. 100/0 of cells)62. Chapsyn-ll0 associates tightly with the post-synaptic density in brain and mediates the clustering of both Kv channels and NMDA receptors (entry 08) in heterologous cells 63 . In native rat brain, chapsyn-ll0 protein shows a somatodendritic expression pattern (see note 1) that overlaps partly with PSD-95 but that contrasts with the axonal distribution of SAP97 (note 2). Chapsyn-ll0 and PSD-95 may form heteromultimers at post-synaptic sites to form a 'scaffold' for clustering of receptors, ion channels, and other signalling proteins 63 . Notes: 1. Compare Kv4.2's predominantly somatodentritic localization64 under Subcellular locations, 51-16. 2. Compare Kvl.4's predominantly axonallocalization64 .

Comparative difficulty of purifying K+ channel proteins to homogeneity from native tissues 48-09-02: In general, K+ channel subunits are expressed at Ilow densities', with more than one subtype co-expressed per cell. These factors make 'direct' purification methods for specific K+ channel proteins technically formidable:

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Whereas 200- to SOO-fold purification can be sufficient to purify Na+ or Ca2+ channel protein to homogeneity from brain65,66, K+ channel subunits require a 10000- to 20 OOO-fold purification for equivalent purity67. In some cases, toxin immunoaffinity t methods have been successfully employed for purification of some classes of K+ channel protein (for reviews see refs 68,69 - see also VLG K Kv-beta, entry 47).

Novel methods for active K+ channel protein production and assay from heterologous cells 48-09-03: Although there is no 'notably rich' source of native vertebrate cell K+ channel protein (see previous paragraph), high-level expression (",,10 7 Shaker K+ channels/transfected cell) driven by an adenovirus promoter t has been suggested as a 'plausible route' for purification of 'milligram-level' amounts of functional K+ channels from heterologous cells 70 . Reconstitution of functional Shaker channels from an insect expression system following their immunoaffinity purification has also been described 71 . Note: This and a previous study 72 described the application of the proton pump bacteriorhodopsin as a light-driven current source for the development and control of transmembrane potentials in reconstituted vesicles. Bacteriorhodopsin is capable of depolarizing the membrane (to at least 0 mV level) within a few seconds, while more rapid depolarizations can be achieved by the application of a brief intense illumination preceding the preset illumination level (supercharging). This light-driven vesicular voltage-control system can be applied to functional assay of Kv (and other) channels, permitting optimization of 'active' protein production for structural studies.

Reported changes in channel densities accompanying thymocyte maturation processes 48-09-04: Kvl.3: The type n K+ channels of native T lymphocytes (encoded by Kvl.3, see Current type, 48-34) show variable densities of expression in parallel with the rate of cell division 73, 74. Immature proliferating thymocytes display ",,200-300 channels/cell, the number decreasing to ",,10-20 channels/cell during differentiation into mature, quiescent T cells 73, 75. Activation of mature T cells results in a 20-fold increase in K+ channel number, the increase being exclusively of type n 76 .

Other phenomena associated with tvariable' Kv channel expression in heterologous cells 48-09-05: Kvl.l/RCKl: Direct correlation between channel density and transcriptional rate of a metallothionein promoter fused to the RCKI K+ channel in stably transfected Sol 8 cells has been reported 77. Mouse L cells stably transfected with Kvl.l eDNA have been estimated to yield 10004000 functional surface channels/cell 78 (see also Protein interactions, 48-31). Note: A number of heterologous expression studies have specifically related variable mRNA expression levels to differences in inactivation kinetics, pharmacological profile and other functions. For example, 'variable' expression levels of a Shaker clone have also been reported to affect membrane potential in oocytes 79 : At high channel densities (Gmax > 10pA/mV) the mean membrane potential was stabilized at

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l_e_n_t_ry_48

---'_

approximately -60 mV and independent of slow Ie-type' inactivation; at lower channel densities, membrane potential was 'very unstable', with its mean value (and amplitude of fluctuations) being strongly influenced by the process of C-type inactivation (see Inactivation, 48-3 7). For further descriptions of differentiated functional properties with 'variable' expression levels, see Current type, 48-34; Inactivation, 48-37 and Blockers, 48-43. See also the fields Activation and Kinetic model under VLG K minK, 54-33 and 54-38, respectively}.

Cloning resource 48-10-01: The ubiquity of Kvl series channel expression and their possession of intronless t coding regions (all except Kvl.7, see Gene organization, 48-20) has enabled retrieval of cDNA and genomic clones containing full-length ('expressible') sequences from a wide range of DNA libraries, as exemplified in Table 2.

Developmental regulation Multiple mechanisms for generating K+ channel diversity 48-11-01: Voltage-gated K+ channels display heterogeneous expression patterns, many of which appear subject to developmental control (see examples, below). Molecular heterogeneity may arise from differential expression of multiple K+ channel subunit genes (see mRNA distribution, 48-13), alternative splicing (see Gene organization, 48-20) and/or the formation of heteromultimers from different subunits (see Protein interactions, 48-31). Spatial- and temporal-selective induction of Kv channel activities in development (developmental heterogeneity) presumably reflects functional specialization within particular 'developmental compartments' or terminally differentiated cell types (for further background, see Developmentalregulation under TUN [connexinsj, 35-11). Localization (segregation) of different Kv proteins to specific subcellular domains may also significantly affect native channel properties, particularly where they are co-localized with modulatory proteins (for examples, see Subcellular locations, 48-16 and Protein interactions, 48-31).

Developmental heterogeneity of Kv channels in brain - early studies 48-11-02: Kvl.I/Kvl.2/Kvl.3/Kvl.4: mRNA transcript distribution patterns of 11 Kv channel genes (encoding both slow- and fast-inactivating K+ channels from four different gene families) have been systematically examined in a developmental context81 . This analysis confirmed that Kv subunit-specific mRNAs are independently expressed and can be characterized by individual but overlapping expression patterns (see mRNA distribution, 48-13). Evidence for heterogeneous expression and post-natal developmental regulation in the hippocampal formation were obtained for multiple transcripts RCK2, RCK3, RCK4, RCKS, Raw3 and Shall gene products. Transcripts present in the hippocampus throughout post-natal life included those encoding RCKI, Rawl, Raw2 and DRKI 82 .

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XShal expression in Rohon-Beard cells, neural crest derivatives and glia 48-11-03: Xenopus-Kvl.l/XShal/Kvl.2/XSha2: In primary sensory neurones of Xenopus laevis, the functional differentiation of delayed rectifier potassium current regulates the waveform of the action potential, while acquisition of A-current (entry 44) also plays a major role in regulating excitability. Within the embryonic nervous system, transcripts encoding XShal and (at a lower level) XSha2 are detectable 83 . The onset of neurogenesis in Xenopus (stage 13) is associated with the induction of Kvl.2 gene expression (isolate XSha2)21. XShal mRNA is expressed in Rohon-Beard cells (primary sensory neurones which exhibit developmentally regulated action potentials - see paragraph 48-11-04) and several structures containing neural crest derivatives including spinal ganglia, the trigeminal ganglion, and branchial arches. Detection of XSha1 in motor nerves and lateral spinal tracts also suggests that both CNS and PNS glia express the mRNA83 . Although many different types of Xenopus spinal neurones exhibit homogeneous development of IKv both in vivo and in culture, transcripts of two genes encoding delayed rectifier current, Kvl.l (this entry) and Kv2.2, are expressed heterogeneously during the period in which the current develops: Kvl.l mRNA is detectable in a maximum of 300/0 of cells while I Kv is immature; Kv2.2 mRNA appears later in approx. 600/0 of mature neurones. For further details of Kv2.2 developmental expression in ventral spinal neurones (as opposed to dorsal spinal neurone distribution of Kvl.l transcripts)84, see Developmental regulation under VLG K Kv2-Shab, 49-11.

In vivo (endogenous) regulatory factors that can 'rescue' perturbed neuronal differentiation 48-11-04: Xenopus-Kvl.l/XSha1: Injection of Shaker-like potassium channel transcripts into two-cell stage Xenopus embryos correlate with larger delayed rectifier current amplitudes and shortened action potential durations 85. XSha1 'overexpression' is associated with reductions in the number of morphologically differentiated Rohon-Beard neurones that appear in cultures prepared from neural plate stage (17.5 h) embryos (see note 1 and paragraph 48-11-03). This suggested that 'premature' modulation of impulses suppresses normal developmental cues in this model in vitro system (ref. 85 ). In comparison, a later study86 showed that morphological differentiation of Rohon-Beard neurones in situ (in vivo) was only 'slightly affected' by overexpression of K+ channels (i.e. endogenous developmental factors (regulatory processes or interactions) appeared to compensate for the effect of channel overexpression). Notably, when cultures are prepared from older neural tube embryos (22-24 h), more neurones containing 'excess' K+ channel RNA differentiate morphologically in vitro. Recovery of differentiation capacity is possible if a minimum of 5 h of further development in vivo is allowed (under conditions in which (i) rapid elevations of [Ca2 +h are permitted and (ii) half of the nervous system has normal levels of potassium channel RNA (see note 1; for general significance of [Ca 2+h transients in development, see this field under ILG Ca Ca RyRCaf, 17-11 and under ILG Ca InsP3, 19-11). Notes: 1. Hypothetically, increased K+ current could oppose long-duration Ca2+ influx and nuclear Ca2+ elevations (see NUC [nuclear, native], entry 38) associated with differentiation

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Table 2. Source tissues for DNA library constructions (From 48-10-01) Kvl.l

MBKl: Mouse brain cDNA library. MKl: Mouse genomic DNA library. RBKl: Rat hippocampal cDNA library. RBKI cDNA and native channels with its properties have been systematically characterized in C6 astrocytoma cells and native astrocytes 80 • RCKl: Rat cerebral cortex cDNA library. RKl: Rat aorta cDNA library.

Kvl.2

RCKS: Rat brain cortex cDNA library. NGKl: Mouse neuroblastoma x rat glioma hybrid cell cDNA library. MK2: Mouse genomic DNA library. RK2: Rat cardiac cDNA library. BK2: Rat brain cDNA libraries. CMSKl: Canine colonic circular smooth muscle cDNA.

Kvl.3

hPCN3: Isolated from human insulinoma cDNA library and from genomic DNA library sources. MK3: Mouse genomic DNA library. RGKS: Isolated from a genomic DNA library. RCK3: Brain, cortex cDNA library. Isolate KV3: Rat brain cDNA library. HLK3: Human brain and T lymphocyte cDNA libraries. Isolate rabK(vl.3): Rabbit kidney medullary cell line GRB-PAPI (see Phenotypic expression, 48-14).

Kvl.4

HKl: Human left ventricular cDNA library. hPCN2: Human foetal skeletal muscle cDNA library. RCK4: Brain, cortex cDNA library. RHKl/RK3: Rat cardiac cDNA library.

Kvl.5

HK2: Human left ventricular cDNA library. hPCNl: Insulinoma cDNA library and genomic library. Isolate KVl: Rat brain cDNA library. RK4: Size-fractionated cardiac cDNA library. RMK2: Rat skeletal muscle cDNA library. BAKS: cDNA library from bovine adrenal medulla, and, subsequently, a bovine genomic library. Rabbit Kvl.S: Portal vein vascular smooth muscle cDNA.

Kvl.6

HBK2: Human foetal brain cortex cDNA library. Isolate KV2: Rat brain cDNA library.

phenotypes such as neurite outgrowth, neurone-myocyte contacts and acquisition of GABA-like immunoreactivity (see ELG Cl GABAA, entry 10). Significantly, the in vitro study described above 85 concluded that larger potassium currents were not compensated by changes in inward currents. 2. In these experiments RNA 'overexpression' is limited to half of the embryo in order to provide an internal control; when K+ channel RNA is overexpressed throughout the embryo, few neurones are observed to differentiate morphologically in vitro (even if cultures are prepared from older neural tube embryos).

Modified endogenous Xenopus Kv channel behaviour by exogenous Kv channel expression 48-11-05: rKvl.l: Rat Kvl.l channel currents have been analyzed in blastomeres during a 12h period prior to stage 15 (early-mid neurula) and in

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entry 48 _

differentiating muscle cells (post-stage 15) following injection of rKvl.l cRNA into fertilized eggs87. In rKvl.l-injected embryos, a high fraction of blastomeres express a delayed rectifier-type current distinguishable from the endogenous muscle delayed potassium current (IK, Ix) by its different voltage dependence. Notably, native IK, Ix appears much earlier in development than in control embryos; this and other pharmacological data suggests an interaction/induced modification between Kvl.l and endogenous channel subunits in the course of development87.

Functional expression of Kvl.3 in myotomal muscle of developing Xenopus embryos 48-11-06: Kvl.3: Injection of cRNA encoding Kvl.3 into fertilized eggs of Xenopus can give rise to functional Kvl.3 channels in dissociated, cultured myotomal muscle cells (i.e. from embryos of stage 20-22 about 40 h post-fertilization at 19°C, cultured in vitro for 2 days)88. Kvl.3 currents were distinguished from endogenous delayed rectifiers (sustained and transient) and an inward rectifier K+ current) by its inactivation properties and its high sensitivity to the non-selective blocker charybdotoxin88.

Kv mRNA repression associated with seizures

48-11-07: Kvl.2/Kv4.2: Seizure t activity induced by the convulsant drug pentylenetetrazole (Metrazole) has been reported to be followed by a reduction of Kvl.2 mRNA (~90min to 6h pj.) and Kv4.2 mRNA (3-6h pj.) in the dentate granule cell layer of the hippocampus, while Kvl.l mRNA levels remained unchanged58 . mRNA signals return to normal (control levels) within 12h. Notably, administration of the anticonvulsant diazepam prior to pentylenetetrazole protects animals from seizure and blocks the suppression of Kvl.2 and Kv4.2 mRNAs, suggesting the mRNA repression is due to induced neuronal activity evoked by seizure (rather than an unrelated side-effect of the drug).

Mechanism of the tantiproliferative' effect of K+ channel blockers in Tcells 48-11-08: Kvl.3: Charybdotoxin (see Blockers under ILG K Ca, 27-43) depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogens t. The scorpion venom peptides noxiustoxin (NxTx) and margatoxin (MgTx) block only the voltage-activated channels, whereas charybdotoxin blocks three types of calcium-activated potassium channel and Kv channels in these cells89 . All three toxins induce 'equivalent' depolarization and block T cell activation in human T cells. On the basis of these and other results 89, it has been concluded that membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to produce depolarization of unstimulated human T cells, thereby preventing mitogenic activation (but see below). Notably, the potent Kvl.3 blocker CP-339,818 (which competitively inhibits [l 25 I]-charybdotoxin from binding to the external vestibule of Kvl.3) suppresses T cell activation in vitr0 90 (see Blockers, 48-43). Supplementary notes: Depolarization attenuates the increase in cytosolic Ca2+ that normally occurs during receptor-ligand coupling91 . Following

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elevation of [Ca2 +h, KCa channels may also contribute to the Tcell membrane potential, as supported by the ability of charybdotoxin to block the hyperpolarization that follows stimulation by mitogens or Ca2 + ionophores 92- 98 (for further details see ILG Ca InsPJ, entry 19 and ILG K Ca, entry 27). More recent work has established an important role for Kvl.3 in maintaining secretion of the lymphokine IL-2 in activated T cells: In this case, following T cell receptor ligation t , Kv1.3 opening prevents the influx of calcium into the (non-excitable t ) cell from 'collapsing' its own electrical gradient (thereby sustaining the IL-2 secretory response). These features have motivated development of blockers for Kvl.3 as potent immunosuppressors by their effects on IL-2 secretion (see Blockers, 48-43 and the 1996 review in ref. 99).

Kvl.l and Kvl.3 in a lymphoid/thymocyte development model 48-11-09: Kvl.l/Kvl.3: Murine foetal and adult CD4-CD8- (immature) thymocytesf display currents with the distinctive rroperties of Kvl.l and Kvl.3, and their mRNA can be detected by RT-PCR in these cells10o. When applied to a murine foetal thymic organ culture system, non-selective peptide blockers of Kvl.l (dendrotoxin, DTx - see Blockers, 48-43) and Kvl.3 (charybdotoxin, CTx - ibid.) decrease thymocyte yields in organ culture without affecting thymocyte viability: In comparison to untreated thymic lobes, DTxtreated thymi contain 56 ± 8% thymocytes (n == 8) while CTx-treated thymi contain 74 ± 4 % thymocytes (n == 7). DTx and CTx also alter the developmental progression of thymocytes in foetal organ culture, consistent with functions critical to thymocyte pre-clonal expansion and/or maturation1OO.

'Parallel developmental onset' of Kv channel expression and voltage-gated Na+ channels 48-11-10: Kvl.3/Kvl.4: There is an 'early onset' of Kvl.3 (RCK3) and Kvl.4 (RCK4) mRNA expression comparable to the temporal mRNA expression pattern of rat sodium channels II and ill in the CNS 101 ,102. Kvl.4 (isolate RCK4)-specific probes detect an mRNA species of "-I4300nt in heart which increases during development 101 .

'Monotonic' development of Kv protein expression patterns in developing hippocampus 48-11-11: Kvl.4/Kvl.5: The 'spatiotemporal' expression patterns of the Kvl.4, Kvl.5, Kv2.1, Kv2.2 and Kv4.2 polypeptides in rat hippocampal neurones developing in situ have been the subject of a comparative study l03. The development of protein expression patterns in situ (see Protein distribution, 48-15) has been described as Imonotonic', i.e. while the 'temporal' and 'spatial' development varies among Kv channels, each subtype initially appears in its adult pattern (taken to suggest that the mechanisms underlying spatial patterning operate through development). Immunoblotting techniques have confirmed the differential temporal expression of K+ channels in the developing hippocampus, and demonstrate developmentally regulated changes in the 'microheterogeneity' of some Kv subtypes 103 (see Subcellular locations, 48-16).

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Hormonal regulation of pituitary excitability via K+channel gene expression 48-11-12: Kv1.5: Glucocorticoidt agonists such as the steroid dexamethasonet

(DEX) can specifically increase Kvl.S mRNA in normal and clonal (GH3) rat pituitary cells104,,105. Dexamethasone application can rapidly induce Kvl.S gene transcription, without affecting Kvl.S mRNA turnovert (tl/2 == O.Sh). These effects are correlated with 3-fold increased expression of a 76 kDa Kvl.S protein (see Protein molecular weight (purified), 48-22) within l2h without altering its half-life (tl/2 == 4h)105. Kvl.S protein induction is also associated with an increase in a non-inactivating component of the voltagegated K+ current. In parallel experiments, dexamethasone did not affect Kvl.4 transcript or protein expression105. DEX treatment has also been demonstrated to increase total cellular and GH3 surface Kvl.5 protein and, in consequence, to modify cell surface homomeric and Kvl.4/Kvl.S heteromeric subunit composition106 . The actions of DEX are markedly tissue-specific: For example, in adrenalectomized rats, DEX rapidly induces Kvl.S mRNA in cardiac ventricle, skeletal muscle and pituitary, but does not affect Kvl.S mRNA levels in hypothalamus or lung107. Methodological note: Dexamethasone induction of the slowly inactivating current in the clonal rat pituitary cell line GH4Cl can be blocked by antisense t phosphorothioate t deoxyoligonucleotides to the Kvl.SmRNA sequence 108. By contrast, antisense deoxyoligonucleotides against Kvl.4 mRNA specifically decrease the expression of the dexamethasone-insensitive rapidly inactivating current in these cells 108.

Induction of Kv channel mRNAs by tstress responses' 48-11-13: Mimicking the 'hormonal stress response' using adrenocorti-

cotrophic hormone has no apparent effect on several Kv mRNA levels58 but the cold stress response of rats has been shown to significantly increase cardiac Kvl.S mRNA expression107. Comparative note: Independent studies on mKvl.l (described in Table 16 under Protein phosphorylation, 48-32) have suggested that decreased basal activities of protein kinase A can 'upregulate' mKvl.l channel expression by changing steady-state levels of RNA and by other post-transcriptional mechanisms 109 (ibid.).

TRH enhances excitability of GH3 cells by inhibition of K+ channel gene expression 48-11-14: Kvl.S: Neuropeptide regulation of K+ channel gene expression is

capable of inducing 'long-term' changes in neuronal action potential activity and synaptic transmission. For example, thyrotrophin-releasing hormone (TRH) downregulates Kvl.S (and Kv2.l) K+ channel mRNAs in G03 pituitary cells by decreasing rates of transcription110. These changes can be correlated with significantly decreased immunoreactivities and K+ current expression within 12 h of TRH application.

Developmental regulation of rKvl.5 by cAMP and K+ -induced depolarization 48-11-15: rKvl.S/rKvl.l: A sequence conforming to a cAMP response elementt (CRE) has been identified in the S'-non-coding region of the rKvl.S

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en t

_

gene at position +636 relative to a transcriptional start site111 (see also Gene organization, 48-20). In primary cardiac cells, cAMP induces a 6-fold increase in the steady-state levels of Kv1.S transcript. In GH3 pitutary cells, by contrast, cAMP induces a S-6-fold decrease in steady state levels of Kvl.S transcript by reduction in Kv1.S gene transcription rate. Progressive deletion of S' non-coding sequences (coupled to a reporter gene assay) have shown that (i) the consensus CRE can confer cAMP inducibility to Kvl.S and (ii) this element is capable of binding CRE-binding protein (CREB) and CRE modulator protein (CREM) in gel shift assays. KCI-induced depolarization has also been shown to increase steady-state levels of Kvl.S transcript in primary atrial cells and decrease it in GH3 clonal pituitary cells111,,112; the KCI treatment has no effect on Kvl.4 or Kv2.l transcription and NaCI cannot suppress Kvl.S transcription l12 .

A K+ channel gene silencer with a dinucleotide repetitive element 48-11-16: Kvl.S: A gene silencert element (alternatively named Kvl.S repressor element or KRE) has been identified by deletion analysis of the Kvl.S gene promoter195. In cell lines that do not express Kvl.S, cis constructs of KRE selectively decreases expression of Kvl.S and reporter genes. KRE contains a dinucleotide repetitive element (polyGT 19 (GAh(CAhs(GAh6h deletion of the repetitive element from reporter constructs abolishes an in vitro selfassociation property and the silencing activity. KRE appears to form a stable nucleoproteint complex with nuclear extracts from GH3 cells (which cannot be detected with CHO and and COS-7 cell extracts)195.

Quantitation of Kv current, mRNA and protein during post-natal heart development 48-11-17: Comparative developmental analysis of the native rat cardiac ventricular currents Ito (entry 44) and I K (entry 45) versus multiple Kva protein/mRNA expression levels (Kvl.2, Kvl.4, Kvl.S, Kv2.l and Kv4.2) has been performed l13 . In this study, mean Ito densities increased 4-fold between birth and P30, whereas IK densities varied only slightly. No variation in either the time- nor the voltage-dependent properties of Ito or IK could be detected over this period. The same study showed ventricular Kvl.4 mRNA levels were 'high' at birth, increased between PO and PIa, and subsequently decreased to 'very low' levels in adult rat hearts (decreases in message were accompanied by a marked reduction in Kvl.4 protein, see note). Notably, mRNA levels of all other Kv channels studied (Kvl.2, Kvl.S, Kv2.l and Kv4.2) increase (3- to S-fold) between birth and adult. Paradoxically however, Kvl.2 and Kv4.2 protein levels increase between PS and adult, whereas Kv1.S protein remained constant while Kv2.l decreased. Note: On the basis of this and earlier data, these authors suggested that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes l13,,114. In earlier studies a 'marked increase' in the amount of mRNA encoding rKvl.S (clones KVl/RMK2) in cardiac tissue has been noted during development and a similar but less-pronounced increase of both Kvl.S and Kvl.6 (isolate KV2) transcript in brain32,,44 (see also Channel density, 48-09).

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Directed transcription of the native hKvl.5 gene within single chromosomal fragments 48-11-18: hKvl.5: A single excised human genomic DNA fragment encompassing the hKvl.5 gene promoter region contiguous to its coding sequence has been used to heterologously express Kvl.5 protein in a mammalian cell line lacking voltage-gated K+ channels l15 . In this study, the hKvl.5 gene fragment was (i) linked downstream of a human ,a-globin locus control region t (LCRt or 'gene-activation element') and (ii) stably incorporated into the host erythroleukaemia cell genomic DNA by recombination following transfection of the construct. hKvl.5 gene locus activation and hKvl.5 current can be induced following treatment with various polar/ apolart chemical inducers of differentiation (for details, see Developmental regulation and Voltage sensitivity under ILG K Ca, 27-11 and 27-42 respectively). Expression levels are characteristically independent of chromosomal integration position and are proportional to locus control region (LCR)-gene construct copy number, a parameter specific for each clonal cell line. These results demonstrate the general utility of LCRdirected gene-activation methods for ion channel gene or cDNA expression l15 .

Moderate induction of Kv gene transcription induced by chronic morphine administration 48-11-19: rKvl.5/rKvl.6: Prolonged opiate administration leads to the development of tolerancet and dependencet, while acute opiate administration differentially affects voltage-dependent K+ currents in vivo. While opiate activation of K+ channels is well established (see INR K G/ACh [native], entry 31), opioid-induced inhibi.tion of K+ conductances has also been described. In separate studies l16, 'significant increases' in abundance for mRNAs encoding Kvl.5 (2.1 ±O.15-fold by ISHt) and Kvl.6 (2.3 ±O.5fold) are induced in the spinal cord of rats following chronic morphine administration (compared with controls). Moreover, Kvl.5 protein level was also increased by 1.9-fold in the spinal cord of morphine-treated rats. These findings have been interpretedl16 as 'cornpensatory' for persistent opioidinduced inhibition of K+ channel activity in motor neurones, perhaps contributing to tolerance/dependence conditions.

Isolation probe tCore region' probes maximize retrieval of related sequences 48-12-01: Generally, vertebrate K+ channel isoforms have been isolated using single or mixed probest homologous to Drosophila Shaker locus or from existing gene family members. Homologous isoformst in different vertebrate species are generally highly conserved at the amino acid level, and have been isolated by low-stringency cross-hybridization. Because of the greater degree of conservation for sequences associated with 'core regions', low-stringencyt hybridization screens with these probes were better able to isolate related genes. Note: 'Core' regions e.ncompass the sequences encoding the transmembrane domains, ionic selectivity and voltage-sensitivity determinants (for further details see Domain functions, 48-29 and the [PDTM], Fig. 6).

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Probes for selective reporting or retrieval of individual family members 48-12-02: Channel 'isoform-specific' probes (either nucleic acid or antibody) are generally derived from C-terminal, N-terminal or untranslated sequences which are divergent amongst the gene family members. Between distantly related species (e.g. vertebrate and fly) K+ channel amino acid sequences can retain strong homology, although randomization of nucleotides at degenerate t positions and different codon usage t underlies divergence at the nucleotide level. Panels of PCR t primer pairs designed to selectively amplify known isoform sequences from genomic or cDNA sources are in common use (e.g. primer sets for Kvl.l, Kvl.2 and Kv4.2 subtype sequences are described in58 and for MK3 in33 ).

mRNA distribution Differential expression patterns of the Kv gene subfamily mRNAs 48-13-01: Many cell types express voltage-gated K+ channel subpopulations that are characterized by different functional and pharmacological properties l17,118. Generally, Kv subunit genes show distinct but overlapping expression patterns within brain and other tissues 58,101. However, the expression pattern of some Kv subunit mRNAs in native tissues appear ubiquitous. Partially overlapping expression patterns in defined regions (e.g. ref. 81, see Developmental regulation, 48-11) indirectly suggest the formation of different heteromultimeric K+ channel complexes; strictly, however mRNA distributions indicate sites of biosynthesis, and do not necessarily indicate the ultimate locus of protein expression (see Protein distribution, 48-15). Furthermore, the roles of different K+ channel proteins could be markedly influenced by their localization to specific subcellular domains (see Subcellular locations, 48-16). Table 3 lists some qualitative (verbal) summaries of Kv subunit mRNA expression patterns, as originally reported. Although of limited value, they can at least identify discrepancies in gross distribution patterns and can serve as a foundation for systematic comparative studies using genotype/phenotype database approaches (see footnotes to Table 3 and Resource H - Index of cell types).

Information derived from mRNA 'expression'surveys covering specified cell types (example) 48-13-02: Since molecular probes 'covering' complete (known) gene families or subfamilies are now available, some studies have attempted characterization of mRNA abundance or distribution within single cell or tissue types. For example, the quantitative assessment of eighteen different voltage-activated potassium channel genes in rat sympathetic ganglia has been described l19 . Comparative studies like this can reveal the range of subtype expression. Thus, sympathetic ganglia were shown to express eleven a subunit genes and two {3 subunit genes l19 . Relative levels of mRNA expression can also be compared between different functional 'lineages' of cells; for example, between the superior cervical ganglion (SCG) and two pre-vertebral sympathetic ganglia, the coeliac ganglion (CG) and the superior mesenteric ganglion (SMG). In these cases, only four mRNA subtypes were shown to be differentially expressed:

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Table 3. Reported tissue distributions of Kvl subfamily mRNAs (From 48-13-01) Kv

Systematic/clone names, original descriptions, notes and references

Kvl.l

hKvl.2: RT-PCR: Human airway smooth muscle120 (see Phenotypic expression, 48-14). rKvl.I-RCKI: Northerns: Localized distribution suggests the channel mediates functions that are concentrated in distinct subregions. Detected in cortex and cerebellum but not in heart, kidney, spleen, lung, testis, liver and muscle10. RCKI mRNA is of generally low abundance (compared to actin mRNA probes which show rvxSOO the signal intensity of RCKI probes). Predominantly expressed in the adult nervous system (rvneurones and/or glial cells) at approx. equal mRNA abundance to RCKS; high mRNA levels found in sciatic and other peripheral nerves 101 . Maximal levels in caudal brain regions, relatively low levels in rostral regions and in retina: Cerebellum + + + +; cerebral cortex + + +; corpus striatum +; hippocampus ++; inferior colliculus + + + +; medulla-pons + + + + +; midbrain (-colliculi) -t- + + +; olfactory bulb +; spinal cord + + + +; superior cOlliculus + + + + + 101. Comparative note: RCKI closely resembles the temporal and regional expression pattern of rat sodium channel I in the CNS 101,102. mRNA not detected/low abundance in adrenal and submandibular glands, kidney, cardiac, skeletal and smooth muscle. ISH: High mRNA levels detected in granule cells of the dentate gyrus, the pyramidal cells of the Ammon horn (CA3 >. CAl), and in the cerebellum. Low expression levels were found in basal ganglia (caudate putamen, globus pallidus and ventral pallidum)121. rKvl.l-RKI: Northerns: Brain » cardiac atrium == skeletal muscle; no signal in liver and cardiac ventricle 17. rKvl.I-BKI: ISH: Expressed at relatively high levels in the hippocampus, thalamus, cerebral cortex and cerebellum. Not expressed in the medial habenula; hybridization relatively strong in the superficial and deep layers (cf. Kvl.2); in hippocampal subregions, the relative distribution of channel mRNA is CA3 ~ dentate gyrus> CAl. Concentrated expression in the cell bodies of the granule cells of the dentate gyrus and the pyramidal cells of CA3 and CAl - therefore expressed in neurones and possibly in CNS glial cells. Expressed in many hilar cells at much higher levels than in the neighbouring dentate granule or CA3 pyramidal cells. Expressed in Purkinje cells and granule cells but not detected in cells within the molecular layer (cf. Kvl.2 and Kv4.2). Seizure t activity does not affect Kvl.l mRNA levels (see Developmental regulation, 48-11) 58. mKvl.I-MBKI: Northerns: Brain +. + + +; heart +; skeletal muscle +. mKvl.l/mKvl.2/mKvl.3: RT-PCR: mRNA from enzymatically dissociated, isolated mouse rod bipolar cells show that these neurones co-express Kvl.l, Kvl.2 and Kvl.3 122 (see Subcellular locations, 48-16).

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1L-_e_n_t_ry_4_8 Table 3. Continued Kv

Systematic/clone names, original descriptions, notes and references

Kvl.l

xKvl.l/XShal: RNAaseP: XShal mRNA is expressed in Rohon-Beard cells (primary sensory neurones which exhibit developmentally regulated action potentials) and several structures containing neural crest derivatives83 (see Developmental regulation, 48-11).

Kvl.2

hKvl.2: RT-PCR: Human airway smooth muscle120 (see Phenotypic expression, 48-14). rKvl.2/BK2: ISH: Generally no localized distribution observed, but resembles cell density patterns determined by Nissl stainingt; suggests the channel mediates functions that are generally distributed. 1s,s8 rKvl.2/RAK: Northerns: Northern blots show RAK mRNA in adult rat atrium (strong) and in rat ventricle (very weak)24 (see Phenotypic expression, 48-14). rKv1.2/RCK5: Northerns: Cerebellum + + + +; cerebral cortex + + +; corpus striatum +; hippocampus + +; inferior colliculus + + + +; medulla-pons + + + + +; midbrain (-colliculi) +; olfactory bulb ++; spinal cord + + + + +; superior colliculus ++101. rKv1.2/RCK5/RK2: Northerns: Relative mRNA abundance brain> cardiac atrium> aorta == ventricle. No signal in skeletal muscle 17. Expression restricted the adult nervous system (rvneurones and/or glial cells) at approx equal mRNA abundance to RCKl; high mRNA levels found in sciatic and other peripheral nerves 101 . Different probes show feint RCK2 signals in embryonal tissue and neonatal heart but not adult heart RNA118. rKvl.2/Kv4.2: ISH: Expressed in the medial habenula; hybridization relatively diffuse in the superficial and deep layers (cf. Kv1.1); in hippocampal subregions, the relative distribution of channel mRNA is CA3 > dentate gyrus ~ CAl. High dentate gyrus granule cell and hilar cell expression - as for Kvl.l. Most abundant in Purkinje cells and in a subset of neurones in the molecular layer, compared to signals in the neighbouring granule cell layer (cf. Kvl.l and Kv4.2). Seizure activity can reduce Kvl.2 and Kv4.2 mRNA levels s8 (see also Developmental regulation, 48-11). rKvl.2/Kvl.4/rKvl.5: Using a quantitative RNAase protectiont assay comparing the abundance of fifteen different potassium channel mRNAs in rat cardiac atrial and ventricular muscle123, only Kvl.2, Kvl.4 and Kvl.5 (plus Kv2.1 and Kv4.2) were judged to be expressed at I significant levels' (see also mRNA distribution under VLC K Kv4-Shal, 51-13). Canine Kv1.2/CSMK1: Northerns: CSMK1 is expressed in a wide variety of gastrointestinal smooth muscles27. Portal vein, renal artery, and uterus do not express CSMK1 mRNA, suggesting that (among smooth muscles) expression of this K+ channel may be restricted to gastrointestinal smooth muscles27 (see also Phenotypic expression, 48-14).

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Table 3. Continued Kv

Systematic/clone names, original descriptions, notes and references

Kvl.2

xKvl.2/XSha2: RNAaseP: Expressed in the nervous system but not detectable in skeletal muscle21 .

Kvl.3

rKvl.3/Isolate KV3/RGK5: RT-PCR: RNA transcripts detected in brain, lung and spleen; also cloned from rat peripheralleukocytes32 . rKvl.3/RCK3: Northerns: Cerebellum (-); cerebral cortex + +; corpus striatum + +; hippocampus + +; inferior colliculus + + + + +; medulla-pons + + + -t-; midbrain (-colliculi) + + +; olfactory bulb + + + +; spinal cord + + +; superior colliculus

+ + +101.

mKvl.3/MK3/HLK3: RT-PCR: MK3 mRNA is expressed in T lymphocytes. Encodes a channel with biophysical and pharmacological properties indistiguishable from those of voltage-gated type n K+ channels in T lymphocytes31 (see Current type, 48-34). Kvl.4

hKvl.4/HKl: Northerns: Atrium + + +; ventricle + + +. rKvl.4/RCK4: Northerns: Cerebellum (-); cerebral cortex + + +; corpus striatum + + + + +; hippocampus + + + +; inferior collicul\, us + + ++; medulla-pons +; midbrain (-colliculi) + + +; olfactory bulb + + + + +; spinal cord (-); superior colliculus + + + + +101 (see also Kv3.4, this table, under VLC K Kv3-Shaw, comparing distributions of RCK4 to Raw3, encoding another A-type K+channel. rKvl.4/RK3: Northerns: Relative mRNA abundance: Brain~cardiac atrium> aorta == skeletal muscle:> cardiac ventricle. 17 Transcripts also detected in skeletal muscle, tongue, stomach, small intestine, uterus and liver. rKvl.4/RGHK9: ISH: mRNA detectable in anterior pituitary; particularly abundant in the hippocampus 124.

Kvl.S

hKvl.5/HK2: Northerns: Atrium + + + + +; ventricle +. hKvl.5/hPCNl: Northerns: Brain (-); kidney (-); liver (-); lung (-); pancreas ++; skeletal muscle (-); ventricle +. RT-PCR: hPCNl is present in normal human pancreatic islets but is 'not detectable' in skin fibroblasts or HepG2 cells. RT-PCR: Human airway smooth muscle 120 (see Phenotypic expression, 48-14). rKv1.5/Isolate KV1: Northerns: Relatively widespread distribution: found in brain, heart, kidney, lung and skeletal muscle32 (see also Developmental regulation, 48-11). Comparative note: Isolate KVl mRNA is expressed at high levels in the GH3 pituitary cell line; although Kvl behaves as a non-inactivating delayed rectifier in oocytes, the voltage-dependent K+ current in GH.3 cells is a relatively rapidly inactivating current but has similar pharmacology to KVl current in oocytes). Rat spinal cord motor neurones are 'highly emiched' in the Kvl.5 and Kvl.6 nlRNAs (see l16 described under Developmental regulation, 48-11). Kvl.5 is also widely expressed in glial cells of brain and spinal cord125 (see Protein distribution, 48-15).

entry48

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Table 3. Continued Kv

Systematic/clone names, original descriptions, notes and references

Kvl.5

rKvl.5/RK4: Northerns: Ubiquitous in excitable tissue, being present at comparable levels in atrium, ventricle, aorta, brain and skeletal muscle. Relative mRNA abundance: Cardiac atrium == cardiac ventricle == aorta == skeletal muscle> brain. No signal in liver. RK4 is the most abundant cardiac channel mRNA of the RKI to RKS group17. rKvl.5/RMK2: RNAaseP: The 3' non-coding regions of the brain, cardiac and skeletal muscle RMK2 transcripts are identical44 . rKvl.5/Kvl.5: RT-PCR: RNA transcripts for Kvl.l, Kvl.2, and Kvl.5 have been detected in adult rat sciatic nerve preparations (see also Kv1.1/Kv1.5 under Protein distribution, 48-15).

Kvl.6

hKvl.6/HBK2: Northerns: Not found in tissues other than brain. rKvl.6/RCK2: Northerns: Detected mainly in midbrain areas and brainstem. ISH: 'Homogeneous' expression levels in most brain regions; slightly elevated message levels in the piriform cortex, the olfactory tubercule, and the dorsal endopiriform nucleus. Low expression in the hippocampus, the central medial thalamic nucleus, the zona incerta, the medial amygdaloid nuclei, and the lateral amygdaloid area. Generally low expression in the cerebellum but 'significant' in the Purkinje celllayerl18 . Rat spinal cord motor neurones (see Kv1.5, above)l16. rKvl.6/Isolate KV2: Northerns: Transcripts detected in brain only32.

Notes: 1. 'Verbal' descriptions of expression patterns are included where they have been specifically reported, but these are a poor substitute for the original in situ hybridization data (ISH:). 2. Relative expression levels (mostly according to schemata in original references) have been indicated by a variable number of '+' signs within a group of tissues (e.g. + + +), where the comparison applies within that group only. 3. Mapping of expression patterns is a complex task and has to take many variables into account, such as in situ localization, developmental regulation, subunit stoichiometry, and factors regulating 'overlapping' or coexpression. For further details of annotated digitized image libraries which permit direct comparison of mRNA/protein expression patterns in relation to biological specialization of tissue regions (e.g. in brain) see Resource TSearch Criteria and Genotype/phenotype models under Resource H - Cell types. 4. To help consolidate multiple in situ hybridization data for Kv subunit expression patterns into a single reference source for availability on the WWW, please forward citation details as described under Feedback, 48-57. 5. Other designations used above include Northems: as determined by Northern blot r analysis (low sensitivity); RNAaseP: as determined by RNAase protectiont analysis (intermediate sensitivity); RT-PCR: as determined by reverse transcription-PCR amplificationt analysis (high sensitivity).

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Kv1.2 and Kv1.4 (this entry), Kv2.2 (entry 49) and Kv{j1 (entry 47); transcripts from all four genes were more abundant in the pre-vertebral ganglia. By comparison with native channel currents (see entries with '[native)' suffix in their sortcode) this type of study can 'propose or reject' candidate subunits underlying native currents. For example, on this basis, it was concluded that (i) members of the Kv4 family are likely to underlie low-threshold A-current in sympathetic neurones (as detailed in entry 51) while (ii) cDNAs encoding two currents prominent in native sympathetic neurones (M-current and D 2 -current, see entry 53) were 'yet to be identified,119.

Phenotypic expression Point mutations in the hKvl.l gene associated with one form of familial episodic ataxia

48-14-01: hKv1.1: One form of familial episodic ataxia t (EA) is characterized by brief episodes of ataxia t with myokymia t (continuous movement or 'rippling' of muscles) evident between attacks consistent with reduced capacity for repolarization in affected nerve cells (for disease phenotype description in relation to developmental onset, see AEMK under OMIM 160120). Using a group of Genethont markers t from a region of human chromosome 12p carrying a paralogous t cluster of K+ channel genes (see Chromosomal location, 48-18) Litt et. al. 126,127 initially described linkage t in four AEMK kindreds t. Chemical cleavage mismatchr mutational analysis of genomic PCR products specifying the coding region of KCNAlled to the description of several additional, different, missense point mutations t in hKv1.1 in these families12S-130. Direct sequencing of products showed that each of the four families had a different missenser mutation, each predicted to affect a highly conserved residue in the hKv1.1 protein (Va1174Phe in the S1transmembrane domain, Arg239Ser in S2, Phe249De in the S2/S3 intracellular loop, and Val408A1a at the border of S6 and the Cterminal domain). All these mutations were present in the heterozygous t state, suggesting that EA/myokymia can result from autosomal dominantr mutations in the KCNA1 gene on chromosome 12p13 (see Chromosomal location, 48-18). Mutant expression studies in Xenopus oocytes have demonstrated that two of the EA subunits form homomeric channels with altered gating properties. For example, Kv1.1 V408A channels (mutated in the region encoding the C-terminal) have voltage dependence similar to wild-type Kv1.1 channels, but have faster kinetics and increased C-type inactivation l29 (see Inactivation, 48-37). Alternatively, the voltage dependence of Kv1.1 F184C channels (mutated in the region encoding TM1) is shifted 20 mV positive. Four other EA subunits studied did not produce functional homomeric channels but reduced the potassium current when co-assembled with wild-type subunits l29.

Roles of Kv channels from subcellular heteromultimeric co-assemblies 48-14-02: Kv1.2; Kv1.4: Subcellular distributions of Kv1.2-containing K+

channels indicate they play diverse functional roles in several neuronal compartments, regulating pre-synaptic Q! post-synaptic membrane excitability, depending on the neuronal cell type (see Subcellular locations, 4816). Kv1.4 protein is targeted to axons and possibly terminals, suggesting a

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pre-synaptic role in synaptic transmission64 (for some exceptions, e.g. olfactory bulb mitral cell dendrites, see ref. 131 ). In contrast to Kvl.4, a second Q subunit forming A-type channel protein (Kv4.2, see VLC K Kv-Shal, entry 51) is concentrated in post-synaptic dendrites and somata, implying distinct roles for these channel types in vivo (see also Subcellular locations, 48-16). Evidence supporting heteromeric co-assembly of rKvl.4 and rKvl.2 in rat brain has been obtained (see Protein distribution, 48-15 and Protein interactions, 48-31). The Kv 1.4/Kv 1.2 heteromultimer localized in axons and nerve terminals combines features of both 'parent' subunits, resulting in an A-type K+ channeI132 ). At presynaptic loci, Kvl.4/Kvl.2 heteromultimers are likely to form native A-type K+ channels regulating neurotransmitter release. For evidence suggesting fast-inactivating channel components (such as Kv1.4) can mediate 'longlasting' after-hyperpolarizations (tending to reduce action potential frequency) during recovery from inactivation, see Table 19 under Inactivation, 48-37. For general phenotypic roles of A-type channels in native neurones, see VLC K A-T [native], entry 44.

Proposed role of Kvl.2 in smooth muscle excitation-contraction coupling 48-14-03: Canine Kvl.2/CMSK1: Kvl.2 is expressed in canine gastrointestinal muscles; its potential role in regulation of electrical slow wave activity has been described27 (see also mRNA distribution, 48-13). In Xenopus oocytes CSMKI cRNA induces the expression of low-conductance K+ channels displaying a linear current-voltage relation in inside-out patches with a slope conductance of 14pS. The channels were blocked in a concentrationdependent manner by 4-aminopyridine (IC so 74 JlM)27.

Kvl.3 in regulatory volume decrease (RVD) in response to hypotonic shock

48-14-04: mKvl.3: Transient transfection t of a Kvl.3 expression construct into cells of the mouse cytotoxic T lymphocyte line CTLL-2 (unable to volume regulate and 'devoid' of voltage-dependent K+ channels) reconstitutes their ability to volume regulate 133 . This property appears to 'critically depend' on (i) volume-induced membrane potential changes and (ii) the Kv subunit isoform used: When transfected with Kv3.1 transient expression constructs, CTLL-2 cells do not show RVD. According to a model for regulatory volume decrease (RVD, see Fig. 2 and ref. 133) the ability of a Kv channel to confer the capacity for RVD may be partially explicable in terms of different voltage dependence of activation for Kv1.3 and Kv3.1 channels (for background to Kv1.3 and Kv3.1 in native T lymphocytes, see Developmental regulation under VLC K DR [native], 45-11; for other properties, see VLC K Kv3-Shaw, entry 50).

No apparent role for Kvl.3 in CSF-l-stimulated proliferation of microglia 48-14-05: rKvl.3: In cultured microglia established from neopallia of newborn rats an anion current and a K+ current (resembling Kv1.3) are activated reversibly under hypo-osmotic (cell swelling) conditions l34 (see paragraph 48-14-04 and Fig. 3, p. 419). Although this study suggested that both Kir and CI- channels are necessary for proliferation of rat microglia stimulated by colony-stimulating

_'--

e_n_try_4_8_

in hypotonic medium (e.g. for CTLL, see text, 25% PBS containing r1L-2 at 8.3 ng/ml)

in hypotonic medium

Cell swelling and retu rn to isotonic volume

1. Volume expansion 2. CI channel opening (depolarizing cell towards Eel, e.g. between -10 and -35 mV)

wale/"

3. Opening of Kvl.3 rc~:~eIS [accompanied by

/

RVe Figure 2. Simplified model for crLL regulatory volume decrease (RVD) following hypotonic shock. (From 48-14-05) factor 1 (CSF-l), it found no evidence that 'even a transient activation' of Kv channels were necessary for the CSF-stimulated proliferation process l34 .

A Kvl.3 homologue with predicted roles in renal medullary K+

transport 48-14-06: Rabbit Kvl.3; Rabbit Kvl.2: The potassium conductance of the kidney medullary (papillary epithelial) cell line GRB-PAPI is composed of Shaker-like potassium channels. From RT-PCRt studies (see notes to Table 1 under Gene family, 48-05) candidate genes encoding these channels include Kvl.l, Kvl.2, Kvl.4 and latterly, the 'glibenclamide-sensitive' rabK(vl.3) clone34, which is also expressed in native renal medulla and brain. The latter encodes an ORF of 513 aa, and is related to but not identical to other Kvl.3 isolates (e.g. in having a different N-terminus and a single-channel conductance that saturates following expression in oocytes). Note: Independent electrophysiological studies 135 have shown that GRBPAP1 cells (grown on permeable supports and capable of developing electrogenic Na+ transport) display a 'dominant' K+current' that is slowly inactivating, time- and voltage-dependent; in this study, RT-PCR of GRBPAPI mRNA amplified a segment of Kvl.2.

l_e_n_t_ry_48

_

IDominant' effects of Kvl.3 on resting membrane potentials in native and heterologous cells 48-14-07: Kvl.3: An outwardly rectifying K+ channel in osteoclasts with many properties resembling those of Kvl.3 has been described136. Application of charybdotoxin (50nM, see Blockers, 48-43) changes osteoclast resting membrane potential (inducing a depolarizing shift of 5-10mV from a typical resting potential of -50mV). Note: RT-PCRt on osteoclast RNA pools has identified expression of both Kvl.3 and Kir2.1/IRKl (entry 33). rKvl.3: Sensitivity to the non-selective blocker charybdotoxin (CTx) (see Blockers, 48-43) has been used to argue a contribution of Kvl.3 to the repolarization of action potentials at pre-synaptic terminals of hippocampal inhibitory neurones 137j CTx-induced facilitation of transmission may be partly explained by its effects on Kv channels rather than large-conductance, calcium-activated channels (BKca, see ILG K Ca, entry 27). hKvl.3: Heterologous expression of human Kvl.3 'resets' the resting potential of chinese hamster ovary (CHO) cells, 'clamping' it within a narrow range close to the threshold of activation of Kvl.3 138. This property partly depends on Kvl.3's intrinsically steep voltage dependence of activation and slow/ incomplete inactivation. Inhibitors of Kvl.3 such as margatoxin depolarize transfected CHO cells to the potential of non-transfected cells 138 .

Membrane potential regulation in excitation-secretion coupling of pancreatic j3 cells 48-14-08: hKvl.5/hPCNl: Stable transgenic t overexpression of the tetraethylammonium (TEA)-insensitive hKvl.5 channel in pancreatic cells of transgenic mice attenuates glucose-activated increases in [Ca 2 +]i and prevents the induction of TEA-dependent [Ca2 +]i oscillations 139 (for further background, see ILG Ca InsP3, entry 19 and ILG K Ca, entry 27). Augmentation of expression in pancreatic /3 cells was confirmed by immunoblot studies of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings showed the presence of high-amplitude TEAresistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycaemia and hypoinsulinaemia. Note: hPCNl was originally derived from a human insulinoma line28 .

Kv channel phenotypes in non-excitable Ltk- mouse fibroblasts 48-14-09: hKvl.5: Following stable t expression in Ltk- mouse fibroblasts (Lcells) the fast-activating, non-inactivating hKvl.5 channel (i) prevents dexamethasone-induced increases in intracellular volume and (ii) inhibits Na+ / K+ -ATPase activity by 25 % (as measured by 86Rb+ uptake)140. Independent measurements of alanine transport was also lower in Kv1.5-expressing cells, indicating that the expression of this channel modifies Na+-dependent amino acid transport. Expression of the rapidly inactivating subunit Kv1.4 did not alter alanine transport relative to wild-type or sham-transfectedt cells. The properties induced by Kv1.5 expression may be related to changes in the resting membrane potential induced by this channel (-30 mV) in contrast to that measured in wild-type sham-transfected, or Kv1.4transfected cells (-2-0mV). Quinidine block of hKv1.5 (60J,!M) negates the effects of Kv1.5 expression on intracellular volume, Na+ /K+ -ATPase, and

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electrogenic Na+ -dependent alanine transport 140. Comparative note: Kvl.5: Although human airway smooth muscle cells express mRNA from several members of the Kvl gene subfamily (see mRNA distribution, 48-13) Kvl.5 appears to 'predominate' voltage-dependent current and the regulation of basal tone 120.

'Closely matching' heterologously expressed Kv subunit channels to 'native' types 48·14·10: The difficulties involved in 'predicting' specified Kv subunit 'contributions' to voltage-gated K+ currents observed in native cells is outlined under Channel designation, 48-03 and in VLG K Kv-beta, entry 47). In the brain, for example, multiple Kv gene products are likely to be involved in regulating excitability of any single neurone. Despite the existence of multiple mechanisms which generate K+ current diversity (see Developmental regulation, 48-11), several descriptions have appeared where native and heterologously expressed channel properties exhibit 'notable similarity' (see Table 4).

Physiological roles deduced from spontaneous mutants and gene knockout (background) 48·14·20: Heritable phenotypes associated with mutated or deleted gene loci provide a potentially powerful method for analysing specific contributions of Kv and other channels to higher organismal function. In humans, patterns of disease or symptomatic disorders (observed in families or populations) may follow laws of Mendelian inheritance t. As such, disease phenotypes may be linked to the specific inheritance of 'defective' gene products or closely linked markers! (ibid.). In this way, a number of physiological disorders have been associated with mutations in specific ion channel genes. The well-established roles of K+ channels in the nervous system makes them important candidates for heritable and sporadic forms of neurological disease both in humans and in animals. For this reason, much effort is being directed to gene mapping/disease linkage studies, and the production of transgenic t animal models of disease carrying targeted disruptions or specified mutations in cloned genes (see Gene knockout t in the on-line glossary). Phenotype analysis of gene knockout mice may not be straightforward, especially for gene products that have important roles in development or that commonly form complexes with other proteins in native cells. Furthermore, many null t mutants have no apparent phenotypic changes, or may require detailed long-term observation, behavioural analysis or extensive histological comparisons to reveal them. To complement these approaches, genetic analysis of several extant mouse mutants (including those exhibiting certain neurological disturbances) is also underway. As described above, these studies attempt to find evidence for linkage of disease markers to known or predicted chromosomal loci/regions encoding specified gene product(s). For comparative purposes, Table 7 (under Chromosomal location, 48-18) also includes some cross-referenced examples of these, relating known mutant phenotype/markers to regions including Kv channel gene loci.

lL...--_ _ _ry_4_8

_

en t

Table 4. Notable similarities between tc10ned' and tnative' voltage-gated K+ channel properties. See general tcautionary' notes in this field and under Channel designation, 48-03. For important contributions of Kv{3 subunits to native channel phenotypes, see VLC K Kv-beta, entry 47. (From 48-14-10) Kv

Properties and cross-references

Kvl.l

48-14-11: Kvl.l-RBKl: RBKI has been cited as contributing to the dominant native voltage-gated K+ current in C6 astrocytoma (glial) cells 80 (compare Kv1.5, below).

Kvl.2

48-14-12: rKvl.2/RAK: IKv 1. 2 in Xenopus oocytes (isolate RAK, one amino acid difference from isolate BK2) 'compare closely' to native rat neuronal delayed rectifier-type currents in cardiac atrium (see Activation, 48-33 and ref.24).

Kvl.3

48-14-13: Kvl.3/KV3: The properties of heterologously expressed Kvl.3 and Kv3.1 subunits correspond closely to the 'predominant' voltage-activated K+ channels of native T lymphocytes (n-type and 1type respectively; for further details see 141 , VLC K Kv3-Shaw, entry 50 and cross-references to Kv1.3 in this entry). 'Kvl.3-like' currents have also been described in a kidney medulla cell line and osteoclast preparations (see Phenotypic expression, 48-14).

Kvl.4

48-14-14: rKvl.4: For prominent immunostaining in axons and terminals, see also Subcellular locations, 48-16. 48-14-15: rKv1.4/RGHK9: The A-type current of rat pituitary tumour cell line GH3 /B-6 (as compared to the biophysical and pharmacological properties of the rKvl.4 isolate RGHK9 cDNA expressed in Xenopus oocytes following RT-PCRt from a GH3 /B-6 poly(A)+ template 124 ). Although the activation kinetics differ, the properties of 'cloned' and 'native' currents are indistinguishable with respect to (i) 4-aminopyridine block; (ii) voltage dependence and slope of steady-state activation and inactivation and (iii) slow recovery from inactivation and time constant values 124. 48-14-16: rKvl.4/RHKl: The isolates RHK1 37 (r-vKvl.4 isolate RCK4 22 ) the clone FKl (ferret Kvl.4)4o and clone HKl (hKvl.4)36 were originally described as displaying fast activation and inactivation 'similar' to the transient outward current (ITO, entry 44) described in native rat ventricular myocytes 142. Notably, however, Kvl.4 has more recently been shown to be a low-abundance protein in rat atrial and ventricular myocytes l14; in consequence, its role in myocyte excitability has been questioned (ibid.). For further determinants of inactivation behaviour in Kv channel complexes, see VLC K Kv-beta, entry 47.

Kvl.5

48-14-17: hKvl.5/fHK: Intact human cardiac atrial myocytes display a rapid delayed rectifying K+ current with properties and kinetics 'identical to' those expressed by a K+ channel isolate (fHK) cloned from human heart eDNA and stably expressed in a human-derived cellline143 (for comparative data, see references to Kv1.5 (isolate

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Table 4. Continued Kv

Properties and cross-references

Kv1.5

fHK) under Activation, 48-33, Single-channel data, 48-41 and Blockers, 48-43). Although there is some 'correspondence' between the cloned and native channel properties, the 'precise' contribution of Kv1.5 channel current to the cardiac action potential remains unclear. See also/compare direct evidence for roles of erg subfamily channels (e.g. HERG) underlying native cardiac currents under Phenotypic expression of VLC K eag/elk/erg, 46-14. 48-14-18: hKv1.5/HK2: Although not conclusive (see above) Ltkcell line stably expressed t HK2 (cloned from human cardiac ventricular cDNA) displays has also been specifically stated to possess many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes l44 . If verified, this would suggest that human Kv 1.5 contributes to the initial fast repolarization and to the K+ conductance during the plateau phase of the cardiac action potential l44 . Note: HK2 current does not resemble either the rapid or the slow components of delayed rectification described in guinea-pig myocytes. 48-14-19: rKv1.5: A specific contribution of Kv1.5 to the delayed rectifying K+ current of spinal cord astrocytes (see Protein distribution, 48-15) has been proposed following incubation of these cells with antisense oligodeoxynucleotides 125 . Antisense treatment (i) reduces delayed rectifier current density and (ii) shifts the potassium current steady-state inactivation (without altering current activation, cell capacitance or cell resting potential) (compare Kvl.l, above).

Notes: Historically, cloning of the Drosophila Shaker (see Related sources and reviews, 48-56) established that altered neurological phenotypes coupled to locomotor dysfunctions could be directly related to mutations in genes encoding voltage-gated potassium channels. Through the extensive use of behavioural mutants, comparative genetic analyses of the major K+ currents in embryonic Drosophila neurones and muscle have been performed, and the 'relative contribution' of the Shaker, Shab, Shaw and Shal loci have been determined (for brief summary, see Phenotypic expression under VLC K Kv2-Shab, 49-14). Detailed comparisons of 'native' versus 'cloned' channels encoded by the Shaker locus in Drosophila are also included in references listed under Related sources and reviews, 48-56; see also properties of novel potassium channels in Drosophila photoreceptors described in ref. 145 . Additional references to Drosophila K+ channel genes in relation to their mammalian counterparts are described under entry 27 (ILC K Ca, Drosophila slo, mouse mslo, human hslo); entry 46 (VLC K eag/elk/ erg, Drosophila eag-related loci including HERG) and entry 47 (Drosophila hyperkinetic, related to mammalian Kv{3 subunits).

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_

Protein distribution KVQ subunit components of immunoprecipitated DTx acceptor complexes 48-15-01: Subtypes of a-dendrotoxint (o-DTx)-susceptible K+ channels are prevalent throughout mammalian brain and are composed of heterogeneous K+ channel protein assemblies l46 that also contain auxiliary Kv/J subunits147 (for details, see VLC K Kv-beta, entry 47). Immunoprecipitationt of the DTx acceptor complext in bovine brain using a monoclonal t antibody selective for Kvl.2 (mAb 5) and polyclonal antibodies for other Kvo subunits indicate that Kvl.l, Kvl.4 and Kvl.6 can be components of the acceptorl46 . While 'virtually all' DTx receptors contain the Kv1.20 subunit l46,148,149, the distribution of other o subunits in the complex can vary considerably across different brain regions, as summarized in Table 5. Autoradiographic co-distribution studies using affinitypurified anti-Kyo and anti-Kv,8 subunit antibodies and 125I-labeled DTx have shown that (i) the areal and laminar distribution of o-DTx acceptors correspond most closely to Kval.2 and Kv{32 immunoreactivity149; (ii) o-DTx binding also corresponds closely to Kvl.l and Kvl.6 immunoreactivity in the cerebral and cerebellar cortices, hippocampus and in fibre pathways and (iii) with the exception of the hippocampus and entorhinal cortex, [125 I]0-DTx binding does not correspond closely to Kvl.4 or Kv,8l staining patterns. Kvl.l, Kvl.4, Kvl.6 and Kv,8l components of o-DTx acceptors may be limited to specific projectiont systems. Note: o-DTx is a 56 amino acid peptide isolated from the venom of the African green mamba. It binds selectively and with high affinity to voltage-gated K+ channels in mammalian brain.

Comparative studies examining multiple Kv protein distribution in heart and brain 48-15-02: Westernt blotting of cardiac atrial versus ventricular membrane proteins with a panel of anti-Kv channel has confirmed the presence of Kvl.2, Kvl.S, Kv2.1 and Kv4.2 in heart and revealed differences in the relative abundances of these subunits in the two membrane preparations l14 . Kvl.5 levels are weaker, but comparable in the two preparations; Kv2.1 (entry 49) and Kv4.2 (entry 51) abundances appear higher in atrial membranes. Anti-Kvl.4 antibodies reveal Kvl.4 is not an abundant protein in adult rat atrial or ventricular myocytes l14 (for further details, see Table 5). In a separate extensive study 131, specific polyclonal antibodies for five Kvl subfamily 0 subunits (Kvl.l, Kvl.2, Kvl.3, Kvl.4, Kvl.6) were used to determine detailed distribution patterns in rat hippocampus, cerebellum, olfactory bulb and spinal cord. This study 131 extended but partly contradicted the previously reported64 'stereotypical' targeting of Kvl.4 channel 0 subunits to axonal compartments (e.g. in reporting a part-dendritic localization of Kvl.4 protein in olfactory bulb mitral cell in addition to axons).

Subcellular locations General significance of Kv protein subcellular locations in neurones 48-16-01: The particular 'functional role' of any given set of ion channels may vary depending on their 'precise location' on the cell surface or within

11II

_L...--

,

e_n_try_4_8_

Table 5. Summary of KvO'. subunit co-distributions as detected in brain by immunoprobes 146 . See also VLC K Kv-beta, entry 47, mRNA distribution, 48-13 and Subcellular locations, 48-16. (From 48-15-01) Kv

Properties and cross-references

Kvl.l

48-15-03: Kvl.l: Variable amounts of Kvl.l subunits are observed in K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex and brainstem, with a notably 'larger proportion' of Kvl.l in DTx acceptor complexes isolated from the cerebral cortex and brainstem. 48-15-04: Kvl.l/Kvl.2: In the hippocampus, both mKvl.l and mKvl.2 proteins are present in axons, often near or at synaptic terminals in the middle molecular layer of the dentate gyrus, while only mKv1.1 is detected in axons and synaptic terminals in the hilar/ CA3 region150. In the cerebellum, both mKvl.l and mKvl.2 are localized to axon terminals and specialized junctions among axons in the plexus region of basket cells. Strong differential staining is observed in the olfactory bulb, where mKvl.2 is localized to cell somata and axons, as well as to proximal dendrites of the mitral cells (see also Subcellular locations, 48-16). 48-15-05: Kvl.l/Kvl.2: Antibodies recognizing Kvl.l, Kvl.2 and synapse-associated protein 90 (SAP90) are predominantly localized (concentrated) in septate-like junctional regions, which connect the basket cell axonal branchlets in the Pinceau (a network of cerebellar basket cell axon branchlets surrounding the initial segment of the Purkinje cell axon)151. These results are consistent with SAP90 assisting the formation of Kvl.l/Kvl.2 heteromultimers in cerebellar Pinceaux junctions. Comparative note: By contrast; (i) Kv3.4 (entry 50) is uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma (i.e. localized to the Purkinje cell axon initial segment) and (ii) voltage-gated Na+ channels (entry 55) are not detectable in the Pinceau, but are localized to the Purkinje cell axon initial segment 151 . 48-15-06: Schwann cell staining ~,.yith anti-Kvl.l antibodies reveals high concentrations of Kvl.l (i) in the axonal membrane at juxtaparanodal regions and (ii) intracellularly, within perinuclear compartments. Although it is likely that intracellular immature forms of these (compartmentalized) signals repn:~sent subunits, comparison with Kvl.5 distributions (this table) indicate that closely related Kv channels (from the same subfamily) need not co-assemble and can be localized differentially in the same cell152 (see also N-glycosylation under Sequence motifs, 48-24 and Protein interactions, 48-31).

Kvl.2

48-15-07: Kvl.2: Kvl.2 subunits are uniformly distributed in brain regions listed under Kvl.l. Kvl.2 co-immunoprecipitates with Kvl.l in rat brain membranes, and has been co-localized with Kvl.l in the juxtaparanodal regions at nodes of Ranvier in myelinated axons and

----l_

l"----e_n_t_ry_48

Table 5. Continued Kv

Properties and cross-references

Kvl.3

in terminal fields of basket cells in mouse cerebellum 153,154 (see above and note below). Kv1.2 is co-localized with Kv1.4 in the dentate gyrus 132 and co-precipitates with Kv1.4 within rat brain membrane preparations. Note: A striking example of co-localization between Kv1.2 and PSD-95 occurs in the basket cells 155 (for significance see Fig. 7 under Protein interactions, 48-31).

Kvl.4

48-15-08: Kv1.4: See Subcellular locations, 48-16 for predominant axonal/terminal distribution of Kv1.4 protein. Variable amounts of Kv1.4 subunits are observed in K+ channels purified from the brain regions listed under Kv1.1, with a larger proportion of Kv1.4 being found in DTx acceptor complexes from the hippocampus. Note: Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes l14 : A 'very faint' band was detected at 97 kDa in atrial and ventricular preparations when an anti-Kv1.4 antibody (that reveals intense Kv1.4 expression in brain) is used at a 5- to 10-fold higher concentration (see Table 4 under Phenotypic expression, 48-14).

Kvl.5

48-15-09: Kv1.5 and Kv1.1 proteins have distinct distributions in Schwann cells. Anti-Kv1.5 antibodies have localized subunit expression to (i) the Schwann cell membrane at the nodes of Ranvier; (ii) 'bands' that run along the outer surface of the myelin and (iii) intracellular distributions in the vicinity of the nucleus 152 (see also Kv1.1, this table). rKv1.5: By immunohistochemistry: Kv1.5 protein is abundant in glial cells of adult rat hippocampal and cerebellar slices, as well as in cultured spinal cord astrocytes. Note: Kv1.5 immunoreactivity was described as particularly intense in the endfoot processes of astrocytes surrounding the microvasculature of the hippocampus 125 (see also Phenotypic expression, 48-14).

Kvl.6

48-15-10: Kv1.6: Kv1.6 subunits are uniformly distributed in those brain regions listed under Kv1.1.

Notes: 1. In this study 146 Kv-specific antibodies generally precipitated a different proportion of the channels detectable with radioiodinatedt a-DTx in every brain region (anti-Kv1.2> 1.1» 1.6> 1.4), consistent with a widespread distribution of hetero-oligomeric subtypes (see Protein interactions, 48-31). 2. For examples of immunohistochemical localizations of Kv1 subfamily channels in single-cell types coupled with functional studies64,156 described under Subcellular locations, 48-16. internal membranes. As pointed out in refs. 64,157, this is of particular significance for K+ channels in neuronal membranes, which have the capacity to respond uniquely to a given input, and where neuronal integration depends on local responses of spatially segregated inputs to the

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cell and the communication of these 'integration centres' via the axon. The subunit diversity shown by the Kv channel family is thus of major significance for supporting a multiplicity of functional roles (via formation of many multiple channel types) and subcellular localizations or 'targeting' of channels may extend this functional diversity still further. For example, differential subcellular and regional distributions of Kv subunits forming Atype t channels (rKv1.4 and rKv4.2) imply distinct roles for these channel proteins in vivo (this field, below, Protein distribution, 48-13 and corresponding entry/fieldnames under VLC K Kv4-Shal (i.e.. 51-13, 51-16). Kv1 subfamily Q subunits interact through their cytoplasmic C-terminus with a family of membrane-associated (putative) guanylate kinases, including PSD95 and SAP97. Heterologous co-expression of either SAP97 or PSD-9S with a number of Shaker-type subunits induces 'co-clustering' of the kinase homologues with the Kv channels 62 (for types of clustering induced by PSD-95 and SAP97, see Protein interactions, 48-31; see also effects following mutation of the C-terminal lEDTV' motif in Kv1.4 under Sequence motifs, 48-24).

Co-location of Kvl.l/Kvl.2 in ;uxtaparanodal regions of myelinated axons 48-16-02: Kv1.1/Kv1.2: mKv1.1 and mKv1.2 polypeptides (probably as heteromultimers) occur in subcellular regions where rapid membrane repolarization may be important (juxtaparanodal r regions of nodes of Ranvier in myelinated axons t and terminal fields t of basket cells t 153. In separate studies, mKv1.1 and mKv1.2 proteins have also been shown to be present in unmyelinated axons, specialized junctions between axons, and proximal dendrites 150 (for broader distributions, see Table 5 under Protein distribution, 48-15).

Kvl.2 subcellular locations appear distinct in different cell types 48-16-03: Kv1.2: Kv1.2 protein immunoreactivity shows a complex differential subcellular distribution within different neurones of rat brain 158 . At certain loci (e.g. hippocampal and cortical pyramidal cell and Purkinje cells) Kv1.2 appears concentrated in dendrites. In other neurones such as cerebellar basket cells, Kv1.2 is predominantly (if not exclusively) localized to nerve terminals 158, although some immunoreactivity is associated with certain axon tracts.

Comparison of temporal Kv protein expression patterns observed in situ versus in vitro 48-16-04: While the timing of expression for Kv1.4, Kvl.S, Kv2.1, Kv2.2 and Kv4.2 polypeptides observed in situ within the hippocampus appear to be retained in vitro, properties such as cellular and subcellular localization appear to be different 103 . While similarities in Kv protein expression in situ and in vitro indicate the same regulatory mechanisms control spatiotemporal patterning, observed differences between levels of expression for all subtypes studied (except Kv2.1) indicate additional mechanisms operating in situ which mediate Kv channel abundance 103 . Kv1.4 protein is targeted to axons and possibly terminals, suggesting a pre-synaptic role in synaptic transmission64 (for some exceptions, e.g. olfactory bulb mitral cell dendrites,

III

lL...--e_n_t_ry_4_8

----I_

see ref. 131 ) (see also the general contrasting subcellular locations between the A-type channel subunits Kv1.4 and Kv4.2 described under VLC K Kv4Shal, entry 51).

Distributions of Kvl subfamily channels in rod bipolar cells of mice 48-16-05: mKv1.1; mKv1.2; mKv1.3: Generally, immunohistochemical localizations in single-cell types show Kv channel subunits to have a unique subcellular distribution (see above). For example, in enzymatically dissociated isolated bipolar cells of mice Kv1.1 immunoreactivity is detected in the dendrites and axon terminals, whereas Kv1.2 and Kv1.3 subunits are localized to the axon and the post-synaptic membrane of the rod ribbon synapse, respectively122,159. Whole-cell patch-clamp studies on the same preparation indicate that the activation voltage of the native delayed rectifier current (IK ) of the isolated bipolar cell and the inhibitory constants for current blockade by TEA, 4-AP, and Ba2 + are 'most similar' to properties measured for Kv1.1 (as expressed in oocytes; TEA and 4-AP inhibitory constants for native I K differ from the inhibitory constants for Kv1.2 or Kv1.3 in oocytes). Despite these differences, all three channels are likely to function in the intact retina to allow complex modulation of retinal synaptic signals 122. On the basis of retinal Kv subunit distribution studies, it has been proposed that each Kv channel subtype is associated with a specific subcellular functional module, and each local K+ conductance responds uniquely to local voltage and second messenger signals 159. Notably, no single Kv channel subtype is expressed over the entire length of retinal neurones.

Role of Kvj32 subunits in promoting surface expression of Kv channel subunit complexes 48-16-06: Kv1.2: Heterologous co-expression of Kval.2 with Kv,B2 has been shown to promote the transport of aKv1.2 to the cell surface (for further details, see Subcellular locations under VLC K Kv-beta, 47-16). Comparative notes: 1. Amongst the Kv,B subunit variants initially isolated and characterized, Kv,B2 was unusual as it did not exert any marked changes on the inactivation properties of co-expressed Kv1 family subunit channels (ibid.). 2. Prolonged treatment of oocytes expressing Kv1.1 with (S-p)-8-BrcAMPS induces channel phosphorylation and can direct channel expression to the plasma membrane 160 (for further details, see Table 16 under Protein

phosphorylation, 48-32).

Polarized expression of Kvl.4 to basolateral membrane of MDCK cells 48-16-07: Kv1.4: Immunocytochemistry and confocal microscopy of the polarized epithelial cell line MDCK shows specific basolateral membrane localization of Kv1.4, detectable in two forms (glycosylated and nonglycosylated)161 (see also the Kv2.1 cytoplasmic domain associated with polarized (lateral membrane) expression and clustering in MDCK cells under Subcellular locations, 49-16).

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Table 6. Data pertaining to RNA transcripts from genes encoding Kv1 subfamily K+ channel proteins (From 48-17-01) Kv1.1

48-17-02: hKv1.1: 9.5kb 120 - human airway smooth muscle (see

Phenotypic expression, 48-14). 48-17-03: mKv1.1: MBK1/MK1: Single ~8kb transcript, probing brain poly (A)+. Note: Relatively large for the channel protein size. RCK1: 1.0-5.0kb detected, 4-5kb being consistent with RCK 1 cDNA size10. RK1: 8 kb, also hybridizes to less-intense bands of 3 and 4kb. Kv1.2

48-17-04: MK2/RK2/RCK5: Hybridizes to a ~10kb transcript (consistent with RCK5-probed blots). RCK2: Restricted expression, chiefly in brain; probes from 3' UTR t detects a 'high level' ~6 kb species in neonatal and adult brain RNA; short RNA species of ~0.6 and 0.8 kb can be seen in brain and adult skeletal muscle respectivelyl18. BK2: ~9.5 kb 15. hKv1.2: 11 kb 120 (human airway smooth muscle)

Kv1.3

48-17-05: RGK5/isolate KV3/MK3: 9.5 kb.

Kv1.4

48-17-06: HK1: Two transcripts of 3.5 kb and 8 kb (both poly(A)+ and total cardiac mRNAs). RCK4: ~4.3 kb in heart and other tissues (see Cell-type expression index, 48-08). RK3: strongest hybridization to a 4.5 kb transcript in heart RNA; 15 kb and 1.5 kb bands also reported. 48-17-07: rKv1.4: rKv1.4 (isolate RCK4)-specific probes detect an mRNA species of ~4300 nt in heart which increases during development 101 . The detection of 'long transcripts' (ca. 80009500nt) from the Kv1.1, Kv1.2 and Kv1.3 (intronless t ) genes indicates that a relatively large proportion of the mRNA is untranslated, e.g. in the isolate rnKv1.1/MBKl this would indicate >6 kb (from approx. 8 kb) is untranslated if the observed polyadenylation sites are used in vivo. 48-17-08: Methodological note: l'ranslation of Kv1.4 employing

an internal ribosome entry sequence (IRESt) has been noted162. IRES elements enable a single RNA transcript encoding two different proteins to be 'driven' from the same promoter (Le. on a single transcript) but translated independently. This has several applications in ion channel expression, for example, in coupling expression of an ion channel subunit to a selectable resistance protein product (e.g. neomycin for stable expression) or to an intrinsic expression reporter protein (e.g. green fluorescent protein t ).

Kv1.5

48-17-09: hKv1.5: 3.5 plus 4.4kb (human airway smooth muscle)120 (see Phenotypic expression, 48-14). HK2: In RNAase protection assays, the HK2 sequence protects 2.5 kb and 1.5 kb messages; neither is sufficient for full-length HK2. hPCN1: A single, low-abundance 4 kb transcript reported in insulinoma, RIN5FS and HIT m2.2 cell RNA. RK4: 3 kb transcript in heart RNA. Isolate KV1: 3.5 kb.

---J_

1L.-_e_n_t _ry_48

Table 6. Continued Kvl.5

48-17-10: mKvl.5: Two alternatively spliced mRNAs of Kvl.5 have been described in mouse heart45 : A long form, encoding a 602 aa protein, and a short form (Kvl-5Ll5'), where the first 200 N-terminal amino acids upstream of the transmembrane segment S1 is deleted. By RNAase protection t both variants are present in a wide range of tissues (e.g. heart, brain and thymus), with the long form being described as 'predominant'. It was deduced that Kvl-5Ll5' arises by an intra-exonic splicing event. An additional short cDNA clone designated as Kvl-5Ll3' encoding a non-functional C-terminal truncated protein (that inhibited expression of the long isoform) was also described in this study45.

Kvl.6

48-17-11: HBK2: Hybridization of an RCK2 probe with total rat brain

mRNA followed by RNAase protection t produced a resistant band of 6800 nt. Isolate KV2: 6.5 kb.

Transcript size Significance of reported transcript sizes 48-17-01: Transcript size ranges as reported for different Kvl channel gene

subfamily members are listed in Table 6. Amongst the subfamily, there are some notable instances of 'long transcripts' comprising much untranslated RNA (e.g. Kvl.l, Kvl.2, Kvl.3) and also evidence for alternative splicing of mRNA transcripts (see Table 9 under Gene organization, 48-20 and Kv1.5, Table 6). Reporting of similar-sized transcripts with small variations in predicted coding region lengths (rvl-lOaa) between separate studies may indicate the isolation of different alleles t of the same Kv gene (see Protein molecular weight (purified), 48-22).

SEQUENCE ANALYSES The symbol [PDTM] denotes an illustrated feature on the generalized Kv channel protein domain topography model (Fig. 6).

Chromosomal location Mechanisms contributing to the origin of the Kv multigene family 48-18-01: The complexity of higher eukaryotic genomes reflects contributions

from many different types of genetic duplication, recombinational exchange and transfer mechanism that have operated at several levels during evolution (for revie~ see Lewin, 1994, under Related sources and reviews, 48-56). As discussed in ref. 54, the 'modern' distribution of K+ channel genes can be partly explained by invoking basic mechanisms such as gene duplicatio~ chromosomal duplication and rearrangement, changes in whole ploidy I number (genome duplicationt) and/or differential gene silencingt . In

III

_1.......-

e_n_try_4_8-----1

particular, localized, tandem gene duplication (followed by sequence divergence) is associated with the generation of clusters of functionally related genes, and this appears typical for Kv channel gene arrangements (this field, below and Fig. 3). Large-scale duplications and/or rearrangements (e.g. affecting whole chromosomes or substantial segments of them) can also generate contiguous segments of linked genes that have a corresponding (paralogoust) region of related, linked genes elsewhere in the genome. The 'clustered' distribution of several groups of Kv channel genes in the mouse has confirmed and extended the existence of such paralogous regions54 (ibid.). Aberrant recombinationt events such as unequal crossing-overr and possibly excision-insertion events mediated by retrovirusest or retrotransposonst 163 may also 'isolate' (separate) genes from clusters and 'place' them at distal genomic loci. Initial analyses of known Kv channel gene chromosome localizations (Table 7) have identified several apparently 'isolated' genes, which may have undergone transplacement by these or other mechanisms.

Kv genes are clustered in paralogous regions of the mouse and human genomes 48-18-02: Striking evidence for Kv gene clustering t at chromosomal loci has been found in the mouse genome (i.e. where more than one K+ channel gene maps to the same chromosomal region)54. In this study, interspecific backcross (IB) analysist (this field, below) was used to identify 'tight' linkage groupst on mouse chromosome 3 (containing six linked Kv genes) and mouse chromosome 6 (containing four linked Kv genes). Some evidence for paralogy also exists for regions on mouse chromosome 7. Notably, Kv genes from distantly related subfamilies can be located within the same cluster (Table 7, below and Fig. 3). A separate analysis2 of Kv channel genes from the Shaker and Shaw subfamilies have been localized to probable clusters on human chromosomes 11 and 19q13, suggesting these regions may also be paralogous. Interspecies homologies between chromosomal segments encompassing Kv channel genes on mouse chromosomes 3, 6 and 7 suggests regions of human chromosomes Ip, 12p and 19q may also be paralogous54 (see also Table 8). These findings have important implications for deducing possible evolutionary mechanisms giving rise to clustering (see Figure 3). The chromosome 3 and 6 clusters are described as part of Table 7. Comparative note: In Drosophila, the genes encoding Shaker, Shab, Shaw, and Shal do not appear to be clustered in the genome l64 .

Supplementary note: Regions of synteny between mouse and human chromosomes 48-18-03: 'Absolute' chromosome locus mapping of all Kv channel gene family members is incomplete (at the time of compilation). In the absence of direct evidence (and because definition of 'exact' map positions in relation to multiple markers in humans is relatively difficult) a number of studies have 'inferred' or 'predicted' human chromosomal loci from known map positions determined in mouse (and vice versa). These predictions rely on maps of linkage t and synteny homologies t between mouse and human (e.g. see refs. 165-167). These maps have been applied in chromosome localization studies of a wide variety of cloned genes in mouse {for background, see

II

r--------------- Present-day 'isolated' Kv genes (unlinked) - ...I

I I

:

Kcn

I

:

I

Encoding primordial channel gene (ancestral)

(see text under Chromosomal location, 48-18)

Aberrant I segmental recombination events promoting gene dispersal

Ken

Yc.

Encoding precursor of the Shaker (Kena) & Shal (Kcnd) subfamilies

I

+

i-----------------~ Hypothetical

'Yc

I

~

""

I

h J :

(approx. 300 million years ago) I I

I I

Encoding precursor of the Shab (Kcnb) & Shaw (Kcnc) subfamilies

I I I I I I I I I

(examples, see text under Chromosomal location, 48-18) Locus: Kcnd1 rs Encoding:?

Kcna3

Kcna8

Kcnc4

mKv1.2

mKv1.3

mKv1.8

mKv3.4

Mouse chromosome 3 (synteny with human 1 p) Locus:

Multiple chromosomal rearrangements

divergence

Segregation to contemporary locations

Encoding:

Kcna1

Kcna6

Kcna5

mKv1.1

mKv1.6

mKv1.5

Mouse chromosome 6 (synteny with human 12p) Locus: Encoding:

·

Kcnb

Kcna

Kcnc

Shab

Shaker

Shaw

Locus:

·

KCNA7

KCNA3

hKv1.7

hKv1.3

Human chromosome 19q

Encoding:

Establishment of four different Kv gene subfamilies w:iQLjQ the divergence of flies and mammals (> 600 million years ago)

II

Kcna2

I

'"

Shal

I

Present-day Kv gene 'clusters' (linked)

Functional divergence

Kcnd

X

-------------------------------------------

t

Local gene duplications Sequence

X

I I I I I

genome duplication event

I

:

Ken

X

I L

hypothetical duplication

X

X X

I

KCNA4

KCNC1

hKv1.4

hKv3.1

Human chromosome 11

Figure 3. Hypothetical events in Kv channel gene evolution based on contemporary chromosomal distributions in mouse and humans. (After Lock et al. (1994) Genomics 20: 354-362.) (From 48-18-01)

('b

='

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~ ~

00

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e_n_try_4_8_

Table 7. Chromosome locations reported for Kvl subfamily genes (From 48-18-02) Consensus/summary Further details (see note 2) (for symbols see note 1) Kv1.1 hKvl.l, KCNA1, 12p13 mKvl.l, Kcnal, chromosome 6 [~]

hKvl.l/HuK(I): (Human locus name KCNA1): Human chromosome 12p1354,173-175. For evidence that mutations in KCNAl cause myokymia with periodic ataxia, see Phenotypic expression, 48-14 and OMIM entry #160120: Myokymia with Periodic Ataxia [Paroxysmal Ataxia with Neuromyotonia, Hereditary; Episodic Ataxia With Myokymia; EAM; Ataxia, episodic, with myokymia; AEM; AEMK; Episodic ataxia, Type 1; EA1]. mKvl.l: (Mouse locus name Kcnal): Mouse chromosome 6, part of a cluster within a paralogous t region (see Fig. 3).

'Kv1.1-related sequences' in the mouse genome 54 Kcnalrsl, chromosome 6 [~] Kcnalrs2, chromosome 2 Kcnalrs3, chromosome 3 [~] Kcnalrs4, chromosome 7 [~]

48-18-03: There are at least four sequences designated as 'mKvl.l-related' in the M. spretus genome (Kcnalrsl, Kcnalrs2, Kcnalrs3, Kcnalrs4,54, see below). It is possible these cross-hybridizing loci are identical to other loci identified with Kv-gene specific probes (ibid.); alternatively, these may represent unidentified K+ channel genes or pseudogenes t. Loci for these sequences have been reported as Kcnalrsl: Mouse chromosome 6, part of a cluster within a paralogous t region (see Fig. 1). Kcnalrs2: Mouse chromosome 2. Note: The locus for recessive anorexia (anx) mutation is in a similar region to that reported for Kcnalrs2 (and possibly mKvl.4) on mouse chromosome 2. Pre-weanling mice homozygous for anx are characterized by anorexia/ reduced body weight, uncoordinated gait, head weaving, hyperactivity and body tremors 176 . The gene encoding follicle-stimulating hormone B is also located in this region 175 . Kcnalrs3: Mouse chromosome 3, part of a cluster within a paralogous t region (see Fig. 1). Kcnalrs4: Mouse chromosome 7. Note: The recessive gene qv associated with the quivering phenotype is located in the same region as Kcnalrs4 and possibly mKvl.7/mKv3.3. Mice homozygous for qv are characterized by pronounced, incessant quivering, locomotor instability, partial hindleg paralysis and deafness 177 (see also mKv4.1-related sequence under Chromosomal location in VLC K Kv4-Shal, 51-18).

l_e_n_t_ry_4_8

_

Table 7. Continued Consensus/summary Further details (see note 2) (see note 1) Kvl.2 hKvl.2/HuK(IV): (Human locus name KCNA2): hKvl.2, KCNA1, [?] 12 First was tentatively assigned to human chromosome 1233; later analyses placed the gene on human or Ip mKvl.2, Kcna2, chromosome Ip54. chromosome 3 [~] mKvl.2: (Mouse locus name Kcna2): Mouse chromosome 3, part of a cluster within a paralogous t region (see Fig. 3). Kvl.3 hKvl.3, KCNA3, Ip13.3 mKvl.3, Kcna3, chromosome 3 [~] (For updates see OMIM 176263)

hKvl.3/HuK(Ill): (Human locus name KCNA3): Human chromosome Ip13.3 29,54. mKvl.3: (Mouse locus name Kcna3): Mouse chromosome 3, part of a cluster within a paralogous t region (see Fig. 3).

Kvl.4 hKvl.4, KCNA4, [?] Ilp14.1 or llq13.4q14.1 mKvl.4, Kcna4, chromosome 2

hKvl.4/hPCN2: (Human locus name KCNA4): Human chromosome Ilp14.1 175,178 or 11p14-p13 179, consistent with Kcna4 localized to the homologous segment on mouse chromosome 2 in ref. 180 (see below) or llq13.4-q14.1 181 . mKvl.4: (Mouse locus name Kcna4): Mouse chromosome 2 (compare Kcna1rs2, this table). Marker order determined as (proximal)-Acra-Kcna4Pax-6-a-Pck-l-Kras-3-Kcnbl-(distal)180.

Kvl.5 hKvl.5, KCNA5, 12p13 mKvl.5, Kcna5, chromosome 6 [~] (For updates see OMIM 176267)

hKvl.5/HuK(VI): (Human locus name KCNA5): Human chromosome 12p13 (i.e. distal short arm band 13)42,173. Note that trisomyt of the human 12p region is associated with an epileptiform disorder (trisomy 12p syndrome182) characterized by 3 Hz spike and wave discharges, but a specific link to altered K+ channel activities has not been reported to date. mKvl.5: (Mouse locus name Kcna5): Mouse chromosome 6, band F, part of a cluster within a paralogoust region (see Fig. 3).

Kvl.6 hKvl.6: (Human locus name KCNA6): Predicted to be hKvl.6, KCNA6, 12p on human chromosome 12p54,180. mKvl.6, Kcna6, mKvl.6: (Mouse locus name Kcna6): Mouse chromosome 6 [~] chromosome 6, part of a cluster within a paralogoust (For updates see region (see Fig. 3). OMIM 176257)

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_8_

Table 7. Continued Consensus/summary Further details (see note 2) (see note 1)

II

Kvl.7 hKvl.7, KCNA7, 19q13.3 mKvl.6, Kcna6, chromosome 7 [~] (For updates see OMIM 176268)

hKvl.7: (Human locus name KCNA7): The mouse clone MK4 sequence has been used as probe to show the location of the human isoform to be located on chromosome 19q13.3 173 (K. Kalman, unpublished). mKvl.7: (Mouse locus name Kcna7): Mouse chromosome 7 (in close proximity to the gene encoding mKv3.3; K. Kalman, unpublished; see VLC K Kv3-Shaw, entry 50) (compare/see also Kcna1rs2, this table).

K(v)I.8 hKvl.8, KCNA8, Ip mKvl.6, Kcna6, chromosome 3 [~] (For updates see OMIM 176269)

hKvl.8: (Human locus name KCNA8): Predicted to be on human chromosome Ip54. mK(v)I.8: (Mouse locus name Kcna8): Mouse chromosome 3, part of a cluster within a parologous t region (see Fig. 3).

Other laltered neurological phenotype' mutants mapping to regions close to known Kcn loci in mice

Comparative notes: Chronic locomotor hyperactivity is observed in several mouse mutants (see paragraphs below and descriptions of anorexia (anx) and quivering (qv) this table; see a1s0 54). Notably, such phenotypes were used to initially identify each of the potassium channel defects defined by, for example, Drosophila Shaker, Shab, Shaw, Sha1, Slo, eag and Hk. For a consolidated, crossreferenced listing of these and other known ion channel defects to specified or candidate mutant loci, see resources such as Online Inheritance in Man (OMIM).

Opisthotonus

48-18-04: The mouse chromosome 6 region encompassing Kcnca1 (Kvl.l), Kcnca5 (Kvl.5), Kcnca6 (Kvl.6) and Kcnca1rs1 (this table) also encompasses the recessive opt (Opisthotonus) mutation54,183. Homozygous opt mice are characterized by loss of motor co-ordination at approx post-natal day 12 and subsequently develop severe seizures, leading to death at approx. 3-4 weeks 183 (but see next paragraph).

Deafwaddler

48-18-05: The mouse chromosome 6 region encompassing opt (previous paragraph) also carries the recessive dfw (deafwaddler) mutation54,184. Homozygous dfw mice are characterized by deafness, head bobbing behaviour and walking with a hesitant 'wobbly' gait l84. A detailed mapping study185 has since established the gene order on the distal arm of

l_e_n_t_ry_48

_

Table 7. Continued Consensus/summary Further details (see note 2) (see note 1) Deafwaddler

chromosome 6 to be cen-opt-dfw-Rho (D6Mit44)Kcna1, Kcna5, Kcna6, suggesting that the neurological mutants opt and dfw (above) affect two different genes, neither of which is caused by a mutation in the clustered K+ channel genes.

Other crossreferences

48-18-06: For known chromosomal locations of vertebrate Shab, Shaw and Shal subfamily genes, see FactsBook fields 49-18, 50-18 and 51-18 respectively. 48-18-07: For details on ion channel genes linking to familial human long QT syndrome, see Chromosomal location under VLC K eag/elk/erg, 46-18. 48-18-08: For trisomy 12p syndrome, see this table,

above. 48-18-09: For spinocerebellar ataxia type 2 (SCA2)

mapping to human 12q23-q24, see ref. 186 and compare to human K+ channel loci, this table. 48-18-10: For benign familial neonatal convulsions

Supplementary note on expanded trinucleotide repeat motifs (of general significance, no relationship to Kv or other ion channel gene loci reported to date).

(epilepsy) linked to genetic markers on chromosome 20q, see ref. 187 and compare to human K+ channel loci, this table. 48-18-11: 'Expansion' of the trinucleotide repeat CAG encoding polyglutamine have been associated with five hereditary neurodegenerative diseases including Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), Machado-Joseph disease (SCA3), spinobulbar muscular atrophy (SBMA) and dentatorubal and pallidolyusian atrophy (DRPLA) (for minireview of

related references, see188 ). Each of these diseases involves a progressive loss of specific neuronal populations, and all are characterized by the phenomenon known as genetic anticipation, where symptoms appear at earlier ages and with greater severity in successive generations. Hypotheses relating expanded trinucleotide repeats to changes in signalling protein conformation have been discussed (ibid.).

Notes: 1. [?] symbol denotes possible conflict; [~] symbol denotes likely paralogous cluster. 2. This column list species equivalents (see also Table 8), notes on phenotypes (if known) and other cross-references. See also references to OMIM (Online Mendelian Inheritance in Man). OMIM entry 176260 presents a general discussion of voltage-gated K+ channels.

II

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e_n_try_4_8_

Table 8. Summary of mouse/human chromosomal syntenies or homologies used to infer or predict 'equivalence' of chromosomal segments containing Kv gene clusters. For further background, see refs165-167~169-172 (From 48-18-02) Affecting Kv localization

Mouse (known)

Kvl.l

Kcna1 on chromosome 6 Region homologous to human chromosome 12p (in paralogous cluster with Kcna5 and Kcna6) (location confirmed for KCNA5, see table 7) (see also ref. 189 in footnote)

Kvl.2

Kcna2 on chromosome 3 Between regions (in paralogous cluster) homologous to human chromosome lq and Ip (designated as 1p in54; see also Kvl.2 in Table 7 and Kv3.4, this table)

Kvl.4

Kcna4 on chromosome 2 Region homologous to human chromosome 11p

Kvl.5

Kcna50n chromosome 6 Region homologous to (in paralogous cluster) human chromosome 12p

Kvl.6

Kcna6 on chromosome 6 Region homologous to (in paralogous cluster) human chromosome 12p

Kvl.8

Kcna8 on chromosome 3 Region homologous to (in paralogous cluster) human chromosome Ip Kcnb1 on distal Region homologous to chromosome 2 human chromosome 20q Kcnc2 on chromosome 10 Region homologous to human chromosome 12q Kcnc4 on chromosome 3 Region known (human (in paralogous cluster) chromosome Ip21) Kcnd2 on chromosome 6 Region homologous to human chromosome 7q

Comparative note only Kv2.1, see entry 49 Comparative note only Kv3.2, see entry 50 Comparative note only Kv3.4, see entry 50 Comparative note only Kv3.4, see entry 50 Comparative note only Kv4.1, see entry 51 Comparative note only Kv4.1-related sequence see entry 51

Kcnd1 on proximal X chromosome Kcnd1rs on chromosome 3

Human (predicted)

Region homologous to the petite arm of human X Region homologous to human chromosome 1p

Note: Ref. 189 described yeast artificial chromosomes (YAC) propagating 1 Mb segments of human chromosome 12p13 with KCNA1, KCNA5, KCNA6 coclustered within a segment of approx. 300 kb size; these genes are organized 'head-to-tail' and are transcribed from the same DNA strand in the order KCNA6-KCNA1-KCNA5. Differential mRNA expression patterns of these genes (field 13) suggest they are transcribed independently of each other189.

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l_e_n_t_ry_48

_

ref. 168). Patterns of co-segregation with specific linked genes within interspecific backcross (IB) mapping panels (e.g. the >1300 gene panel used for m analyses of between C57BL/6J x M. spretus strains) have major applications in deduction of syntenic t and paralogous t regions between the murine and human genomes 165,166,169-172. For further technical background and references to mapping of cloned DNAs in the mouse, see Chromosomal location under Resource D - Diagnostic tests.

Encoding A tglobal' sequence alignment of Kvl subfamily members 48-19-01: An amino acid sequence alignment, representing the majority of Kvl subfamily proteins is shown in Fig. 4. This figure denotes several general features of Kvl sequences described in other fields, and references to this figure are given under the most pertinent fieldname. Note that individual nucleotide and/or amino acid sequences may be retrieved for local analysis using the accession numbers listed under Database listings, 48-53.

Gene organization The majority of vertebrate Kvl genes are intronless in their proteincoding regions 48-20-01: The protein-coding regions of most Kvl subfamily genes are uninterrupted in the genome (Le. are intronless 19 ) according to presently recognized splice site t consensus sequences. Notably, the Kvl.7 gene possesses an intron t in the region of its sequence encoding the SI-S2 extracellular loop52. The lack of introns in coding sequences of the vertebrate Kv genes contrasts with Drosophila Shaker, where multiple exonst spanning > 120 kb can be alternatively spliced t to generate at least five functional transcripts 190,191. Different Drosophila Shaker channels inactivate over different time courses (see references in special section on Drosophila Shaker under Related sources and Reviews, 48-56). Most (but not all) members of the vertebrate Shaker, Shab, Shaw and Shal-related K+ channel gene subfamilies mostly yield single mRNA species (see field 17), each of which exhibits a distinct pattern of tissue-specific expression. Kv subunit genes are generally single copyt within the genome (see Southerns, 48-25). Related genes arise principally by mutation-selection t acting on duplicated genes (for proposed mechanisms, see Chromosomal location, 48-18). See also the 'head-to-tail' arrangements described for multiple Kvl subfamily genes in mammalian genomes (ibid.) which can appear in clusters (e.g. the KCNA6-KCNA1-KCNA5 cluster on human chromosome 12p189 (described in footnote to Table 8).

Summary of intron sequences found in Kvl subfamily non-coding regions 48-20-02: Kvl.l/Kvl.2/Kvl.3: The detection of 'long transcripts' (ca. 80009500nt) from the Kvl.l, Kvl.2 and Kvl.3 genes (see Transcript size, 48-17) together with several shorter ones with the same probe suggest that splicing of primary transcripts from these genes occurs in vivo. To date, the majority

II

II

mKv1. 1(MK1) rKv1.1 (RBK1) hKv1.1 (HUK( I» xKv1. 1()(Sha1) mKv1. 2(MK2) rKv1.2(RCK5) hKv1.2(HUK( IV» dKv1.2 bKv1. 2( BGK5) xKv1.2(XSha2) mKv1.3(MK3) rKv1.3(RGK5) hKv1.3(HPCN3) mKv1.4 rKv1.4(RCK4) hKv1.4( HPCN2) bKv1.4(BAK4) rKv1. 5(KV1) hKv1.5(HPCN1 ) mKv1.6(MK6) rKv1.6(RCK2) hKv1.6(HBK2) APLK Shaker

mKv1.1(MK1 ) rKv1.1CP.&K1) hKv1. 1(HUK( I» x~v1.1(XSha1 )

~~~ j~:~~~)

hKv1.2(HUK(lV» dKv1.2 bKv1.2(BGK5) xKv1.2(XSha2) mKv1.3(MK3) rKv1.3(RGKS) hKv1.3(HPCN3) mKv1.4 rKv1.4(RCK4) hKv1.4(HPCN2) bKv1.4(BAK4) rKv1.5(KV1 ) hKv1.5(HPCN1 ) mKv1.6(MK6) rKv1.6(RCK2) hKv1.6(HBK2) APLK Shaker

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Figure 4. Amino acid sequences of Shaker-related Kv1 subfamily isoforms together with that of Drosophila Shaker. Reported ORF lengths in the Kv1 subfamily include Kvl.l: MBK1MK1/RBK1/RCK1/RK1: 485 aa. Kvl.2: MK2/BK2: 499 aa; RCK5: 498 aa. Kvl.3: hPCN3: 524 aa. MK3: 530 aa. Isolate KV3/RGK5/RCK3: 525 aa. Kvl.4: hPCN2/HK1: 653 aa. RCK4: 655 aa. Kvl.5: hk2: 605 aa. hPCN1: 613 aa. Isolate/clone KV1/RK4: 602 aa. HCK1: 584 aa. Kvl.6: HBK2: 529 aa. Isolate/clone KV2: 530 aa. RCK2: 500 aa. (Alignment kindly provided by George Gutman, University of California at Irvine.) (From 48-19-01)

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_

of these events have been related to the presence of introns in 5' untranslated (non-translated) regions t (designated as 5'-UTRs or 5'_NTRs)18,19 as described in Table 9. Alternative splicing t in untranslated regions could conceivably play a role in controlling tissue specificity or quantitative expression levels of Kv proteins. See also Kv3.1 (isolate KV4) and Kv3.3 under VLC K Kv3Shaw, entry 50.

Multiple polyadenylation signals in the Kvl.4 gene

48-20-07: Kv1.4: KCNA4 contains three conserved polyadenylation t signals in the 3' UTR. Although the 'precise' employment of these signals is unclear, their differential utilization may partly account for the different transcript sizes (2.4kb, 3.5kb, 4.5kb) detected in Northern t assays using Kv1.4 probes 175 (see also Transcript size, 48-17 and predicted relationship of transcript size to stability in paragraph 48-20-08).

Possible role of 3' UTR motifs in control of channel mRNA stability 48-20-08: Kv1.4: The 3' untranslated region of KCNA4 and the rat, bovine and human KCNA4/Kv1.4 cDNAs contain multiple ATTTA and ATTTG motifs 175 (e.g in rKv1.4 (RCK4) 5'-ATTTAATAG ATATAGGTCACAATTTAATCTTGGATTTA ATTAAA-3'). These sequences are reminiscent of conserved AU sequences from the 3' untranslated region of (cytokine) GMCSF mRNA that have been shown to 'destabilize' cytokine messages (via selective mRNA degradation) when present in 3' UTRs 193 . By analogy, message stability of K+ channels would be increased when the number of these repeats are minimized. In the case of the KCNA4 gene (see Table 9 and Transcript size, 48-17) the shortest detected messages (2.4 kb in length) are likely to contain no more than 400 nt of UTR (i.e. assuming the ",2 kb segment comprising the 654 aa coding region is intact). This implies that the shortest (2.4 kb) transcript would have the highest stability, because the ATTTA motifs would be excluded from the 2.4 kb transcript 175. In Xenopus oocytes, mKv1.4 equivalent amounts of 3.5 kb transcripts produce ",4 to 5fold larger currents than 4.5 kb mRNAs 192. Note: tUA-rich sequences' have also been shown to induce ttranslational blockade' independently from these effects on message stabili tY 94.

Comparative note: Alternative splicing in the Drosophila Shaker gene 48-20-09: For initial descriptions of alternative mRNA splicing producing K+ channel diversity from the Drosophila Shaker gene see refs 190,191,196-198. An immunological characterization of specific K+ channel components produced by alternative splicing from the Shaker locus has confirmed that different subtypes of A-channels are formed in different tissues 199: On immunoblots, the protein products from the locus are in the size range 65000-85000 Da with no smaller products being evident (compare Protein molecular weight (purified), 49-22). In agreement with in situ hybridization studies, immunocytochemical techniques reveal a 'non-uniform' distribution of Shaker products in the brain of the adult fly199.

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_nt_ry_4_8_

Table 9. Predicted introns in Kv1 subfamily non-coding regions (From 48-20-02) Kvl.l

Kvl.2

Kvl.4

Kvl.5

48-20-03: mKvl.l/MBKI genomic: Alternative splicing of a short intron in the 5' UTR of mKvl.l can give rise to both spliced and unspliced polyadenylated RNA 18 . The 5' intron (position -886 to -514 relative to the start codon of the protein product shown in Encoding, 48-19) includes three potential initiation codons conforming to the consensus sequence PNNATG (where P is A or G and N is any nucleotide). 48-20-04: mKvl.2/MK2 genomic: Comparison of mouse Kvl.2 genomic sequences with rat Kvl.2 cDNA sequences (see Methodological notes, this field) reveals (at least) two introns in the 5' UTR. According to the analysis of Chandy and Gutman (1994, see Related sources and reviews, 48-56), an intron closest to the 'authentic' translational start site (positions -607 to -136) contains six possible translational start sites producing ORFs ranging from 18 to 156 bp (two of which meet the preferred Kozak initiation sequence PNNATG, see above). Only the splice acceptor site t of the other (upstream) intron has been detected at position -920. 48-20-05: mKvl.4/genomic: The Kvl.4 basal promoter is GC-rich, contains three SPI repeats (CCGCCC, -65 to -35), lacks canonical TATAAA and GGCAATCT motifs, and has no apparent tissue specificity. One region enhances activity of this promoter192. Analysis of the mKvl.4 genomic sequence (KCNA4) has revealed a single large intron ("-I3.4kb) in its 5' UTR 175. A single exon contains the remaining "-10.8 kb of the 5' UTR, a large open reading frame t ("-12.0 kb encoding the 654aa mKvl.4 protein), and all of the known 3' UTR ("-11.1 kb). The 5' UTR contains eight sequences conforming to PNNATG (see above). The longest detectable transcript from this gene (4.5 kb, see Transcript size, 48-17) can be accounted for by the sequence of the genomic DNA, although the precise ends of this transcript have not been defined. Note: The shortest detectable Kv1.4 transcript (2.4 kb, ibid.) is predicted to contain (at most) 400bp of non-coding region and lacks any 3' ATTTA motifs (see paragraph 48-20-08). Furthermore, the majority of the 5' ATG motifs (ibid.) would not be present in a transcript of this size, and in consequence, the 2.4 kb transcript may be ex~ected to have both increased stability and translational efficiency l 5. 48-20-06: rKvl.5: The rKvl.5 gene lacks a canonical t TATA boxt , has several transcription start sites t, and the 5' non-coding sequence is intronless 111 . For details of the cAMP-response element (CRE) identified in the 5' UTR of rKv1.5, see Developmental regulation, 48-11.

Notes: 1. The presence of 'upstream' initiating codons (ATGs) or in 5' UTR sequences (see, for example, MBKI and mKv1.4 in Table) may inhibit translation of K+ channel and other cDNAs in heterologous cell expression systems (probably by interference with ribosome'scanning' processes for the'authentic' start site, the competing initiation sites 'delaying' authentic codon binding). 2. Comprehensive analysis of alternative splicing events generally requires direct comparison (alignment) of genomic t (chromosomal) sequences and all cDNA t sequences (for further background,. see Gene family and Gene organization under ILG K Ca, 27-05 and 27-20 respectively).

l_e_n_t_ry_4_8

_

Homologous isoforms Individual Kvl subfamily sequences are generally highly conserved between species 48-21-01: For a full listing of cDNA isolate names (i.e. clone names crossreferenced to species of origin) falling into the Kvl subfamily, see Cene family, 48-05. For further background to Kv channel interspecies identities and Kv gene evolution, see Domain conservation, 48-28 and Chromosomal location, 48-18.

Protein molecular weight (purified) Kv subunit M r values following in vitro versus in vivo translation 48-22-01: rKvl.4 j hKvl.3: Specific polyclonal antibodies raised to a synthetic peptide sequence (residues 13-37) from the N-terminal of rat Kvl.4 (antibody Kvl.4N) detects a full-length monomeric polypeptide of ",88000 Da when Kv 1.4 mRNA is translated in vitro and subjected to SDSPAGEt analysis 64 . On immunoblots of native rat brain membranes, the Kv 1.4N antibody precipitates a diffuse band of ",95000 Da. The 7 kDa difference between in vitro and in vivo translation products is probably due to additional post-translatory glycosylationt in the latter system (see Protein molecular weight (calc.), 48-23 and Sequence motifs, 48-24). Similarly, the apparent molecular mass of the immunoprecipitated type n channel from Jurkat cells (~ hKv 1.3) is approximately 65 000 Da (significantly greater than that of the 58 000 Da in vitro translated product, and suggestive of post-translational modification events)200. Specific immunoprecipitation in both native and cell-free preparations can be blocked by an excess of the Kvl.4 peptide immunogens but not by unrelated competitor pepides64 . For details of molecular weight variants of Kv1.1 during early biosynthesis and subunit assembly, see Protein interactions, 48-31. A 'very faint' band is detected at 97 kDa in cardiac atrial and ventricular preparations when an anti-Kvl.4 antibody (that reveals intense Kvl.4 expression in brain) is used at a 5- to la-fold higher concentration in Westernt blots l14 (see also Protein distribution, 48-15). Mrvalues for affinity-purified Kv channel complexes from native cells 48-22-02: Native Kv channel assemblies (e.g. those immunoprecipitated with high-affinity toxins) are octameric (04.84) complexes as first established by Oliver Dolly and co-workers (for details, see VLC K Kv-beta, entry 47). Protein assemblies such as the DMB K+ channel sialated glycoproteint complex (described under Blockers, 48-43) has a total molecular weight ",450 kDa and possesses toxin-binding subunits between 65 and 95 kDa. Rat brain DMB protein is composed of polypeptides of 80, 42 and 38 kDa, whereas in bovine brain peptides of 74, 42 and 38 kDa are found 201,202. The larger 80/74 kDa peptides generally form the toxin-binding subunits and the smaller subunits do not appear to be glycosylated (for significance, see Protein molecular weight (purified) under VLC K Kv-beta, 47-22). Comparative notes: 1. Kvl.l/RCKI expression in oocytes can be detected by immunoprecipitation as a 57 kDa polypeptide by SDS-PAGEt (representing

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Table 10. Calculated molecular weights (From 48-23-01) Kvl.l

MBK1: 56.4kDa. RBK1: 54.6kDa. RCK1: 56.4kDa (56379). RK1: 54.6kDa

Kvl.2

BK2: 56.7 kDa (56706). RCK5: 56.8 kDa (56 768).

Kvl.3

Isolate KV3: rv58.4 kDa (rv 55 kDa when translated in vitro). RCK3: 58.4 kDa (58397) (see domain structure model [PDTMj, Fig. 6). HK1: 73.2kDa (73211). RCK~: 73.4kDa (73398)

Kvl.4 Kvl.5

HK2: 66.6kDa (66640). hPCN1: 67.1 kDa (67097). Isolate KV1: rv66.6 kDa (rv 67 kDa when translated in vitro). RK4: rv66.6 kDa. HCK1: rv64.3 kDa

Kvl.6

HBK2: 58.9 kDa (58891). Isolate KV2: rv58.8 kDa (rv 56 kDa when translated in vitro)

functional channel subunit monomers that are N-glycosylated)203. 2. Kv1.3 purified from African green monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system has an estimated size of approx. 64 kDa by SDS-PAGE204 . By sucrose gradient sedimentation, purified Kv1.3 is approx. 270 kDa, consistent with it being a homotetrameric complex of 64 kDa subunits under these conditions 204 . Negative-staining electron microscopy reveals that Kv1.3 protein can form small crystalline domains consisting of tetramers (approx. dimensions 65 x 65 A) with the centre of each tetramer comprising a stained depression possibly representing the ion conduction pathway204.

Comparative note: Drosophila Shaker 48-22-03: Recombinant baculovirus t constructs expressing the Drosophila Shaker H4 cDNA induce a band migrating at approx. 75 kDa on SDS-PAGE gels stained with Coomassie blue205 . As indicated by the lack of requirement for radiolabelling, the 75 kDa species (confirmed to be Shaker protein by Western blotting l ) accounts for a 'substantial fraction' of the membrane protein in Shaker-infected Sf9 cells205 .

Protein molecular weight (calc.) 48-23-01: Voltage-gated K+ channel of mammalian brain are oligomers of glycosylated polypeptides of typical predicted molecular weight of 6595 kDa (monomeric). The molecular weight values quoted in Table 10 are generally derived from sum totals of constituent amino acids (i.e. from predicted amino acid sequences derived from cDNA sequences).

Sequence motifs 48-24-01: Entry cross-references to predicted 'functions' for a number of common sequence motifs described in the literature are listed in Table 11.

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Table 11. Cross-references for sequence motifs described in the Kv channel family (From 48-24-01) Motif, site or function

Described under

{E/L)TDV C-terminal motif

This field (Sequence motifs) and Fig. 7 under Protein interactions, 48-31

H5 (pore) motif (K+ channel signature sequence)

In Kv channels, see Selectivity, field 40 of Kventries. Variation in CNG channels, see entries ILG (CAT) cAMP, entry 21 and ILG CAT cGMP, entry 22. In calcium-activated K+ channels, see ILG K Ca, entry 27. Variation in eag-related channels, see VLG K eag/elk/erg, entry 46. Absence in minK channels, see VLK (K) minK, entry 54

HMMEEM~ALS motif (N-terminals of Kv2, Kv3, Kv4, not Kvl)

This field (Sequence motifs)

Intron/exon splice site motifs

Gene organization, field 20

IYLESCCQARY motif (N-terminals of Kv2, Kv3, Kv4, not Kvl)

This field (Sequence motifs)

N-Glycosylation motifs

This field (Sequence motifs)

NAB Kv assembly motifs

Protein interactions, field 31

O-Glycosylation motif (Kvl.6)

This field (Sequence motifs)

Phosphorylation motifs

Protein phosphorylation, 48-32

PNNATG motif (nucleotide)

Gene organization, 48-20

Proline-rich motif

Interacts with SH3 domains of Src (see Protein phosphorylation, 48-32)

Repetitive sequence motifs

This field (Sequence motifs)

Serine/cysteine motif in ball domain of A-type channels

Table 19 under Inactivation, 48-37; see also sequence alignments under VLG K Kv-beta, entry 47

Signal sequence motifs (absent)

This field (Sequence motifs)

Tl assembly motifs (domains A and B)

Protein interactions, field 31

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Table 11. Continued

Motif, site or function

Described under

Voltage-sensor motifs including: leucine heptad (zipper) motif conserved positively charged residues conserved negatively charged residues

Voltage sensitivity, field 42

YFFDR N-terminal motif

Protein interactions, 48-31

Other specific sites determined by structure/ function analysis

e.g. Other structural determinants contributing to Kv channel assembly, block, expression, inactivation, selectivity, voltage sensing and modulation; see crossreferences in Table 14 under Domain functions, 48-29

A number of other 'motifs' are described within the Kv1 subfamily sequence alignments in Fig. 4 under Encoding, 48-19, cross-references in Table 14 under Domain functions, 48-29 and listings in Resource G - Consensus sites and motifs. Phosphorylation motifs are described under Protein phosphorylation, 48-32.

C-terminal deletion/mutation including the conserved (E/L)TDV motif 48-24-02: hKv1.5: The C-terminal regions of Kv1 subfamily K+ channels show little conservation between isoforms (see Domain conservation, 48-28). An exception to this is the last four C-terminal residues {E/L)TDV, which are well-conserved from Drosophila to human. Deletions of 4, 16, and 57 Cterminal residues of hKv1.5 do not affect whole-cell current amplitude, midpoint of activation, degree of inactivation, or activation kinetics compared to wild-type hKvl.5 following heterologous expression in mouse L-cells 206 . Deletion experiments removing C-terminal residues of rKv1.1 channels have shown similar results 206 . Mutation of the C-terminal sequence (-ETDV) of Kv1.4 abolishes its binding to, and prevents its clustering with the membrane-associated (putative) guanylate kinases SAP97 and PSD-95 62 (see Subcellular locations, 48-16 and Fig. 7 under Protein interactions, 48-31).

Repetitive sequences in Kv subunits 48-24-03: Figure 4 (under Encoding, 48-19) shows several simple alanine (A), glutamine (Q), glycine (G), histidine (H) and lysine (K) repeats in several Kv protein-coding regions. This feature is most marked in the N-terminal of the Kv1.4 subfamily proteins which show rapid inactivation properties. In this

II

l_e_n_t_ry_4_8

_

Table 12. Sequence motifs specifically reported in Kvl subfamily proteins (see also Protein phosphorylation, 48-32) (From 48-24-04) Kvl.l

MBK1: In common with all known Kv channels, no N-terminal signal peptide sequence is apparent. RBK1: N-gly: Five potential Nglycosylation sites (Asn-X-Thr-Ser); glycosylation in vivo could potentially increase the Mr of RBK1 protein to values close to affinity-purified dendrotoxin receptor ('"'-'76kDa). RCK1: N-gly: A single N-glycosylation site at Asn207.

Kvl.2

RCK5: N-gly: N-glycosylation sites at Asn38, 207, 465, 480 and 490 conforming to NXT/S. Note: Most of these sites are at analogous positions in the RCK series proteins. BK2: N-gly: Five possible Nglycosylation sites t 15. t

Kvl.3

RCK3: N-gly: Two putative N-glycosylation sites at Asn59 and 229 (see [PDTM], Fig. 6). Isolate KV3: N-gly: Six potential Nglycosylation sites (one in the N-terminal region, two in the Sl-S2 loop, and three within the C-terminal domain32 .

Kvl.4

HK1: N-gly: Asn-X-Ser/Thr N-glycosylation t motif at aa 181 and 352 (Note: only the 352 position is extracellular). hPCN2: N-gly: See also Domain conservation, 48-28. RCK4: N-gly: Three putative Nglycosylation sites at Asn183, 354 and 644.

Kvl.S

HK2: N-gly: Putative N-glycosylation site ataa 124. hPCN1: N-gly: A single N-glycosylation motif conforming to Asn-Xaa-(Ser or Thr) at aa 121. A putative leucine zipper t sequence is conserved in the S4H4 region. hPCNl shows the Shaker-type (Arg or Lys-Xaa-Xaa) motif repeated seven times in the S4 transmembrane domain. Isolate KV1: Potential leucine zipper t sequence found between the S4 and S5 domains (Leu402-Leu430). N-gly: There are five potential sites for N-glycosylation (N-X-5/T); four are located in the N-termina1 region, and one (Asn290) is within the 51-S2 loop. HCK1: N-gly: Asn125 and 190.

Kvl.6

HBK2: This and other Kv1.6 isolates do not have an N-glycosylation site in the loop between the Sl and S2 transmembrane domains (as seen in the 10-30aa long SI-S21oop of other cloned K+ channels). The single N-glycosylation motif at Asn46 is intracellular, and therefore not N-glycosylated. Notably, the Sl-S2 bend is unusually long ('"'-'70 aa) and is rich in glycine/serine residues, reflecting a possible O-glycosylation t. Isolate KV2: Potential leucine zipper t (Leu356 to Leu384); N-gly: one (non-functional) N-glycosylation site in the N-terminal domain32 .

case, the repeats begin just after a stretch of positive charges thought to form part of the inactivating tbal}, domain, as underlined in Fig. 4 (ibid.). Note: Such simple repeated sequences may provide a 'spacer' function within channel tertiary structures and/or playa role in channel assembly.

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Reported N-glycosylation motifs Kvl-Kv4 subfamilies 48-24-04: With the exception of mKvl.6 and hKvl.6, an extracellular N-glycosylation motif is present between the 51 and 52 transmembrane domains in all Kvl subfamily sequences (as indicated by asterisks in Fig. 4 under Encoding, 48-19; actual motif sequences are underlined). This conservation is notable considering the relative divergence of the 51-52 loop regional sequences compared to the remainder of the 'hydrophobic core' domains. Further reported sites are indicated in Table 12. Note, however, that motifs located intracellularly or in transmembrane domains are not utilized (see Resource G - Consensus sites and motifs). Assembly and maturation of Shaker K+ channels 48-24-05: Voltage-dependent K+ channel a subunits generally assemble to form tetrameric membrane protein complexes. Glycosylation occurs at two positions in the 51-52 loop: Shaker protein is made as a partially glycosylated precursor (immature form) that is converted to a fully glycosylated product207. Further studies208 have indicated that the immature protein is coreglycosylated in the endoplasmic reticulum (ER) whereas the mature protein is further modified in the Golgi apparatus. Shaker channels appear to assemble on the ER, while maturation can be inhibited by blockers of ERto-Golgi transport by (i) treatment with brefeldin A or (ii) incubation at Is o C208 . Glycosylation of Shaker channels in oocytes occurs in two stages, generating an immature and a mature form of Shaker protein. Notably, however, glycosylation does not appear essential either for assembly of functional channels or for their transport to the cell surface 209 .

Southerns 48-25-01: Generally, Kvl-subfamily genes are single-copy in the genome (see Gene organization, 48-25).

STRUCTURE AND FUNCTIONS Because of the large volume of structure/function data available for Kv channels, general similarities derived fronl multiple sequence alignments of all Kv family proteins are tabulated under Domain conservation, 48-28 (Table 13) and Domain functions, 48-29 (Table 14). Further information on each subtopic is then cross-referenced to relevant entry/field name loci as part of these tabulations. In-press update: The study of K+ channel structure-function relationships has been considerably advanced by the availability of the Streptomyces lividans crystal structure494 .

Amino acid composition Composition of K+ channel protein domains based on hydropathicity analyses 48-26-01: In general terms, protein subunits encoded by all Kv gene subfamilies can be divided into three broad regions of approximately equal

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size with regard to amino acid composition: The 'N-terminal third' [PDTM] and 'e-terminal third' [PDTM] are hydrophilic, whereas the 'middle third' (the core region, [PDTM]) incorporates six stretches of hydrophobic t residues forming a-helical transmembrane domains (SI-S6, [PDTM])210 plus an H5 sequence t (also designated as the P-region or pore-lining domain) between SS and S6 segments (for 'mapping' of 'functions' to K+ channel subunit domains, see the [PDTM), Fig. 6, Domain arrangement, 48-27, Domain conservation, 48-28 and Domain functions, 48-29).

Domain arrangement Continual refinement of K+ channel structural models 48-27-01: Prior to the availability of high-resolution K+ channel molecular

structure information494, modelling approaches reliant on interpretation of structure-function t data (e.g. 'iterative' testing of structural predictions by means of site-directed mutagenesis t followed by functional analysis of mutant proteins) had been a dominant approach. These models are subject to continual refinement; hence, where available on internet resources, contemporary models of K+ channel protein structure or related datasets will be cross-referenced from entry update pages via the CSN (www.le.ac.uk/csn/). The impact of cysteine scanning mutagenesis (note 1) and toxin-channel interaction site mapping (note 2) are reviewed in ref. 211 . These experimental approaches have been the key for proposing that ion conduction pathways in K+ channels are short, and that permeating ions appear to traverse channel proteins via short canals ('canaliculi') of 10 A or less (note 3). Notes: 1. Cysteine scanning mutagenesis employs substitution of a natural channel amino acid residue by cysteine to make it reactive with sulphydryl-specific probes. 2. Toxin-channel interaction site mapping employs peptide neurotoxins of known three-dimensional structure to map contours of contact sites in channel pores. 3. 'Canaliculi' are implied from identification of channel residues in pore regions that are a few residues apart and yet are on opposite sides of the membrane; similar structures have been proposed for the movement of gating charges in voltage-sensing domains (as reviewed in ref. 211).

Oxidative induction of intersubunit disulphide bonds 48-27-02: Several voltage-activated K+ channel primary sequences contain

two conserved cysteine residues in putative transmembrane segments S2 and S6. A proposal that these cysteines form an intrasubunit disulphide bond210 was originally tested207 using site-directed mutagenesis followed by electrophysiological and biochemical analysis of the Shaker B K+ channel. Each Shaker B subunit contains seven cysteine residues, including the conserved residues Cys286 and Cys462 and a less conserved cysteine, Cys24S. Each cysteine in the Shaker B protein can be mutated individually without eliminating functional activity, suggesting that the protein does not contain an 'essential' disulphide bond for protein folding or the assembly of active channels (at least under most conditions, see below). Furthermore, limited proteolysis, electrophoresis and immunoblotting under reducing and non-reducing conditions indicate (i) that the two conserved

II

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residues Cys286 and Cys462 do not form a lnon-essential' disulphide bond with each other or with Cys24S 207. Subsequently, however, it was shown that disulphide bonds are formed between Shaker subunits in intact cells exposed to oxidizing conditions (detectable by the presence of four high molecular weight adducts on Shaker protein purified under non-reducing conditions). In these cases, intersubunit disulphide bond formation can be eliminated upon serine substitution of either Cys96 in the N-terminal or CysSOS in the C-terminal of the protein212 . These patterns of intersubunit disulphide bond formation (between Cys96 and CysSOS) thus provide important direct biochemical evidence for (i) the N- and C-terminal regions of adjacent subunits being proximal within native channel structures and (ii) Shaker K+ channels contain four pore-forming subunits :S-S bond adducts representing dimers, trimers, and two forms of tetramer (one linear, one circular containing one, two, three or four disulphide bonds, respectively)212.

Domain conservation Interspecies identities (see also Gene family, 48-05) 48-28-01: In general, the mammalian Shaker-related isoforms show a high degree of interspecies identity at the amino acid level. For example, as derived from the sequence alignments in Fig. 4 (under Encoding, 48-19) human, rat, mouse, dog and bovine Kv1.2 amino acid sequences show >98% identity over their entire protein-coding regions. Xenopus Kv1.2 shows >91 % identity to the mouse equivalent. Notably, rKv1.5 and mKv1.5 show greater divergence (870/0 overall identity, discounting an II-residue size difference); this diversity increases in the first 100 N-terminal residues «640/0 identity, as compared to ",,99.40/0 identity between rKv1.2 and hKv1.2 in the same segment). Relatively high amino acid conservation is seen in Shaker-related channels across large evolutionary distances (e.g. aligned sequences of Drosophila Shaker and rKv1.1 (RCK1) still exhibit ",,82 % identityl0). For further background to Kv channel gene evolution, see Chromosomal location, 48-18.

Regions showing high conservation across all Kv subfamily members 48-28-02: The 'core region' sequences comprising a-helical domains 51-56 (see [PDTMj, Fig. 6) show ",,400/0 amino acid identity between any two subfamilies with ",,700/0 identity within each subfamily, irrespective of species. Within this region, conservation is generally higher for amino acid residues facing the intracellular side of the membrane and on transmembrane domains S4, S5 and S6 10 .

K+ selectivity determinants (the P-region) is highly conserved across all K+ channel subfamilies 48-28-03: The P-region (approximately 22 residues, as indicated in the sequence alignment under Encoding, 48-19) can be identified in all known K+ selective channel subunit sequences, including all Kv subunit channels (entries 48 to 51 inclusive), calcium-activated K+ channel subunits (see ILG K Ca, entry 27), inward rectifier K+ channel subunits (see INR K subunits,

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1_ _

_

entry 33) and evolutionary antecedents (e.g. Drosophila K+ channels, see Related sources and reviews, 48-56 and bacteria such as E.coli, see Miscellaneous information, 48-55). Figure 5 shows an enlarged alignment of the P-region with additional features marked including consensus residues across presently known subunit sequences (see also Selectivity, 48-40). In press update: see also the Streptomyces lividans K+ channel crystal structure study494.

tlnternal repeat' arrangements in voltage-gated channels 48-28-04: In comparison to voltage-sensitive sodium and calcium channels which show four internal repeats (each forming six transmembrane segments, see VLC Ca, entry 42 and VLG Na, entry 55), voltage-gated potassium channels consist of one internal Irepeat', with the functional channel arising from the assembly of four monomers and in some demonstrated cases, association of Kv,8 subunits (for details see Predicted protein topography, 48-30, Protein interactions, 48-31, VLC K Kv-beta, entry 47 and VLG Key facts, entry 41). The 'single-repeat arrangement' of K+ channel subunit genes (domains Sl-S6 + HS loop, see VLG key facts, entry 41) probably formed the prototype for other members of the voltagegated channel family via gene duplication events.

Cross-species conservation - overview 48-28-05: Voltage-dependent potassium channels are generally highly conserved from Drosophila to vertebrate central nervous systems (see Gene family, 48-05). Mechanisms partly explaining known patterns of Kv channel gene loci in the murine and human genomes are outlined under Chromosomal location, 48-18. Hypotheses which invoke chromosomal or genomic duplication affecting primordial (ancient) forms of K+ channel genes (see ref.54 and Fig. 1, under Abstract/general description, 48-01) predict that murine Shaker-like genes on mouse chromosomes 3 and 6 have a Ipairwise' parology t (ibid.). This hypothesis is supported by the sequence relatedness of (i) Kcna1 (encoding mKvl.l on chromosome 6) with Kcna2 (encoding mKvl.2 on chromosome 3) and (ii) Kcnca5 (encoding mKvl.S on chromosome 6) with Kcnca8 (encoding mKvl.8 on chromosome 3). Intragenic recombination processes (i.e. exchanging limited stretches of protein-coding regions between highly related genes during homologous pairing t ) would be expected to have important roles in promoting 'functional specialization' of Kv channels during genome evolution. These processes (and larger scale rearrangements) may also act in non-coding regions to alter features such as cell-type expression range (i.e. susceptibility to cis-acting gene regulatory factors t including second messengers and transcriptional activator t /silencer t complexes), subcellular targeting (see Subcellular locations, 48-16) and intron/exon splice site choice (see Gene organization, 48-20).

Poorly conserved sequences in the extracellular loop 51-52 48-28-06: The loop between putative transmembrane domains Sl and S2 is the most variable within the core region of the RCK proteins22 . It has been shown for an Aplysia K+ channel APLK that the 'Sl-S2 loop' is glycosylated t ,

II

II [2] * symbols indicate identical amino acid residues to Drosophila Shaker (top line)

[1] Alignment of putative P-regions of Kv subunit family members (as indicated) against that of Drosophila Shaker (top line) illustrates the high degree of conservation within this region.

r---- P-region

~

[3] ~ symbols indicate conservative substitutions from Drosophila Shaker

Drosophila Shaker NSFFKSIPDAFWWA~

~1.1

E*H*S***********~****~**~*Y**TIG****

Drosophila Shab

~K*V***~*****~****

r~2.1

~K*****AS****~****

Drosophila Shaw

HND*N***LGL***~*****

r~3.1

H~H**N**IG**********

Drosophila Shal

A*K*T***A***~*****

r~4.1

K~*T***A***~*****

rK(v)S.l (:IK8) rK(v)6.1 (K13)

~L*****QS****~****

All Irv-series All K channels

F sIP

} - - See VLG K Kv2-Shab, entry 49

} - - See VLG K Kv3-Shaw, entry 50

} - - See VLG K Kv4-Shal, entry 51

} - - See VLG K Kvx, entry 52

SPE*T***~****~****

fWwa vt:MTTvGYCnn britt

G

~

[4] In consensus sequences, capitals indicate residues that are completely conserved; lower case residues ·predominate '

[5] The IGYG 1 motif (boxed, see text) is 'near-universally I conserved in potassium-selective channels. Notable exceptions are minK channels (see VLG K minK, entry 54) and mammalian K channels related to Drosophila eag (possessing a GFG sequence at the positions homologous to GYG - for details see VLG K eag, entry 46) ("D

Figure 5. Alignment of P (pore) regions from multiple K+ channel subtypes. (From 48-28-03)

=

~

,J::l..

00

1'--_e_n_t_ry_4_8

---'_

providing direct evidence for its extracellular location t (see Fig. 4 under Encoding, 48-19 and ref.213).

IIsoform-specific' properties 48-28-07: The N- and C-terminal regions may vary considerably, generating isoform-specific properties as for K+ channel variants in Drosophila. Further notable examples of domain conservation are given in Table 13.

Domain functions (predicted) Structure-function studies for Shaker-type channels 48-29-01: Special note: The extraordinary range and depth of studies

concerning structure-function analysis of known voltage-gated K+ channels makes it difficult to compile comprehensive summaries without extensive substructuring of the entry, hence the following is a limited overview. Protein 'domain functions' which show similarities across the entire superfamily of voltage-gated channels are also described in VLC key facts, entry 41, VLC Ca, entry 42 and VLC Na, entry 55. Table 14 lists studies which have employed mutational analysis to test hypotheses concerning various functional attributes of Shaker-related K+ channels, subject to the limitations described above. Where applicable, the list is indexed by defined structural loci, cross-referenced to supplementary information in specified paragraphs or entries. See also the additional conclusions from the Streptomyces lividans K+ channel crystal structure study494.

Predicted protein topography Predicted, observed and modelled voltage-gated K channel structures and assemblies 48-30-01: As introduced in VLC key facts, entry 41, protein domain models for Kv channel subunits are broadly similar to those described for voltagedependent Ca 2 + and Na+ channels (see VLC Ca, entry 42 and VLC Na, entry 55). Voltage-gated K+ channel genes represent a single repeat compared to the four repeats typical of the Ca2 + and Na+ channels. Early expectations that four interacting Kvo: subunits form the ion-conducting pathway have largely been borne out by (i) studies on native immunoprecipitated O:4{34 channel complexes (see entry 47 and Protein molecular weight (purified), 48-22); (ii) visualization of tetrameric subunit assemblies in plan view by electron microscopy228 (65 x 65A for Kv1.3)204 and (iii) production of homomultimeric t or heteromultimeric t aggregates with defined composition6,229 (see also ref.s and oxidative induction of intersubunit disulphide bonds under Domain arrangement, 48-27). Heteromultimeric associations contribute to the functional diversity of voltagegated potassium channels by generating distinct properties from the 'parent' homomultimers (e.g. RCK1/RMK2 heteromultimers 44 ). A predicted topography of the Kv1.3 pore region using NMR t -derived structures of scorpion toxins that interact with the pore and complementary toxin and channel mutagenic cycles has also appeared230 . This study predicted that

II

_ entry 48 - - - - - - - Table 13. Cross-references for patterns of domain conservation/divergence in Kv1 subfamily K+ channel proteins derived from amino acid sequence alignments (From 48-28-07) Domain, motif or defined region (ordered by position in primary sequence)

Summary, cross-reference for further details (see note 1)

Intracellular NVariability in Kv1 subfamily (see Fig. 4 under terminus (N) Encoding, 48-19). Kv1 subfamily, specific Role in rapid (N-type) inactivation (see Table 19 examples under Inactivation, 48-37). mKv1.1/MBK1: Residues 71-123 and 287-349 show >95 % identical residues to the Drosophila Shaker protein sequence; nucleotide sequences are divergent (see Isolation probe, 48-12). rKv1.2/BK2: A 136 residue hydrophilic region leading into the first hydrophobic domain contains 94 % identical residues with Drosophila Shaker. Extracellular loop SI/S2 Generally poorly conserved between different (eI2) isoforms, e.g. hKv1.6/HBK2: Currents expressed in oocytes 'closely resemble' those of RCK1/RCK3/ RCK5 despite large variation in the extracellular loop domain between domains 51 and 52. rKvl.6/clone KV2: Homologous to rKvl.5/clone KVl, differing predominantly in the N- and Cterminal regions and in the loop linking the 51 and 52 domains. In rKvl.6/clone KV2, this region is highly acidic, whereas in isolate KVl it is enriched in proline. Transmembrane domain S4 (S4)

Role in voltage-sensing (see Voltage sensitivity, 4842).

rKv1.2/BK2: (example) An uninterrupted sequence of 63 amino acids corresponding to the 54 sequence and the adjacent 55 domain are completely conserved between rKv1.2 and Drosophila Shaker. hKv1.4/hPCN2: (example) Shows conservation of the positively charged (Arg or Lys-Xaa-Xaa) 7-repeat motif of Shaker-type K+ channels in the 54 transmembrane segment. Pore-lining region H5 High conservation in Kv and other K+ -selective (P) and flanking regions subfamilies (see Fig. 4 under Encoding, 48-19). Variations within other ion channel families (see cross-references within Table 11 under Sequence motifs, 48-24).

IL-_e_n_t_ry_4_8

_

Table 13. Continued Transmembrane domain S6 (S6)

High conservation in Kvl subfamily; contributes to part of the pore (see Fig. 4 under Encoding, 48-19).

Hydrophobic core, spanning S1 to S6 (above rows)

Conservation in Kvl subfamily; in particular, all transmembrane regions are (relatively) highly conserved between different Kv1 channels and Drosophila Shaker (see Fig. 4 under Encoding, 48-19).

Intracellular Cterminus (C)

Variability in Kvl subfamily (see Fig. 4 under Encoding, 48-19). Involvement in 'C-type' inactivation (see Inactivation, 48-37). hKvl.5: Stably expressed 32 amino acid C-terminal extensions of hPCNl/Kvl.5 channels by an epitope-fusion tag t can be made without any significant effects on channel activity, monomeric structure or N-glycosylation214 .

Note: 1. Examples only, as taken from the literature; compare alignment in Fig. 4 under Encoding, 48-19 and the PDTM, Fig. 6. For evolutionary antecedents in flies and bacteria see Gene family, 48-05, Chromosomal location, 48-18 and Miscellaneous information, 48-55. Kvl.3 has a shallow vestibule (4-8A deep, ,,-,28-32 A outer dimension, ,,-,2834A at its base) at the external entrance to the pore. The pore (9-14A external entrance) tapers to a width of 4-5 A at a depth of ,,-,5-7 A from the vestibule. High-resolution NMR has also been used to determine structure of the N-terminal inactivation ('ball') domain peptides from rKvl.4 (this entry) and rKv3.4 (entry 50). The compact rKv3.4 ball folds to permit the formation of an intramolecular disulphide bridge structure and exposure of two phosphorylation sites. Both ball domains possess surface regions that are positively charged, hydrophobic and negatively charged. Other atomic scale structural models for Kv channels231-236 are subject to continual refinement, as supported by crystal structures (e.g. ref, 494). Where available on internet resources, contemporary models of K+ channel protein structure or related datasets will be cross-referenced from entry update pages via the CSN website (www.1e.ac.uk/csn/).

'Outer shell' plus 'inner core' structure 48-30-02: A typical monomeric protein domain topography model [PDTM] for Kv 1 subfamily channels is shown in Fig. 6. This broad'S I-S6 + H5' arrangement is preserved in other Kv subfamilies (Kv2, Kv3, Kv4 - entries 49 to 51). Molecular cloning of Kir channel family members led to the distinction of an tinner core structure' necessary for K+ -selective permeation where Ml, H5 and M2 in Kirs are 'equivalent' to the S5, H5 and S6 domains in Kv family channels (compare the [PDTM) in INR K [subunits}, entry 33). Thus Kir channels lack the touter shell' of SI, S2 and

_'--

e_n_try_4_8_

Table 14. Cross-references for structure/function studies in Kv1 subfamily channels based on analysis of defined mutant proteins (From 48-29-01) Function/feature/motif (alphabetical order)

Summary of subtopics and cross-references for further details (see note 1)

Assembly mechanisms

Listed under Protein interactions, 48-31, including: Expression-suppression by truncated channels Kv subfamily-specific assembly Channel assembly as an early event in channel biosynthesis Shaker K+ channels folding and assembling in the endoplasmic reticulum208 (see also review in Isacoff et al. (1993) under Related sources and reviews, 48-56).

Block, determinants

Listed under Blockers, 48-43, including: Clofilium215 Charybdotoxin receptor mappint16 Toxin receptor transfer17 External TEA +- and K+ -binding site Internal TEA+-binding site Dendrotoxin-binding site Mast cell degranulation peptide-binding site 4-Aminopyridine-binding site

Domain functions, comparative

Cross-referenced from Table 13 under Domain conservation, 48-28.

Expression, determinants

Listed under Protein interactions, 48-31, including: Expression-suppression by truncated channels218 Role of certain Kv{3 subunits in modifying expression properties (see also VLC K Kv-beta, entry 47 and Subcellular locations, 48-16).

Motifs, phosphorylation

Listed under Protein phosphorylation, 48-32.

Motifs, N-glycosylation

Listed under Sequence motifs, 48-24.

Motifs, leucine zipper

See Voltage sensitivity, 48-42.

Inactivation, determinants

Listed under Inactivation, 48-37, including: Fast, N-type inactivation; N-terminal'ball' domains 219 'Ball' domain receptor Slow, C-type inactivation Role of Kv{3 subunits in inactivation (see also VLC K Kv-beta, entry 47).

Intersubunit disulphide bond formation

Intersubunit S-S adducts from oxidized (neighbouring) cysteines in assembled native channels (see Domain arrangement, 48-27).

1'---_e_n_t_ry_4_8

_

Table 14. Continued Function/feature/motif (alphabetical order)

Summary of subtopics and cross-references for further details (see note 1)

In vivo mutant analysis

Biological functions inferred from phenotypes of (i) spontaneous or transgenic mutant constructs in Kv channel genes or (ii) transgenic Kv gene deletions ('knockouts') are summarized under Phenotypic expression, 48-14. Mutant gene locit which are associated with (candidates for) the incidence of transmissible phenotypes consistent with defects in Kv channel function are summarized in Chromosomal location, field 18 of relevant entries.

Selectivity, determinants

Listed under Selectivity, 48-40, including: Pore region/ion selectivity determinants (Pregion)22o-223; K+ channel signature sequence mutations224 .

Structural models

Contemporary K+ channel protein structure models or related datasets will be cross-referenced from entry update pages via the CSN (for details, see Feedback &. CSN access, entry 12).

Rectification properties

Listed under Current-voltage relation, 48-35, including: Modification of rectification by domain deletions225,226.

Voltage sensing

Listed under Voltage sensitivity, 48-42, including: S4 voltage sensor mutations Electrostatic interactions in S4 Recovery of 'non-expressible' S4 charge neutralization mutants227.

Voltage-activation

For 'conversion' of depolarization-activated, outwardly rectifying channels into hyperpolarization-activated, inwardly rectifying channels225, see Current-voltage relations (this table).

Zn2+modulation

Listed under Channel modulation, 48-44.

Note: 1. Examples only, as taken from the literature; compare alignment in Fig. 4 under Encoding, 48-19 and the PDTM, Fig. 6.

II

II I (a) Monomeric domains ('unfolded') I Voltage-sensor domain (one of four in assembled multimer)

l

I (b) Monomeric subunit, schematic ('folded')

N-glycosylation site (motif NXT/5) Approximate position of N-glycosylation site in all Kv subfamily members (except Kv1.6)

Extracellular

!I l ~ i!lI lt !~

54-55 loop forming 'receptor' for 'ball' domain

~1[s]]1[Sj

Intracellular

I

?1; )

+++

N H2

Amphipathic 'ball' domain at N-termlnus of fast-Inactivating Kv channel subumts

P-region

~

~

Part of 'ball' receptor

(H5)

Approximate position of consensus PKC site in all known Kv subunits Part of N-terminus , (contiguous With 51 ' \ / not shown; relatlvei y +++ long in Kv1.4; ~ : C-termlnal not shown)

..;

Approximate position of homophilic interaction domain (see Protein interactions, 48-31)

For locations of phosphomodulation motifs see Protein phosphorylation, 48·32; For regions affecting block. see Blockers. 48-43

Amphipathic 'ball' domain at N-terminus of fast-inactivating Kv channel subunits

K

::::::(-----1) :+-J:::':' ::.:::::.: Kv1 : + :::::::.

(c) Tetrameric a-subunit assembly, schematic, in plan see also Protein interactions, 48-31 and VLG K Kv-beta. entry 47

::.::::._'~:::::::

Channel symbol ('b

~

t"'t"

Figure 6. Protein domain topography model [PDTM] for a Kv series potassium channel

Q

subunit monomer. (From 48-30-01)

~ ~

00

1L.-_e_n_t_ry_4_8

_

Table 15. Summary of interactions characterized within Shaker-related channel domains and interactions between channels and other classes of protein. For earlier functional studies on incidence of Kv channel heteromultimer formation, see field text. (From 48-31-01) Interacting domains or discrete proteins

Description of interaction and cross-references

Kvl subfamily a subunits interacting with Kv{3 (accessory) subunits

48-31-02: Native a-DTx-sensitive Kv channels occur as tightly associated oligomeric structures in a stoichiometry t of four a to four {3 subunits (for details, see VLC K Kv-beta, entry 47). Although the large numbers of potential combinations of such a4{34 complexes have not been systematically described at the level of single neurones, they are likely to contain different a subunit and {3 subunit isoforms. The subfamily-specific binding site of K v{31 has been mapped to a region overlapping NABKvl, a N-terminal domain (this table, below); NAB Kv1 is essential for the KV{31-mediated inactivation, as well as a-a and a-{3 interactions 237. Further details on Kva/{3 interactions, physiological roles, modulatory properties, gene family relationships, and other features of Kv{3 subunits are also discussed in entry 47 under appropriate fields.

Kv{31-binding site

Kv1 subfamily a 48-31-03: Kvl.4: Selective protein interactions subunits interacting between the discs large (dIg) family of with SAP97 and PSD-95 membrane-associated guanylate kinases with a Kvl.4 channel C-terminal segment were originally detected following screening of a human brain eDNA-derived yeast two-hybrid t system library155. The key features of these domain interactions are illustrated in Fig. 7 (see also chapsyn-ll0 under Channel density, 48-09). The PDZ 1 +2 structural module

48-31-04: The PDZ domains of the human dIg product (a tumour suppressor t ) are organized into two conformationally stable modules, one consisting of PDZ domains '1 + 2' which binds K+ channels, and the other (PDZ3) corresponding to the third PDZ domain (see Fig. 7). The purified PDZ(1 + 2) module, but not the PDZ3 domain can also specifically bind ATp238 (see also Subcellular locations, 48-16 and Sequence motifs,

See also legend to Fig. 7 48-24). a-Dendrotoxin

a-DTx complexing with Kv subunits, see Protein distribution, 48-15.

III

_~

e_n_try_4_8_

Table 15. Continued Interacting domains or discrete proteins

Description of interaction and cross-references

48-31-05: Associations of different N-terminal domains of Kva subunits are strictly 'subfamily specific' (see Domain functions, 48-29). As shown in Fig. 8, the amino acid sequences of known Shaker subfamily genes from different vertebrate species (short names at left, defined in Gene family, 48-05) and the corresponding region of Drosophila Shaker B (ShB) channels show high sequence conservation in this region. The hydrophilic Nterminal domains of Shaker Band RCKI expressed as bacterial fusion proteins t show homophilic binding visualized by Western blots t of crude protein239. Essentially, this region determines Designation of molecular determinants subunit compatibility for co-assembly. of Kv-subfamily-specific Subsequently, conserved structural motifs in each of the four subfamilies (following detailed subunit associations deletion analysis) have been named NABKvl, NABKv2, NABKv3 and NABKv4240. Note, however, NAB domain some studies242l'243 have found nomenclature electrophysiolog.ical data consistent with heteromultimeric assemblies (i.e. producing 'novel currents') following injection of Xenopus oocytes with a 1: 1 mixture of mRNAs encoding channels from distinct subfamilies (e.g. NGK1/Kvl.2 and NGK2/Kv3.1a)242-244.

N-terminal homophilic interaction domains Selected independent studies defining tetrameric assembly domains of Kva subunits

Tl domain nomenclature

Tl subdomains

48-31-06: In independent studies which assayed post-translational processing and assembly of Aplysia AKOI (Akvl.la) K+ channel subunit proteins through deletion mutagenesis t, only deletions retaining the SI domain [PDTM] (Fig. 6) were inserted into the membrane213 . By means of these deletion analyses, subunit assembly has been shown to be 'critically driven' by a conserved sequence in the N-terminal cytoplasmic region, designated the tetramerization 1 or Tl assembly domain213l'245. Subsequent studies determined that if a subunit protein was to heteromultimerize with a Shaker subunit protein, two regions within the Tl domain, Tl subdomains A and B, must be of the Shaker subtype245 . Moreover, incompatibility of a Shaw A region for assembly with a Shaker protein was shown to depend upon the composition of a 30 amino acid conserved sequence in the A region245 .

"---e_n_t_ry_4_8

--'_

Table 15. Continued Interacting domains or discrete proteins

Description of interaction and cross-references Functional Shaker AKvl.la channels are not formed when a glycine is substituted for a serine in the seventh position of subdomain A, indicating a critical role for the serine in wild-type channel assembly245.

Stable, selftetramerization of Tl domain in isolation

48-31-07: Purified T 1 domain self-assembles to form a highly stable tetramer, most probably a closed ring structure246i no separate dimeric or trimeric components can be observed following the assembly process.

Evidence for 'core' assembly domains

48-31-08: Note: A deletion mutant of Kvl.3lacking the first 141 amino acids (Kvl.3, tetramerization domain deleted, Tl-) can form functional channels. A further deletion analytical study has suggested that additional association sites in the central core of Kvl.3 can also mediate oligomerization247. 48-31-09: Deletion of 255 amino acids in the N-

terminal domain of hKvl.4 prevents the formation of hybrid channels within the subfamily but has no effect on homomultimerization or voltagedependent gating. Co-expression of N-terminal deletion mutants of hKvl.4 and Kv2.l result in the formation of functional hybrid channels, suggesting that the N -terminus serves as a recognition site necessary for hetero- but not homomultimeric channel assembly within a subfamily248. Role of Sl segment in assembly

48-31-10: Note: Data maintaining an important role for the 51 segment in the co-assembly of homo- and heterotetrameric K+ channels has been presented, together with a discussion/erratum249 .

Early events in Kv channel biosynthesis and subunit assembly

48-31-11: When Kvl.l and Kvl.4 are co-translated in vitro, isoform-specific antisera co-purifies both proteins even at early time points, suggesting rapid subunit assembly78. Kv subunits belonging to a different subfamily (Kv2.1, see VLC K Kv2-Shab, entry 49) do not assemble with Kvl.l or Kvl.4 using similar co-translation assays. Immune purification with Kv1.1 antisera of surface channels on mouse L-cells transfected with Kvl.l eDNA (see Channel density, 48-09) identify a two-species 57 kDa and 59 kDa doublet band as analysed by SDS-PAGEt (which is absent in precipitates from

Cotranslational assays

II

_L...--

e_n_try_4_8----J

Table 15. Continued Interacting domains or discrete proteins

Lack of effect of Nglycosylation on assembly

II

Description of interaction and cross-references sham-transfected cells). Pulse-chase t metabolic labelling with [35 S]3-Met supported the interpretation that the (precursor) 57 kDa species gave rise to the 59 kDa (product) protein within several minutes of initiation synthesis. At longer chase t times, a 57 kDa species reappeared, - indicating both the early precursor and a mature protein had identical electrophoretic mobilities t. Notably, mutation of the consensus extracellular glycosylation t site (N207, see alignment under Encoding, 48-19 and Sequence motifs, 48-24) yielded two proteins at steady state: (i) a 55 kDa core peptide and a 57 kDa species. Loss of the ability to N-glycosylate at N207 had little effect on channel synthesis, turnover, or function. In summary, these and other results 78 suggested (i) heteromeric assembly of Shaker-like channels is co-translational, and (ii) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.

Subcellular targeting dependent upon Kv protein interactions

48-31-12: Differential 'sorting' of Kv1.2 in different cell types and subcellular locations has been hypothesized 158 to occur following its association with varying K+ channel subunits, implying that Kv 1.2 may participate in distinct heteromultimeric K+ channels within different subcellular domains (see Subcellular locations, 48-16).

Co-assembly with dominant negative mutants

48-31-13: Some studies250 have shown that, at least in heterologous systems like the Xenopus oocyte, co-expression of wild-type and dominant negative mutant Kv1.1 subunits mainly results in loss-ofactive channels rather than producing channels with altered conductance25o .

Comparative note: Common co-existence of multiple current systems in single Drosophila cells

48-31-14: In Drosophila, Shaker, Shal, Shab and Shaw express independent K+ current systems, and a 'molecular barrier' to heteropolymerization is present 9 . Co-expression of all four K+ channel products does not alter their individual properties. The ability to express multiple, independent 'Acurrent' types together with multiple, independent delayed rectifier types is thus common to both lower and higher eukaryotic cells 9 .

_ _ _ry_4_8 en t

_

L...--

(a)

Kv1.4 C-terminal bait See note 3 and interaction of SH3 domains with proline-rich

.,'.,'

(b)

PSD~5 ~SAP9~~~~~~~~-m~~ld/l:i:i:i:J:i~13_~ I :

I I

I I

I I

I I

I I

I aa 224·405

I

I

I

I

I

I I

aa 736-914

I

hdlg Guanylate kinase domain (puatatlve)

shaded box alternative splice variants of hdlg

Note: A similar domain alignment can be made for SAP-97 and other PDZ-containing famify members

(c)

I

Kv1.4 56

I

Kv1.4 C-terminal

_

intermediate deletions - - - - - - - - - - - - - -

In vitro binding of PSD-95

Yeast 2hybrid assay

+

+

+

+

+

+

CSNAKAVETDV 11

terminal aa - 'sufficient'

TOV 3

terminal aa • 'essential'

+

Figure 7. Clustering of Kvl.4 channels via C-terminal interaction with PDZ domains within PSD-95 and hdlg, membrane-associated putative guanylate kinases. (a) Using Kvl.4 C-terminal as Ibait', screening of a human brain cDNA-derived yeast two-hybrid library identified selective protein interactions with several overlapping clones. [b] Kvl.4C-interacting proteins included PSD-95 and hdlg (== SAP97, human homologue of the Drosophila discs large), both members of a membrane-associated guanylate kinase family, shown aligned here. By two-hybrid assay, binding affinity to Kvl.4C was determined to be in the order PDZl + PDZ2 > PDZ2 alone » PDZl alone. PDZ3 domain binding was undetectable. [c] Progressive deletion analysis revealed that the C-terminalll residues of Kv1.4C (645655) are sufficient, and the last four amino acids are essential for PSD-95 interaction. The C-terminal three amino acids of Kv1.4 (TDV) is highlyconserved in the Kv1 but not Kv2, Kv3 or Kv4 subfamilies (see Encoding, 48-19). Notes: 1. PDZ domains were named from the conjunction of membrane-associated proteins they were first described in: fSD-95, 12iscslarge, the tight junction proteins ZO-l and syntrophin. 2. Mutations of Drosophila Discs large result in profound disorganization of synaptic structure, thus supporting a central role for the PDZ-containing families of

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_8_

S3 segments, which may function as an electrostatic shield between the hydrophobic membrane interior and the. highly charged S4 voltage-sensor sequence.

Protein interactions See also fields of VLG K Kv-beta, entry 47.

Multimeric assemblies and subcellular targetting of Kv channel subunits 48-31-01: A large number of protein-protein and multiprotein functional interactions have been studied with respect to Shaker channel and Kvl family members. These studies have unveiled structural bases for homomultimerism and heteromultimerism, showing their dependence upon interactions within Kv channel molecular assemblies. These studies have also shown that targeting of channels within the cell can critically depend upon interactions between Kv channel domains and other classes of protein (see Subcellular locations, 48-16). Table 15 summarizes some key findings related to intra- and intersubunit interactions which have included references to Shaker-related channels.

Incidence and properties of heteromultimeric channels 48-31-15: The formation of hetero-oligomeric channels containing multiple Kvo: subunit and Kvj1 subunit components (see VLC K Kv-beta, entry 47) has been proposed as an important mechanism for generating functional diversity among voltage-gated K+ channels in situ 132,146,153. Formation of heteromultimeric K+ channels by eDNA co-transfection t and cRNA t coinjection has been demonstrated experimentally for Drosophila 253 and mammalian254,255 Shaker-related proteins. Studies of Shaker channels in Drosophila photoreceptor cells suggests that different members of the Kvl subfamily assemble heteromultimeric channels in ViV0 145 . The generation of channel diversity in vertebrates by this mechanism appears to be restricted (see VLC key facts, entry 41 and Domain arrangement under VLC K Kv3-

Shaw, 50-27).

Properties of heteromultimers compared to constituent homomultimers (examples) 48-31-16: RCKl!RCK4: Heterologously expressed RCKI and RCK4 subunits can co-assemble to form RCK1,4 channels with 'intermediate' properties: Currents mediated by RCKI channels (IKv 1.1) do not inactivate in the

proteins in clustering (see Channel density~ 48-09). 3. The SH3 domain (Src homology domain 3) has been shown to bind to a proline-rich motif in Kv1.5 - see Protein phosphorylation, 48-32. 4. The ESDV motif is found at the C-terminus of the NMDA receptor subunits NR2A and NR2B (see entry 08, Vol. I); C-terminal baits of these proteins (116 aa/122 aa respectively) also bind multiple members of the PSD-95 family. (Figures based on data in Kim et al. (1995) Nature 378: 85-88.) (From 48-31-03)

II

(l)

=

t"1"

~ 90 I

ShB Ak01 XSha2 RCK1 RCK2 RCK3

RCK4 RCK5 Kv1 HBK1

100 I

110 I

120 I

130 I

140 I

150 I

160 I

170 I

180 I

190 I

PQHFEPI PRDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGF NGMGV-GSDYDCS-----------------K-----------N-QK-N--Y---N F EN_FERY_------DSYDPEP--EC-------I---------K--S---E------KK-M------N F I R EE_MEI ---SYPRQADHD--EC-------I---------K--A---H----N-KK-M------N M EE_ME__Y_---EFQEAEGGGGCCSS--L---I------------SL---------G--V-F------------N I_M R__Q E_LAA C LPPAL-AAGEQ-C-------I---------K--C---E------K--M--------N L I I_I R__Q-_EE_ME__y _ GGGGYSSVRyS-C----------------MK--A---E------EK-TQ-----------__N K F_I_T__V Q-_EE_LL__y _ --DTYDPEA--EC-------I---------K--A---E------KK-M--------N I R EE_MEM y EEDQA-QDAGSLHHQ--L--I---------G--A---N-------K--R-----N G S AD__R_-Q-__E_MER ---

SYPRQADH---EC-------I---------K--A---N----N-KK-M--------------D-------------------F------M-----------EE-ME-P-----hPCN1 TVEDQALGTASLHHC--H--I---------G--A---N-------K-LP-------------N-----G-----------------S----AD--R--Q---E-MER------hPCN2 GGGGYSSVRYS-S----------------MK--A---E------EK-TQ-------------N------------------------F-I-T--V---Q--EE-LL-------hPCN3 -PSLPAAGEQDCCG------I--I------K--C---E------K--M-----V--------N------------------------I-I-----R--Q--EE-ME----- _ DFPEAGGGGGCCSS--L---I----------S-SL---------G--V-F------------N--------------------------I-L---R--Q---E-LAA------C HBK2 DRK1

RRVRLNVGGLAREVLWRTLDRLPRTRLGK--DC-TB -S-- QVCDDYSLE-

~

00

196 168 (77) 134 (78)

137(n)

140 (73) 154 (75) 278 (72)

133 (78) 210 (72) 176 (n) 199 (70) 275 (74) 251 (74) 140 (72)

-------RPGA-TS--NF-RT ---BMMEEMCALS--Q-LDYWGIDEIYLESCCQARYB 134(19)

Figure 8. Initial identification of a structural element for homophilic interaction in Shaker-subfamily K+ channels. The homophilic interaction domain was originally defined as a 114 amino acid fragment in the N-terminal (aa 83-196 in the ShE sequence as shown). This sequence is shared by all known Drosophila Shaker alternative splice variants 190?191?197?251 and is highly conserved (> 70% identity) within the Kv1-Shaker-related subunit gene subfamily. Included for comparison in the alignment are the Shaker-related K+ channel gene AK01a from Aplysia252 and the rat Shah-subfamily gene DRK1 (see VLC K Kv2). Dashes (- - - -) indicate runs of amino acids identical to the ShE sequence at equivalent positions. The position of the last amino acid is given by the number on the right? with percentage identities to the ShE sequence in parentheses. The demonstration of a subunit compatibility region is consistent with the ability of ShE and RCK1 (for example) to co-assemble and form heteromultimeric channels253 in contrast to subunits from different gene subfamilies (cf. DRK1 sequence from subfamily Kv2.1 in alignment). (Alignment from Li et a1. (1992) Science 257: 1225-1230.) (From 48-31-05)

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millisecond time range and are sensitive to DTx and TEA. Currents mediated by RCK4 channels (JKv 1.4 ) inactivate rapidly, but are 'practically insensitive' to DTx and TEA. The RCK1,4 channels inherit properties from both RCK1 and RCK4 subunits mediating transient currents that are sensitive to DTx and TEA. The RCK1,4 channels have a single-channel conductance that resembles RCK1 channels but their gating resembles RCK4 channels255 (see also the heteromultimeric channel RCK1,4 under Current-voltage relation, 48-35).

tExpression suppression' by interaction of functional with truncated channel proteins 48-31-17: Certain deletion mutants of Kv1.3 have been shown to specifically suppress Kv1.3 currents (arising from full-length sequences) following their heterologous co-expression218 . Suppression of 'full-length' Kv1.3 currents requires the truncated Kv1.3 sequence to contain both the N-terminal and 81 domains. In general, N-terminal-truncated DNA sequences from a given K+ channel subfamily can suppress expression of members of (only) the same subfamilr18. Deletion of the first 141 amino acids of wild-type Kv1.3 yields a current identical to that of full-length Kv1.3, but it cannot be suppressed by a truncated Kv1.3 containing the N-terminus and Sl. Furthermore, suppression of native cell (endogenous) currents appears conditional (e.g. constitutively expressed K+ currents in Jurkat or GH3 cell lines are not suppressible; upregulated Shaker-like K+ currents in GH3 cells are suppressible)218.

Protein phosphorylation General perspective on phosphomodulation of K+ channel activities 48-32-01: Phosphorylation of K+ channel Q subunits (and/or their accessory protein components) can alter characteristics such as channel current amplitude, voltage-dependence of gating and the kinetics of activation. Phosphorylation of K+ channels is thus an important general mechanism for modulating calcium entry, action potential firing patterns (threshold, frequency, height, width) and 'effector' responses coupling membrane excitability to secretion, muscular contraction or gene transcription (see Phenotypic expression, 48-14). Activation of neurotransmitter receptors commonly alters excitability and synaptic efficacyt by generating intracellular second messengers t, with several second messengers acting through protein kinaset and phosphataset proteins that alter properties of K+ channels. Specific examples of Kv1 subfamily phosphomodulation are described in the paragraphs below and in Table 16. For further examples and general perspectives, see the review refs256-259, JLG key facts, entry 14, Resource A - G protein-linked receptors and supplementary notes under Receptor/transducer interactions, 48-49. Comparative note: The location of sites for phosphomodulation motifs may vary between studies of the same gene, reflecting slight differences in residue numbering or specification of motif Iconsensus'. In general, 'functional' motifs are highly conserved across species in multiple amino acid sequence alignments; some motif positions are conserved within subfamilies or even families (see following paragraphs).

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Table 16. Potential phosphorylation motifs and regulatory mechanisms applicable to Kv1 gene subfamily members (for abbreviations see note 1) (From 48-32-03) Kvl.l

48-32-05: mKvl.l/MBKl/rKvl.lRBKl: PKA: Motif at aa 443-446 (cAMP-dependent protein kinase).

'Basal' phosphorylation state - multiple effects of prolonged cAMP analogues 48-32-06: rKvl.l/RCKl: PKA: Ser445; one motif (Ser322) is extracellular and therefore non-functional. RCKI is a substrate for in vitro phosphorylation by the catalytic subunit of protein kinase A 203; Kv1.1 appears to be partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated upon treatment for a short time with a cAMP analogue203 . Site-directed mutagenesis studies demonstrate that phosphorylation of a single site on the C-terminus of Kvl.l can 'fully account' for these phosphorylations 2 0 3 . Further studies 160 showed that although treatment for a short time with various cAMP analogues do not markedly affect the channel function in oocytes, prolonged treatment (lO-16h) with the membrane-permeant cAMP analogue S-{p)-8-Br-cAMPS (8-bromoadenosine 3',5'-cyclic monophosphorothioate) enhances current amplitude through (i) wild-type Kvl.l channels and (ii) mutant Kvl.l channels that cannot be phosphorylated by pkA activation. This enhancement can be inhibited in the presence of the membrane-permeant protein kinase A inhibitor R-(p)-8-Br-cAMPS. These and other experiments support the interpretation that prolonged treatment with S-(p)-8-Br-cAMPS regulates RCKI function via two mechanisms: (i) a pathway leading to enhanced channel synthesis (inhibited by cycloheximide) and (ii) a pathway involving channel phosphorylation that directs channels to the plasma membrane 160 (see also Subcellular locations, 48-16). mKv1.1: PKA: Long-term effects of PKA on mKv1.1 channels have been studied using Chinese hamster ovary (CHO) cell lines stablyt transfected with (i) mKvl.l alone and (ii) co-transfected with both mKv1.l and a plasmid construct expressing a dominant negative t mutation in the regulatory subunit of PKA (PKA-unresponsive, inducing a chronic reduction in the basal PKA activity, following binding to endogenous catalytic subunits of PKA)l09. In the cell lines expressing mutant PKA regulatory subunit (condition ii, reducing basal PKA activity) mKvl.l current density is 3.4-fold higher under condition ii, although current kinetics are unaltered. RNAase protection t assays indicate that levels mKvl.l RNA are marginally increased (by approx. 2-fold) under condition ii while protein levels are increased by approx. 3-fold. These and other results were taken to suggest that PKA can regulate mKvl.l channel expression by changing steady-state levels of RNA and by other post-transcriptional mechanisms 109.

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Table 16. Continued Kvl.2

48-32-07: rKvl.2/RCK5: An N-terminal mutation (Thr46Val) eliminates stimulatory effects of injected PKA (see below); motif at aa Ser448.

Mechanism of PKA stimulation of Kvl.2 in oocytes 48-32-08: Kvl.2: PKA: Activation of PKA by (i) application of isoproterenol to oocytes expressing a ,B-adrenergic receptor or (ii) direct injection of the catalytic subunit of PKA induces an increase in Kvl.2 current261 . The effect has been localized to phosphorylation at an N-terminal residue (Thr46, see above); single-channel recordings show that of three comnductance levels of Kvl.2, PKA modulation increases the time spent in the higher conductance states (converting previously 'silent' channel proteins to activate on depolarization).

Tyrosine phosphorylation 'downstream' of 7-TD receptor activation 48-32-09: rKvl.2/RAK: TyrK/PKC: Activation of Ml muscarinic acetylcholine receptors in Xenopus oocytes potently and acutely suppresses co-expressed Kvl.2 current amplitude through a pathway involving [Ca2 +Ji elevation, phospholipase C activation, and direct tyrosine phosphorylation of the channel262 (see note 2). N-terminal Tyr132Phe mutant channels display qualitatively less suppression than wild-type Kvl.2. Further analysis of neuroblastoma cells showed that a similar tyrosine kinase-dependent pathway links endogenous G protein-coupled receptors to suppression of the RAK channel native to these cells262 . The suppression effect of Kvl.2 could be mimicked by activation of PKC. Note: Identification of a novel tyrosine kinase (PYK2) was followed by a direct demonstration of Kvl.2 phosphorylation and rapid suppression of current263 . Notably the PYK2 kinase is activated either by PKC or by [Ca2+]i elevation (see above) and also regulates MAP kinase t functions.

Comparative note on native channel complexes usually containing Kvl.2 (see 48-22) 48-32-10: Patch-clamp analysis of liposomes containing dendrotoxin-binding proteins have been reported to yield K+ channels (Mr rv80 kDa, Shaker-like) whose activity is enhanced by (i) cAMP-dependent protein kinase (see Activation, 48-33) and by (ii) and an 'endogenous kinase' that co-purifies with the channel complex (Mr rv38 kDa, which was not phosphorylated itself in this study)264. Note: This is similar in molecular weight to a Kv,B subunit, see Protein molecular weight (purified), 48-22; no kinase activity has since been reported for Kv,B subunits, but they have been determined to act as protein kinase substrates (see Protein phosphorylation under VLC K Kv-beta, 47-32).

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en t

Table 16. Continued Kvl.3

(Cross-modulation' of Kvl.3 by different protein kinases

48-32-11: hKvl.3: PKA/PKC: Phosphoamino acid analysis t of the 32P-metabolically labelled Jurkat Tcell type n channel (~hKvl.3) has revealed exclusive phosphorylation of serine residues20o . In vitro phosphorylation studies have shown that the Jurkat type n channel can act as a substrate for both PKA and PKC. Note: PKA also phosphorylates the 40 kDa (,8 subunit) protein which co-immunoprecipitates with the type n channel20o, suggesting that Kv,8 forms part of a regulatory mechanism for Kvl.3 in vivo (see VLC K Kv-beta, 47-22 and 47-32 respectively). 48-32-12: HKvl.3/HLK3; rKvl.3/RCK3: PKA/PKC: Application of 5-HT/cloned 5-HT2 receptor suppresses Kvl.3 265 (for further details see Receptor/transducer interactions, 48-49). Suggestions of 'cross-modulation' by PKA and PKC have appeared266 : Phorbol ester and PKA suppression267 are reversed by PKC inhibitors (H7, staurosporine, polymixin Band anti-PKC antibody). AlkPhos: Application of alkaline phosphatase t (via the patch pipette) increases n type channel conductance under basal conditions and reverses the inhibition produced by PKA266 . 48-32-13: mKvl.3: PKA/PKC: In oocytes co-expressing the mouse 5-HT 1c receptor and mKvl.3 channel, addition of 5-HT (serotonin, 100nM) causes a complete and sustained suppression of Kvl.3 currents in approx. 20min268 . The 5-HT-mediated suppression of Kv1.3 currents proceeds via activation of a pertussis toxin-sensitive G protein and a subsequent rise in intracellular Ca2+, but Ca2+ does not directly block the channel. The mechanism of this suppression is not clear - deletion of the first 146 amino acids from the N -terminal, PKC, calmodulin or phosphatase inhibition do not alter the effect268 . Comparative note: 5-HT has no effect on mKv3.1 currents when co-expressed with 5-HT 1c recepto~68.

C-type inactivation modulated by phosphorylation 48-32-14: There is evidence that the rate of C-type inactivation of Kvl.3 is markedly slowed by simultaneous mutation of three putative phosphorylation sites 269 (see Inactivation, 48-37).

Kvl.3 as a potential regulator in T lymphocyte apoptosis 48-32-15: rKvl.3/RCK3: TyrK: Activation of the Jurkat T lymphocyte Fas receptor associated with induction of apoptosis inhibits Kvl.3 current in Tcells. Inhibition of Kvl.3 current has been correlated with tyrosine phosphorylation of immunoprecipitated and blotted Kvl.3 (see note 1). The TyrK inhibitor herbimycin A (which prevents Fas-induced apoptosis) and p56(lck) tyrosine kinase-minus Jurkat cells abolish Kvl.3 inhibition and phosphorylation by anti-Fas antibody (restoration of the p56(lck) kinase partly restores the inhibitory effect270 .

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Table 16. Continued

A constitutive phosphorylation/dephosphorylation cycle profoundly influencing Kvl.3 activity 48-32-16: Kvl.3: Co-expression of Kvl.3 with (i) a constitutively active tyrosine kinase (v-src) or (ii) a receptor tyrosine kinase (human epidermal growth factor receptor) in HEK-293 cells results in a large increase in the tyrosine phosphorylation of the channel protein (reversible by alkaline phosphatase treatment before Western blot analysis )271. Inhibition of native tyrosine phosphatases with the membrane-permeant agent pervanadate induces a 'time- and concentration-dependent increase in the tyrosine phosphorylation of Kvl.3, associated with a time-dependent decrease in Kvl.3 current'. TyrK phosphomodulations are eliminated in a Tyr449Phe mutant Kvl.3 271 . Notes: 1. Tyrosine kinases have been shown critical in Fas-induced cell death. 2. Notably, an earlier study reported that deletion of the consensus TyrK region from the mouse Kvl.3 cDNA had no apparent effect on channel expression in heterologous cells (for details, see ref. 268, below and Receptor/transducer interactions, 48-49). Kvl.4

48-32-17: rKvl.4/RCK4: A single putative phosphorylation site at Ser601. PKC: hKvl.4 activity is depressed by stimulation of PKC and hKvl.4 subunits can be phosphorylated by PKC in vitr0 272 .

Kvl.5

48-32-18: hKvl.5/HK2: Putative phosphorylation site at aa 547-550; hKvl.5/HCKI: PKA: cAMP-dependent PKA at Ser556 (HCKI). 48-32-19: hKvl.5/hPCNl: CaMKII: Ca2+ /calmodulin protein kinase II motif at Thrl33 conforming to Arg-Xaa-Xaa-(Ser or Thr). PKA: PKA phosphorylation motif at Ser557 and Ser580 conforming to (Arg or Lys)-(Arg or Lys)-Xaa-(Xaa)-(Ser or Thr)28. 48-32-20: rKvl.5/Isolate KVI: PKA: Four potential sites for phosphorylation by cAMP-dependent protein kinase (PKA); three are in the carboxy-terminal region and one is in the N-terminal region32. CaMKII: The N-terminal phosphorylation site (Ser81) also lies within a casein kinase IT recognition sequence32 .

Tyrosine kinase interactions with Kvl.5 48-32-21: hKvl.5/hPCNI: TyrK: Qirect association of the Src tyrosine kinase with cloned hKvl.5 and native hKvl.5 in human myocardium has been demonstrated273, mediated by an interaction/ binding between the proline-rich motif of hKvl.5and the SH3 domain of Src (see SH3 in Fig. 7 under Protein interactions, 48-31). Tyrosine phosphorylation of Kvl.5 suppresses channel current in cells co-expressing v-Src in a manner similar to Kvl.3 273 (see this table, above). Note: Modulation (suppression) of Kvl.5 by coexpression with receptors for the peptide growth factors FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) have been reported274 . Similar suppression was observed following co-expression of thrombin or rat 5-HTlc receptors.

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Table 16. Continued Kvl.6

48-32-22: hKvl.6/HBK2: A single intracellular phosphorylation motif at SerSII. 48-32-23: rKvl.6/Isolate KV2: PKA: Two potential sites for cAMPdependent protein kinase (both in the C-terminal region); CaMKII: One potential casein kinase II phosphorylation site within the SI-S2 loop (Ser222 )32.

Shaker 48-32-24: Comparative note: Cytoplasmic application of phosphatases in excised inside-out patches of oocytes expressing Drosophila Shaker channels slows N-type inactivationt gating (see Inactivation, 47-37). Subsequent application of purified catalytic subunit of protein kinase A and ATP reverses the effect, accelerating N-type inactivation (back to its initial rapid rate)275. A C-terminal consensus site for PKA phosphorylation appears responsible for this modulation (for further details see 275). Notes: 1. Information applicable to a given subunit is denoted by field tagging in bold according to the following convention: PKA: protein kinase A; PKC: protein kinase C; CamKII: Ca2 + -dependent calmodulin kinase II; TyrK: tyrosine kinase; AlkPhos: alkaline phosphatase. 2. This study did not identify the tyrosine kinase that was phosphorylating the channel following M 1 receptor activation, but some kinases can be activated by intracellular calcium elevations or protein kinase C (e.g. PYK2).

Conserved protein kinase C modification sites in the S4-S5 cytoplasmic loop 48-32-02: As exemplified by the listings in Table 16, activation of protein kinase C is associated with suppression of Kvl subfamily channel activities. All Kvl subfamily protein sequences possess one or two consensus t sites for modification by protein kinase C (PKC consensus S/T-X-R/L, see Resource G - Consensus sites and motifs). Moreover, such a site is present in the S4SS loop of all known Kv subunits (in a region proposed in some models236,26o to form part of the internal surface of the ion-conducting pathway). Several Kv channels have been shown to act as PKC phosphorylation substrates t in vitro.

Phosphorylation of Kvl subfamily members by protein kinase A isoforms 48-32-03: A number of consensus sites for phosphorylation by cAMPdependent protein kinase (PKA) and other kinases occur in the Kv series (see Table 16 and Resource G - Consensus sites and motifs), mainly in the Nand C-termini. The position of one of these sites in the C-terminus (approx. 30 aa distant from domain S6) is conserved in the Shaker subfamily, while the others appear to be specific to individual channel proteins. In Drosophila Shaker, phosphatase treatment slows the rate of N-type inactivation, and can be reversed by applying the catalytic subunit of PKA; elimination of the C-

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terminal consensus phosphorylation site (KKKS to KKAA) did not alter normal N-type inactivation, but did prevent the action of the phosphatase. By analogy, the possibility that C-terminal PKA phosphorylation sites may regulate mammalian Kv{3 subunit-Kvo subunit associations (and hence (paradoxically) N-type inactivation and channel membrane targeting functions) has been discussed256 (see also entry 47).

A conserved consensus site for tyrosine kinase phosphorylation in

Kvl subfamily proteins 48-32-04: The motif BPSFQAILY, which conforms to a 'consensus' for phosphorylation by protein tyrosine kinase ([R or K or L]-gap of 2 or 3 aa-[D or E]gap of 2 or 3 aa-[Y*]) is present in the N-terminus of all Kvl subfamily channels (for a 'local' sequence alignment of this region see 'Tyrosine kinase' under Resource G - Consensus sites and motifs; for a 'global' sequence alignment, see Fig. 4 under Encoding, 48-19). The high degree of motif conservation strongly suggests a role for protein tyrosine kinase modulation of Kvl subunits in native cells (for examples, see 'TyrK:' headings in Table 16).

ELECTROPHYSIOLOGY

Activation Studies of Drosophila Shaker channels 48-33-01: As further described under Voltage sensitivity, 48-42, the molecular mechanism of channel activation following displacement of voltage-sensing domains has been most extensively studied in Drosophila Shaker channels. There is a large literature on the origins of gating current t (Le. movements of electronic charge across the membrane field during voltage activation e.g. refs276-282). For an overview of these topics, see ref. 283 and other reviews cited under field 56; see also Kinetic model, 48-38. Note: Similarities have been discussed284 between gating mechanisms of Shaker-type channels and ligand-activated ion channels, comparing opening of the NMDA receptorchannel (entry 08) arising from repulsion between negatively charged W590s (analogous to W435s of the Shaker K+ channel).

Voltage activation of Kv subfamily channels 48-33-02: Comparative studies of voltage activation of vertebrate Kvl subfamily currents in oocytes (e.g. rKvl.l, rKvl.4, rKvl.5 and rKvl.6285 ) show a broadly similar voltage dependence of activation, with halfactivation voltages ranging between -50 and -11 mV and maximum steepness (yielding an e-fold change for voltage increments between 3.8 and 7.0mV)285. Kvl.4 has a shallower activation curve, most likely due to coupling with its fast-inactivation process. Table 17 compares some published values of Kvl channel activation parameters, although 'absolute' values may vary between expression sytems or species, and/or show some temperature dependence (see note 2 in Table 17).

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Table 17. Activation properties of Kv1 subfamily homomultimeric channels (From 48-33-02) Kvl.l

48-33-03: rKv1.1/RCK1: Activates at potentials positive to -30mV; for Kv1.1/RCK1 in oocytes, V n,1/2 - 29.7 ± 7mV; an - 6.5 ± 1.8 mV; 22 t n 15.5 ± 4.4 ms (see note 1; other published values for V n include mKv1.l: -27mV, -34mV; hKvl.l: -30mV, see note 2). For mKvl.l in L929 cells V 1/ 2 was determined as -32±2mV286 . Gating behaviour of single channels is 'not homogeneous': channels either occupy open, conducting states (with only brief closures at irregular intervals) or a gating behaviour which consists of a rapid succession of brief openings and closures, both with average durations of milliseconds22 .

Kvl.2

48-33-04: rKvl.2/RAK: Activates with time constants ranging from 58ms at -20mV to 6ms at +60mV; does not show significant inactivation over 800ms. For Kvl.2/RCK5 in oocytes, V n,1/2 - 34.3 ± 9.7mV; an - 4.5 ± 1.3 mV; t n 6.3 ± 1.8 ms22 (see note 1; other published values for V n include rKvl.2: +3mV and +4.8mV; hKvl.2: -5mV, see note 2). For rKvl.2 in B82 cells V 1/ 2 was determined as -27 ± 6mV286 .

Kvl.3

48-33-05: rKvl.3/Isolate KV3: For Kvl.3/RCK3 in oocytes, V n,1/2 - 25.2 ± 7mV; an - 6.6 ± 1.9mV; t n 13.7 ± 6.5ms22 (see note 1; other published values for V n include mKvl.3: -23mV and -35mV; rKvl.3: -23mVand -14mV; hKvl.3: -13 mV and -20mV, see note 2). For mKvl.3 in L929 cellsV l / 2 was determined as - 26 ± 8 m V 286 .

Kvl.4

48-33-06: rKvl.4/RCK 4: Activates in the subthreshold range 22 (~-60mV) similar to native K+ channels l17; for Kvl.4/RCK4 in oocytes, V n,1/2 - 21.7 ± 7mV; an - 16.9 ±3mV; t n 3.2 ± O.8ms22 (see note 1; other published values for V n include mKv1.4: -27mV and -35mV; rKv1.4: -22mV; hKv1.4: -5mV and -34mV, see note 2).

Kvl.5

48-33-07: hKv1.5/hPCN1: -25mV activation threshold; overall, similar to isolate KV1. Isolate KV1: Activation threshold of ~ -40 mV. Half-maximal activation occurred at -3 mV. 48-33-08: hKv1.5/HK2: Ltk- cell line stably expressed t HK2 (cloned from human cardiac ventricular eDNA) has an activation time course that is fast and sigmoidal (time constants declining from 10ms to <2ms between 0 and +60mV). The midpoint and slope factor t of the activation curve was Eh == - 14 ± S mV and k == 5.9 ± 0.9, respectively144. Note that both activation and inactivation kinetics of Ltk- -expressed t HK2 both display a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperatures 144. Other published values for V n include rKvl.S: -13mV and -3mV; hKvl.5: -25mV (see note 2). For hKvl.5 in MEL cells V 1/ 2 was determined as -14 ±3 mV286 .

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Table 17. Continued Kvl.6

48-33-09: rKvl/6 Isolate KV2: Activation threshold of approx. -40mV. Half-maximal activation approx. -13mV. 48-33-10: Kvl.6/HBK2/RCK2: Currents conducted by these two isoforms do not differ significantly. In their time course and voltage dependence of activation they resemble RCKI and RCKS channels; however the threshold of activation was more positive (rv -30mV) compared to other RCK channels (i.e. they do not activate following -80 to -40 m V depolarizing steps)48. Other published values for V n include rKvl.6: -13 mVand -17mV; hKvl.6: -21 mV (see note 2).

Notes: 1. V n,1/2: Test potential in millivolts where the conductance increase has reached half its maximal value. an: Slope of normalized conductancevoltage relation. The value corresponds to the mV change in test potential to cause an e-fold increase in conductance. t n : Rise time of ensemble t patch currents in milliseconds (the time to rise from 100/0 to 900/0 of final value). 2. 'Absolute' values of activation parameters may vary between studies (see temperature dependence for Kvl.S, above) or between species; these values are taken from the review of Chandy and Gutman (1994, see Related sources and reviews, 48-56) which contains a comprehensive bibliography for the data sources.

Current type 48-34-10: Other than the A-type (transient) current mediated by Kvl.4 and Kvl.7 subunits, homomultimeric Kvl subfamily channels conduct very slow or non-inactivating outward rectifier currents. Kvl.S/HK2: Ltk- cell line stably expressed t HK2 (cloned from human cardiac ventricular eDNA) displays a fully activated current-voltage relationship with outward rectification (in 4 mM [K+]o), but becomes 'more linear' at higher external K+ concentrations. These changes can be explained in part on the basis of constant field rectification t .

Apparent dependence of current properties on cRNA concentration and expression system 48-34-02: Kvl.3: A single mRNA species for Kvl.3 (isolate HLK3) has been reported to express either transient K+ currents or non-inactivating K+ currents depending on cRNA concentration following injection into Xenopus oocytes, with intermediate effects being observed290 . Both current types were also observed (by both whole-cell and single-channel patchclamp recording) from HLK3-transfected IM9 B lymphocyte cell line29o . Table 18 summarizes the reported effects on current type in both transient t and stable expression t systems. Comparative note: These results pre-date the discovery of Kv,B subunits287, some of which are known to affect Kv

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Table 18. Variable Kvl.3 properties following transient and stable expression 29o . Note that these results pre-dated the discovery of Kvf3 subunits287, some of which are known to affect Kv channel inactivation properties (see entry 47) (From 48-34-02) Transient expression (Xenopus oocytes)

Stable expression (IM9 B lymphocytes)

Low cRNA injection (~0.11 ngj oocyte) Condition produces a transient K+ current with a saturating I-V relationship abolished by repetitive stimulations due to a slow recovery from inactivation. The current was fully inhibited by 10nM CTx with a main conductance 12-14pS.

Control (non-transfected) IM9 cells (does not express voltage-dependent K+currents). In stably transfected IM9 B lymphocytes (clone IM9-HLK3, a 'high expressor') the most commonly observed current was transient (initial main component lasting
High cRNA concentration (~35 ngj oocyte) Condition produces a non-inactivating K+current with a linear I- V relationship which did not undergo use-dependent inactivation. This current was insensitive to 10 nM CTx with a main conductance 12-14pS (unchanged).

Notes: 1. Injection of between ~0.11 and ~35 ng cRNAjoocyte produced intermediate behaviour to above. 2. Destruction of cytoskeletal elements (cytochalasin D, colchicine, botulinium C2 toxin) prevented the sustained (non-inactivating mode). 3. The n-type K+ channel in human T lymphocytes has a typical unitary conductance of 10-18 pS and is blocked by nanomolar concentrations of CTx. However other types of voltage-sensitive, non-inactivating, CTxinsensitive channels are also observed in human Jurkat T lymphocytes291 (see also Inactivation, 48-37).

channel inactivation properties (see entry 47). Levels of cRNA expression have also been specifically reported to affect gating properties of Shaker H4 288 and KAT1 channels289 . Functional changes dependent upon expression of variable amounts of Iminimal K' channel mRNAs in oocytes have also been reported (for brief accounts, see the fields Activation and Kinetic model under VLC K minK, 54-33 and 54-38 respectively).

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Current-voltage relation Modification of rectification properties by domain deletions and amino acid substitutions 48-35-01: Mutant (deletant) Kv1.1 channels missing transmembrane segments 51 and 52, 52 and 53, or 51 to 53 (retaining the hydrophilic N-terminal domain in each case) are non-functional following heterologous expression225,226 (see Assembly mechanisms under Protein interactions, 48-31). Notably, however, retaining the deletion of transmembrane segments 51 to 54 (isolate ~SI-S4, N-terminus) can 'convert' the wild-type Kv1.1 channel (depolarization activated with outward rectification) into an inwardly rectifying, hyperpolarization-activated channe1225,226 (compare with the PDTM and characteristics of Kir gene family channels under INR K subunits, entry 33). Although the pore region of ~51-54 is identical to the wild-type channel, it is cation non-selective and has an altered pharmacology226. In separate studies292, it has been shown that certain point mutations can progressively shift Shaker activation to more hyperpolarized potentials, resulting in an increase in the fraction of inactivated channels at negative resting voltages. This conversion (from outward rectifier to inward rectifier) does not rely on a difference in sign or direction of charge movement of the voltage sensor92 .

Modified 1-V relations in heteromultimers 48-35-02: Kv1 subfamily channels display gating and conductance properties characteristic of the subunit compositions, and heteromultimers produced following co-expression of two Kv1 mRNAs in oocytes can show unique properties (see Protein interactions, 48-31). For example, comparison of current responses to depolarizing test pulses in a heteromultimeric RCKl,4 (Kv1.1:Kv1.4) channel to homomultimeric RCK4 (Kv1.4) channels in oocytes reveals differences in their instantaneous 1-V relations t, with RCK1,4 (Kv1.1:Kv1.4) combinations sho"\\ring saturation of peak-transient currents. These differences were postulated to result from different openchannel characteristics and not from different activation kinetics 255 .

Inactivation Kvl subunit domain interactions to different types of inactivation 48-37-01: A large number of studies have provided insights into the molecular determinants of inactivation t properties in Shaker and Kv family channels (as referenced from within Table 19). Kv subunit domains that contribute to distinguishable mechanisms of inactivation have been characterized. First, 'fast' N-type t inactivation293,294, where a 'tethered' N-terminal domain plugs the intracellular mouth of the pore wjth prolonged depolarization and shuts off the current over tens to hundreds of milliseconds. The property of 'intrinsic' rapid N-type inactivation is shared by Kv1.4, Kv1.?, Kv3.3, Kv3.4, Kv4.1, Kv4.2 and Kv4.3 channels (for further details, see VLC K Kv3-Shaw and VLC K Kv4-Shal, entries 50 and 51 respectively). Certain lauxiliary' (,8) subunits of Kv channels that can induce fast inactivation in non-inactivating a subunit complexes also share sequence homology to N-termini of Kv channels that undergo N-type inactivation (for details, see VLC K Kv-beta,

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entry 48 _ - - - - - - Table 19. Inactivation properties of Kv channels, cross-referenced by subtopic. Roles of Kv{3 subunits modifying inactivation properties are described under VLC K Kv-beta, entry 47. Inactivation properties of individual KVQ subunit channels are listed in Table 20 (From 48-37-01) Subtopics or features

Description, cross-references

Terminology

48-37-02: Initial discrimination of N-, P- and C-type inactivation mechanisms298,302-304. For details see separate sections, this table.

48-37-03: N-type inactivation generally refers to rapid inactivationt via physical 'plugging' (occlusion) of intracellular pores by an N-terminal inactivating domain. Thus, descriptions of N-type inactivation have generally been interpreted in The 'ball-and-chain' terms of the ball-and-chain model305, first applied to model N-terminal variants of Drosophila Shaker channels 293,302,306. In the model, an N-terminal inactivation domain (the 'gate' or 'ball') is tethered to the remainder of the channel molecule by a flexible protein 'chain' (see the [PDTMj, Fig. 6). The model states that the N-terminus 'swings' upon Depolarization induces depolarization of the membrane to occlude the blocking particle to channels in an open state (or at least in a state which swing into its receptor permits the Kv channel to open at resting potential in an apparently voltage-independent manner)307. site The ball binds its receptor domain (this table, below) at a position which is in the membrane electrical field close to the inner entrance of the Kv channel pore293,302,308-310. As predicted by the model, inactivating domains do not bind to their receptor in the closed (resting) state. See also 'bipartite' structure of inactivating domains, below. N-type inactivation Defining features

Kvl.4 channel subunit 48-37-04: As an example of a vertebrate channel inactivation domain displaying N-type inactivation, Kvl.4 homomultimeric Q subunit channels- display rapid inactivation and possess the longest N-terminal region of all the Kv 1 subfamily channels (see Encoding, 48-19). Kvl.4 also displays repetitive sequences reminiscent of glutamine repeats in the N-terminal portion of Drosophila Shaker (see Domain conservation, 48-28). A serine/cysteine motif followed by a series of positively charged amino acids is considered to be the inactivating domain (compare the Kv{31 subunit which confers N-type inactivation properties under VLC K Kv-beta, entry 47).

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_'---

e_n_try_4_8_

Table 19. Continued Subtopics or features

Description, cross-references

Sequence homology of 48-37-05: Alignment of the N-terminal domain of Kv,8 Kv,81 subunits with those of KVQ subunits that undergo N-type inactivation suggests some broad structural conservation (illustrated in Fig. 3 under Domain functions in VLC Kv1-beta, 47-29). The inactivating ball domain in the N-terminus of Kv,81 promotes rapid closure of open Kv channels which cannot otherwise inactivate rapidly287 (see fields 4714 and 47-37). See also next paragraph. Inactivating ball domains have been described as having a Composition of ball 'bipartite' structure. The 'first half' of the structure domains contains hydrophobic and hydrophilic amino acids (e.g. a serine/cysteine motif in A-type channels appears to determine the affinity of the inactivating domain to its receptor-binding site (the 'offrate,)311,312 (see also oxidation sensitivity, below). The 'second half' of the ball domain contains a high density of the positively charged residues lysine and arginine. These residues appear to determine 'on-rate' for binding inactivation domains to their receptor. The positively charged residues are likely to enter the electrical field of the membrane, and this accounts for observations of the 'on-rate' having a small voltage dependence. The N-termini Homologous Nof Kvl.4, Kv3.3, Kv3.4, Kv4.1 and Kv4.2 (see Current terminal domains type, 48-34) contain residues that constitute inactivation ball domains conforming to the above description. Sensitivity of N-type 48-37-06: The activity of inactivating ball domains inactivation to protein of vertebrate KVQ channels are highly sensitive to oxidation at a 'critical' cysteine residue308 . oxidation/reduction Oxidation of these cysteine residues (e.g. Cys13 in rKvl.4 and Cys6 in Kv3.4) causes loss of inactivating domain activity, while substitutions by serine eliminates oxidation sensitivity. Notably, positions of 'redox-·sensitive' cysteine residues are conserved in those Kv,8 subunits that induce fast inactivation properties when co-expressed with Oxidation-susceptible non-inactivating KvlQ subfamily channels (e.g. Cys7 in Kv,81, ref.287, see specific comparisions under residues VLC K Kv,8, entry 47). Consistent with these features, oocytes expressing inactivating Q subunit channels such as Kvl.4/RCK4 or Kv3.4/Raw3 lose their fast component of inactivation within 500 ms of patch excision into an oxidizing

II

l_e_n_t_ry_48

---l_

Table 19. Continued Subtopics or features

Description, cross-references

Fast-inactivation restored by antioxidants

environment308 • In these cases, fast-inactivation can be restored by (i) application of antioxidants such as the reduced form of glutathione (GSH) or dithiothreitol (DTT) or (ii) 'patch-cramming' procedures (i.e. pushing inside-out patches back through the membrane of the oocyte). Regulation of inactivation by this type of intracellular mechanism might have potentially important roles in (i) linking metabolism to excitability t and (ii) local regulation of excitability in specific areas of mammalian neuronal membranes 308 (see also Channel modulation under VLC K Kv-beta, 47-44).

Sensitivity of N-type inactivation to protein phosphorylation/ dephosphorylation

48-37-07: For modulation of rate of inactivation of Shaker channels by protein kinase A275, see Table 16 under Protein phosphorylation, 48-32. Comparative note: Protein kinase C (PKC) treatment has been shown to specifically eliminate rapid (N-type) inactivation of hKv3.4, converting this channel to a non-inactivating delayed rectifier313 (see Protein phosphorylation under VLC K Kv3-Shaw, 50-32).

48-37-08: In tandem tetrameric Shaker channels carrying a specific mutation in single subunits, it has been established that only a single gate is necessary to produce inactivation314. Inactivation rate constants for channels with a single gate are one-fourth that of channels with four gates. In contrast, the rate of recovery from inactivation is independent of the number of gates. Statistical analyses based on these and other studies314,315 predict that each of the four Independent operation identical open inactivation gates operate independently, but only one of the four gates closes (in of gates a mutually exclusive manner). Notably, the stability of N-type inactivation is not markedly affected by removing some of the ball domains315.

Number of gates required for fast inactivation in Shaker and vertebrate channels

Single gates can close Kvl.4:Kvl.5 hybrid channels

48-37-09: Analysis of gating properties in Kvl.4:Kvl.5 heteromultimeric channel fusion constructs confirmed a single inactivating subunit can confer N-type inactivation in vertebrate Kv channels316 . The rate of inactivation cannot be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit. Overall, these results further suggested the Kvl.4 inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state316 .

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_4_8_

Table 19. Continued Subtopics or features

Description, cross-references

Effects of deleting inactivating domains

48-37-10: Deletion of N-terminal inactivating domains from fast-inactivating channels leads to a loss of N-type inactivation (e.g. residues 3-25 from Kvl.4)317. Progressive shortening of putative 'chain' regions results in faster inactivation while lengthening has been shown to 'slow down' inactivation rate. Cytoplasmic application of proteases can also remove fast components of inactivation293,318.

'Restoration' of defective N-type inactivation by peptides

Induction of N-type inactivation by peptides

Ball receptor domain location in Shaker channels

Ball receptor domain location in Kv channels

II

48-37-11: Fast inactivation in N-terminal deletants (above) can be restored in a concentrationdependent manner following addition of synthetic ball peptides to inside-out patches (e.g. the first 20 residues of the alternatively spliced variant ShB)294. Peptides corresponding to the ball domain in Kvl.4 (RARERERLAHSR) and Kv3.4 (RGKKSGNKPPSKTCLK) function as inactivation gates308 . This procedure has been able to confer rapid inactivation to otherwise non-inactivating Kv channels (e.g. Kvl.1,319) or Ca2 +-activated K+ channels (see Blockers under JLG K Ca, 27-43). Peptide segments critical for sodium channel inactivation (i.e. the ill-IV linker connecting domains ill and I\r of voltage-gated sodium channels, see VLG Na, entry 55) can also confer Shaker A-type characteristics on non-inactivating potassium channels320 . Differences between inactivation produced by 'free' peptides and 'intact' N-terminal domains have been discussed312 . 48-37-12: Amino acids in the 84-85 intracellular loop contribute to the ball receptor-binding site260,321 which is predicted to be in the electrical Heldt at or near the intracellular cytoplasmic mouth of the ion conducting pathway (see the [PDTM), Fig. 6). Several mutations in the Shaker S4-S5 linker (LQILGRTLKASM~RL) alter inactivation kinetics: (i) replacement of the conserved glutamate residue (underlined, above) with glutamine or aspartic acid markedly slows inactivation; (ii) small, hydrophobic side-chain substituents (alanine or valine) at the position of the conserved leucine (as in the QI1GR segment) accelerates inactivation26o . Note: The Leu-Ala mutation in the ball receptor domain of the 'delayed rectifier' rKv2.1/DRKl is also able to induce rapid inactivation26o . Experiments with hybrid

II...-__ _ _ry_48

_

en t

Table 19. Continued Subtopics or features

Description, cross-references Kvl.4/Kvl.2 K+ channels suggest S4-SS loops of both Kvl.4 and Kvl.2 can serve as the acceptor sites for inactivation gates. In heterotetramers, this analysis predicted all four sets of S4-S5loops would be functional in rapid inactivation322 .

Ball receptor residue interactions

48-37-13: For Shaker K+ channels, it has been proposed that the state-dependent negatively charged site (i.e. the site that I attracts' the positively charged inactivation ball) is W435, which becomes negatively charged after receiving an electron from Y445. This model suggests that the final rapid voltage-independent transition to the open state is due to the deprotonation of Y44S 323 .

Competition for the ball domain receptor by intracellular TEA+

48-37-14: Application of internal tetraethylammonium ions (TEA+) markedly slows N-type inactivation of the Shaker channel, consistent with competition of the ball domain and TEA+ for a common site (see Blockers, 48-43). Mutations at conserved leucine and glutamic acid residues in the S4-SS linker (this table, above) alter [TEA+h sensitivity. Part of the HS region of Shaker B was shown to form part of the inactivation particle receptor (ShB-IP); hydroxylamine ion trapped near this site can stabilize the HS-IP interaction324 . Mutations at residues T441 and T442 (local to the internal mouth of the pore) produce opposite effects on inactivation: the inactivated state is stabilized by T441S and destabilized by T442S. The ammonium derivative, hydroxylamine (OH-(NH!)), binds in the pore region of T441S and further decreases the rate of recovery from N-type inactivation (an effect dependent upon the presence of the N-terminal)324.

Modulation of inactivation recovery by hydroxylamine

Concurrent immobilization of gating charges when ball receptor is occupied

48-37-15: Under depolarization (i.e. when the ball receptor is occupied), the gating charges t in the voltage sensor may be immobilized and the S4 segment may be prevented from returning to its original position325 (see Voltage sensitivity, 48-42).

Recovery from N-type inactivation (i): Channel ire-opening' during recovery

48-37-16: Recovery from N-type inactivation appears to require removal of the inactivating domain from the occluded, but still open, Kv channel pore. Macroscopic tail currents through RCKl-4 channels decay very slowly during hyperpolarization following fast application of synthetic ball peptides (consistent with channel

II

_'--

e_n_try_4_8__

Table 19. Continued Subtopics or features

Description, cross-references reopening upon repolarization)307. Thus 'reopening' current can produce long-lasting afterhyperpolarizations t (AHPt) under current clamp conditions, coinciding with a voltage-dependent 'release' or 'unbinding' of the inactivation gate from its receptor. An independent study applying conditional probability analysis t to single-channel tail currents through Drosophila Shaker channels with intact N -type inactivation also concluded that A-type channels 'reopen' from the inactivated state upon repolarization326 . These reopenings were usually required for recovery, as though the blocking particle must exit the pore before the channel can close. Functional note: Long-lasting AHPs through lA-type channels would be predicted to occur even if the channels were completely 'inactivated' by a preceding depolarization. In native neurones327-330, long-lasting AHPs would tend to reduce action potential frequency, an effect most commonly associated with calcium-activated K+ channels (for examples, see ILe; K Ca, entry 27).

Recovery from N-type inactivation (ii): Relations between permeation and recovery

C-type inactivation Summary of features; domain movements

II

48-37-17: N-type inactivation exhibits several

distinctive properties of open pore blockade326 . Recovery is speeded by increased external K+ concentrations, and blockade can be relieved by trans-permeant ions. In separate studies with ShakerB, the fast phase of recovery can be enhanced by raising external permeant ions (K+, Rb+) and by the blocking ion Cs+, whereas the impermeant ions (Na+, Tris+, choline+) are ineffective331 . Ion permeation through the channels does not· appear to be essential for recovery. These results also suggested that cations influence the fast phase of recovery by binding at a site with an electrical distance greater than 0.5, with recovery from fast inactivation being voltage dependent331 . 48-37-18: 'Slow' C-type t inactivation29s,296 involves a closure ('pinching together') the external mouth of the pore involving residues on adjacent monomers within a tetramer. C-type inactivation can occur alone (e.g. Kvl.3 within native T cells332,333) or in combination with N-type inactivation.

entry 48 _ '----------Table 19. Continued Subtopics or features

Description, cross-references

Possible role of alternate splice variation in Shaker

48-37-19: The kinetics of C-type inactivation are distinct for Shaker channels with different alternatively spliced C-terminal regions; differences in C-type inactivation between the ShB and ShA variants have been localized to a single amino acid in the transmembrane domain S6 302 . For Shaker channels, C-type inactivation is independent of voltage over a range of -25 to +50mV302.

Isolation of C-type inactivation

48-37-20: In Shaker channels, C-type inactivation can be studied in isolation by removing the N-type inactivation domain (e.g. as in the mutant Shaker H4:~6-46)293. C-type inactivation can also be studied in detail within channels where cysteines have been engineered into all four subunits (e.g. Shaker position 449); high-affinity Cd2 + ion binding occurs in the inactivated state as a result of conformational changes at the outer mouth.

Origin of 'C-type' nomenclature

48-37-21 Note: The 'C-type' designation arose that inactivation that remained following deletion of the fast-inactivation gate was sensitive to mutations neighbouring the C-terminal (since then, a large number of mutations in S5, P and S6 segments have been shown to influence C-type inactivation rate).

Observations supporting outer mouth constriction during C-type inactivation

48-37-22: Alternative models for Kvl.3 C-type inactivation in which each subunit acts either independently or co-operatively to produce the observed inactivation kinetics give best fits to data based on heteromultimeric assemblies (thus supporting a co-operative mechanism)334. Independent studies 335 confirmed that the relationship between the inactivation rate and the number of fast subunits (in fast- and slowinactivating heteromultimer combinations) was exponential, as predicted by a co-operative mechanism296 .

Evidence for co-operative gating

Cysteine-reactive reagent probes for detecting domain rearrangements

48-37-23: Probing cysteine substituted channels with thiol-specific methanethiosulphonate (MTS) reagents (e.g. MMTS, MTSES) and cross-linking studies have also helped characterize the dynamic rearrangements of the outer mouth ('constriction') that occurs during C-type inactivation336 (see this table, below).

II

_

entry 48

1....----

_

Table 19. Continued

II

Subtopics or features

Description, cross-references

Bound extracellular K+ accelerates recovery from C-type inactivation

48-37-24: Recovery of C-type-inactivated Kvl.3 in human T lymphocytes is accelerated six-fold by shifting [K+]o from 5 to lSOmM at a holding potential of -90 mV337. This effect has been interpreted as involving a low-affinity extracellular K+ -binding site, at which a bound K+ ion destabilizes the inactivated state, thereby providing a mechanism for Kvl.3 autoregulation. Other results338 have shown that [K+]o can bind to either open or inactivated channels at an effective membrane electrical field distance of 300/0 338 .

Role of His401 in extracellular K+ modulation

48-37-25: Chemical modification of the histidine in the pore motif GYGDMH of Kvl.3 increases the rate of channel inactivation and sensitivity to extracellular potassium339 . An early study340 identified His401 of RGKS/Kvl.3 as being involved in slow inactivation. Note: When Kvl.3 is recorded in oocytes, the rate of C-type inactivation increases when the patch is detached from the cell; His401 has also been determined to be essential for this 'on -celli off-cell' change in inactivation kinetics 341 .

Interactions between C-type and N-type inactivation in Shaker channels

48-37-26: Frequency-dependent cumulative inactivation of Shaker channels is highly sensitive to changes of [K+]o in the physiological range (i.e. greater inactivation at low [K+ ]0' consistent with the findings described above). N-type inactivation has been shown to enhance C-type inactivation by (i) inhibiting outward K+ flux (which normally would occupy the external K+ -binding site and prevent C-type inactivation) and (ii) maintaining the open state of the Shaker channel's activation gate (even after repolarization) thus permitting C-type inactivation to occur over a longer time course297.

Multiple phosphorylation sites influencing C-type inactivation

48-37-27: Kvl.3: Site-directed mutagenesis has been used to show that several putative sites for phosphorylation by protein kinases A and C can influence the basal rate of C-type inactivationt in Kvl.3 269,341. For example, mutation of (i) anyone of four serine and threonine residues to alanines or (ii) two or three of them simultaneously does not eliminate the change in C-type inactivation. Triple-phosphorylation site mutants exhibit a markedly slower basal rate of C-type inactivation in cell-attached patches341 .

l_e_n_t_ry_48

---'_

Table 19. Continued Subtopics or features

Description, cross-references

Chemical modulation of C-type inactivation (examples)

48-37-28: Classically, C-type inactivation is retarded by extracellular (but not intracellular) TEA+ 295?332; by contrast, N-type inactivation is sensitive only to intracellular application of TEA+.

CP-339,818

48-37-29: The non-peptide Kv1.3 blocker CP-339,818 preferentially blocks the C-type inactivated state of the channe1 90 (see Blockers, 4843). For details of modulating C-type inactivation by metal ion binding (following cysteine substitution), see Ref.296 and Channel modulation, 48-44.

Chloramine-T

48-37-30: The cysteine/methionine residueoxidizing agent chloramine-T (Chl-T) induces an irreversible loss of current for Shaker pore or near-pore mutants which display C-type inactivation342 . Sensitivity of several site-directed mutants ion the pore region indicate that Met448 is one of the target residues for Chl-T.

entry 47). By contrast, 'slow' C-type t inactivation295,296, typified by Kv1.3, involves a closure ('pinching-together') of the external mouth of the pore. In some channels, this process has been shown to involve dynamic rearrangement of residues on neighbouring 86 domains on adjacent monomers within a tetramer. Significantly, there is evidence for interaction of N- and C-type inactivation mechanisms in Shaker channels 297 (see Table 19). A third mechanism (P-type inactivation) has been postulated as having characteristics distinguishable from both N- and C-type inactivation (for details, see ref. 298). Determinants of N-, P- and C-type inactivation are discussed as part of review refs299-301 and Chandy and Gutman (1994) (for further distinctions and examples, see Table 19). Further inactivation properties are listed in order of Kv 1 subfamily subtype in Table 20.

Kinetic model A state-dependent model accounting for Kvl.3 inactivation properties

48-38-01: Kv1.3 (isolate/clone KV3): A model for macroscopic t membrane currents through Kv1.3-type channels accounting for state-dependent inactivation t (as opposed to voltage-dependent inactivation t) has appeared345 . This model provides an extension to Hodgkin-Huxley-type descriptions for the cumulative inactivation properties of Kv 1.3 channels as well as a general framework for modelling macroscopic currents when statedependent t processes are involved (see also Inactivation, 48-37). In a model neurone, the macroscopic Kv3 current described in this work produces a

II

_ entry 48 '--------------

Table 20. Inactivation properties listed by subunit (From 48-37-01)

II

Kvl.l

Kv1.1/RCK1: Vhl/2 - 47.0 ± 1.4mV; ah 4.1 ± 0.5 mV (see note 1)22. Current magnitude decreases <200/0 during 10 s voltage steps, as observed343 . During frequent and repetitive stimulation at 1 Hz, a type of response where the channel does not open tends to occur in groups, suggesting the operation of a very slow inactivation process343 .

Kvl.2

Kv1.2/RCK5/BK2/RAK etc.: Non-inactivating. Injection of 0.2ng of cRNA encoding the brain Kvl.2 channel into oocytes has been reported to express a 'very slowly inactivating' K+ current344 . Notably, this inactivation is absent in oocytes injected with 20 ng of cRNA although activation properties remain unchanged (note that this observation pre-dated the discovery of Kv{3 subunits that can alter inactivation properties - see entry 47). RAK: Shows little or no inactivation within 800 ms. Compares closely to the inactivation observed for the intrinsic adult rat atrial delayed rectifie~4.

Kvl.3

Kvl.3/MK3/RCK3: V h1 / 2 - 44.7::i: 4.2 mV; ah 16.0 ±3.2 mV (see note 1)22. Channels in oocytes and type n channels in T lymphocytes inactivate at a rate in between classically defined A-currents, which inactivate in < 100 ms, and delayed rectifiers, which inactivate with time constants> 1 s (reviewed in ref.117 and Hille, 1992, see Related sources and reviews, 48-56). The accumulation of inactivation at 1 Hz depolarizing pulses (due to incomplete recovery during the interpulse interval) is also characteristic of both channel types. RGK5: Slow inactivating. Th for RGK5 is ",>500 ms versus ",50 ms typical of many A-currents. Similar to (but not identical to) the T lymphocyte n channel (the mouse n channel 7b is 107 ms, cf. 7b of RGK5 of 612 ms); these differences are attributed to the differences between oocyte and native cell expression31 . HLK3: Inactivation kinetics (inactivating or non-inactivating) are dependent upon mRNA expression level (summarized under Current type, 48-34). Kvl.3 (isolate/clone KV3): A model for state-dependent inactivation properties applicable to Kv 1.3 channels is described under Kinetic model, 48-38.

Kvl.4

Kv1.4/RCK4Kv1.4/RCK4: V h1 / 2 -73.6±5.4mV; ah 12.8±2.2mV (note 1)22. Inactivating, transient outward K+ currents. RCKl,4 heteromultimeric channels recover more than 2 times faster from inactivation caused by depolarizing pulses than those mediated by RCK4 homomultimeric channels 255 . RCK4 and other cloned IK(A) channels have been reported to Ire-open' after repolarization of the membrane (see ref. 307 and Inactivation under VLC K Kv3-Shaw, 50-37).

_e_n_t_ry_4_8

_

Table 20. Inactivation properties listed by subunit (From 48-37-01)

Kvl.5

Kvl.5/HK2/hPCNI/isolate KVl: Vhl/2 - 44.8 ± 1.4mV; ah 5.6mV (note 1)22; delayed rectifiers, time-dependent outward K+ currents. Inactivation closely resembles RCKI (mean time constant for inactivation 1155 ± 54 ms at 30 mV and 690 ± 17 ms at 60 mV)28 (see note 2). I".J

Kvl.6

Kvl.6/HBK2/RCK2: Resemble Kvl.l/RCKI and Kvl.2/RCK5 channels 48 .

Notes: 1. Vh1 / 2 is the pre-pulse membrane potential at which the current response to a step to 0 mV is 500/0 of its maximal value (pre-pulse duration 25 s, holding potential -80 mV; note that RCKI and RCK5 channels do inactivate over a period of several minutes - therefore the value for a 25 s pre-pulse may not reflect the true steady-state equilibrium potential for inactivation). ah is the slope of the steady-state inactivation (h) curve. It is the change in pre-pulse membrane potential (in mV) necessary to cause an e-fold reduction in the size of the response to a test pulse of 0 mV. Inactivation parameter definitions, conditions and comparative data from ref. 22 . 2. Co-expression of hKvl.5 'n subunits' with hKv{j3 induces inactivation behaviour, hyperpolarizing shifts in the activation curve and slowing of deactivation kinetics (see Fig. 4 in Inactivation under VLC K Kv-beta, 47-37). 3. A study of [K+]o elevations on three inactivating channels (rKvl.4/RHKl, Shaker H4 and H37, the latter mediated by C-type inactivation) has appeared317. For all three channels, elevating [K+]o caused an increase in the channels' chord conductances and a negative shift in the calculated activation curves. Several differences related to the channels' inactivation processes were determined317. 4. Recovery from inactivation of ShakerB K+ channels occurs in two phases, a fast phase (lasting for approx. 200 ms) followed by a slow phase (often requiring several seconds for completion). With Na+, choline+, or Tris+ outside, approx. 150/0 of the channels recover in the fast phase (-80mV), and the other 850/0 enter a second inactivated state from which recovery is very slow. Recovery in the slow phase is not influenced by external ions, but is speeded by hyperpolarization331 . 5. The Drosophila Shaker channel lacks a redox-sensitive cysteine at an equivalent position to the mammalian A-type Kv channels and fast inactivation of this channel does not disappear in excized patches308 .

novel 'short-term memory effect' and firing delays similar to those seen in native hippocampal neurones345 . 48-38-02: A review on the kinetics of voltage-gated ion channels has appeared346; see also evaluations of kinetic models for activation of the Shaker channel347, approaches to parameter optimization of gating models for Shaker channels (based on non-ideal voltage-clamp data)348 and citations under Related sources and reviews, 48-56.

II

_ entry 48

- - - - - - - -

Selectivity See note on atomic scale structural models for Kv channels (including pore domains) under Predicted protein topography, 48-30. In-press update: The availability of crystal structure information for the Streptomyces lividans K+ channel (ref. 494 ) has clarified many of the K+ ion selectivity mechanisms described here and in Table 21.

The S4-S5 linker, P-region and S6 segments 48-40-01: A large number of studies (Table 21) have determined that ion selectivity functions in voltage-dependent K+ channels are predominantly associated with a segment of 21 contiguous residues known as the P-region (i.e. the pore region between domains SS and S6, see [PDTM), Fig. 6). The P-region, along with the 86 segment and the 84-85 linker appear to contain most of the pore determinants (see also Table 21). The great majority of pore structure-function studies have used the Drosophila Shaker channel as their experimental system, although parallel studies of Kv channels have provided new insights on pore architecture (particularly by means of channel-toxin interactions - see Blockers, 48-43). A number of biochemical approaches have been key in studying selectivity determinants (e.g. the use of silver ion22o or membrane-impermeant methanethiosulphonate349,35o to probe reactivities with reporter cysteines placed at specified positions in the pore). The topography of the external pore and vestibule of the Shaker K+ channel as initially predicted by silver ion probing in ref. 220 is shown in Fig. 9. Note: Many of the detailed predictions of these approaches are subject to continual refinement, and so are not described in detail in this field. Where they exist, pointers to contemporary

pore (structural) models on the Internet may be available from the CSN website (see entry 12).

A highly conserved amino acid sequence segment across all known K+ -selective channels 48-40-11: In physiological ionic gradients, Kv channels show high selectivity for K+ over Na+ ions. No exceptions to the expected dependence of reversal potentials t on the external K+ concentration have been reported (55 m V/ decade t from tail current analysis t ). As illustrated on the [PDTM] (Fig. 6), all potassium-selective channel proteins comprise external and internal mouths leading to a narrow transmembrane pore that may be simultaneously occupied by several K+ ions. Ion conduction pathways in K+ channels are short (relative to those of ELC channels, Volume I). Permeating ions appear to traverse channel proteins via short 'canals' ('canaliculi') of 10 A or less. For links to structural models for K+ channels, see Domain arrangement, 48-27. Amino acids in the P-region, the only recognizably conserved segment in all K+ -selective channels, irrespective of subfamily or species (see previous paragraph and Miscellaneous information, 48-55) influence ion permeation, selectivity and sensitivity to pore blockers. Drugs and toxins that occlude the channel pore (e.g. extracellular/intracellular TEA+, DTx and CTx, see Blockers, 48-43) interact with residues in the loop linking the S5 and S6 segments and the P-region (see also Table 21).

II

I

entry48

_

~------

Table 21. Protein domains affecting ionic selectivity in Kv1 channels (From 48-40-01) In-press update: descriptions of selectivity determinants from K+ channels protein crystallography 494.

Feature

Description and cross-references

Principal determinants 48-40-02: Experimental approaches using point mutations352,353 or chimaeric constructs354 have of ionic selectivity provided evidence that the ion-selective pore of voltage-sensitive K+ channels is chiefly determined by a sequence of ",20 amino acids located between the highly conserved S5-S6linker (the H5 sequence or The P-region latterly P-region, see the [PDTMj, Fig. 6). This sequence contains amino acids that determine properties such as organic ion and toxin blocker sensitivitr24,303,355,356, selectivity towards NHt and Rb+ 353 and single-channel conductance354.

The P-region 'invagination'

48-40-03: In many K+ channel models (see the [PDTMj, Fig. 6) the polypeptide segment between S5 and S6 forms a deep invagination into the membrane, entering and exiting the lipid layer at the same (extracellular) site210 . 48-40-04: A single-site mutation (T441S) in the H5 region of Shaker has been shown to increase the apparent relative permeability of the channel to NHt, an effect that is sensitive to small changes in external K+ 357. This behaviour is consistent with an anomalous mole fraction t effect which is not apparent in the wild-type channel, and supports the view that T441S alters the affinity of a putative ion-binding site for NHt and ammonium derivatives.

The 'YG' motif

Interconversion of ionic selectivity by point mutations

48-40-05: The property of K+ selectivity in homomeric voltage-gated K+ channels is related to the presence of two 'extra' amino acids, typically YG (Tyr-Gly) that are absent from the pore-forming region of cation-non-selective cyclic nucleotide-gated channels358 (see feature labelled K+ selectivity determinant in the sequence alignment under Encoding, 48-19 and compare the aligned sequence figure in Domain conservation under ILG CAT cAM~ 21-28). Mutations have been introduced into the pore region of voltage-activated K+ channel coding sequences which confer the essential features of ion conduction in the cyclic nucleotide-gated ion channels, i.e. poor selectivity among monovalent cations and divalent cation block358 .

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Table 21. Continued Feature

Description and cross-references 48-40-06: Comparative note: Ca2 + channel

characteristics can be 'transferred' to voltage-gated Na+ channels when residues Lys (in domain In) and/or Ala (in domain IV) in the P-region of the Na+ channel are replaced by Glu residues (cited in ref.221, see also VLC Na, entry 55). When Shaker P-region residues are mutated in order to 'match' the P-region of the Na+ channel, the mutants can be expressed, whereas when analogous mutants are made in Kvl.l channels, the mutants failed to express221 . 'Non-P-region' domains in ion permeation

48-40-07: Single amino acid substitutions in the 84-85 loop alter Rb+ selectivity, single-channel K+ and Rb+ conductances, and the sensitivity to open channel block produced by intracellular TEA+, Ba2 + and Mg2 + have been described359 . Note: For residues that 'switch' the 'preferred' ionic conductances of K+ to Rb+ in Kv channel pores, see ref. 360 in Selectivity under VLC K Kv-Shab, 49-40.

K+ /Rb+ 'switching'

48-40-08: Evidence supporting a role for the 86 segment in K+ ion permeation (and in governing the sensitivity to internal TEA+ and Ba2 +) has been presented following studies of Shaker-NGK2 (Kv3.1a, entry 50) chimaeras. Transfer of the Kv3.1a S6 segment into Shaker induces the S6 chimaera to adopt the single-channel conductance and blocker sensitivity of the Kv3.1a channel223 .

'Post-S6' region

48-40-09: In ShakerB, exchanging a short stretch of nine cytoplasmic amino acids located just past the S6 domain, ('post-86'), produces a large increase in potassium conductance with no effect on internal or external TEA blockade222 .

Inconsistency of silver ion probe data with eight-stranded {3-barrel models of pores

48-40-10: Early proposals of a ,a-barrel model for a K+ channel pore structure composed of four {3 hairpins 6,352-354 have been contradicted by results derived from reactivities of mutated Cys residues with silver ion22o (see also the [PDTMj, Fig. 6).

Depolarization-induced assay for Kv channels

86

Rb+ efflux as a prototype high-throughput

48-40-12: CHO cells expressing Kvl.5 have been used in a prototype highthroughput assay that measures depolarization-stimulated 86Rb+ efflux as an indicator of K+ channel activation351 . Importantly, there is a high

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Figure 9. Topography of the external pore and vestibule of the Shaker K+ channel. A hypothetical single Shaker subunit, a 90° sector of the outer pore region and vestibule is displayed. Residues studied here are indicated by P-region numbering; residues F425 and K427 are also indicated by Shaker numbering. Arrows indicate residues whose positions relative to the pore axis were previously established by mapping with reversible pore blockers. Shaded residues have side-chains that are proposed to project into the aqueous pore of vestibule. Unshaded residues are Ag+ -unresponsive positions that are proposed to project away from the aqueous phase. Diagonal stripes indicate residues whose insensitivity to Ag+ cannot be clearly interpreted in terms of side-chain projection. The reactivity of residue A2 (horizontal stripes) is slower than that of the other Ag+responsive residues. (Reproduced with permission from Lii, Science (1995) 268: 304-7.) (From 48-40-01). In-press update: Compare with details of the P-region/selectivity filter of the Streptomyces lividans K+ channel revealed by protein crystallography 494.

signal:noise ratio in this system, as non-transfected or vector-transfected control cells do not display measurable 86Rb+ efflux under depolarizing conditions. Applications of the assay in discriminating isoform-specific K+ channel modulators has been discussed351 .

I

Single-channel data 48-41-01: A summary of single-channel properties derived for Kvl subfamily channels appears in Table 22.

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_ entry 48 "------------

Table 22. Single-channel properties of Kv1 subfamily members (From 48-41-01) Kvl.l

RCK1: Slope conductances t of RCKI in its main open state over -60mV to 40mV were 8.9 and 9.3pS with 100mM K+ (cytoplasmic) and normal Ringers (pipette), rising to a 22 pS main state with 100 mM extracellular K+ 343 . RCKl: Chord conductance t (see note 1) assuming a reversal potential of -lOOmV was rv8.7pS22. Stably expressed RCKI channels in Sol-8 cells display a unitary conductance of 14pS77. Mouse Kvl.l stably expressed in L929 cells rvl0pS286.

Kvl.2

RCKS: Rat Kvl.2 stably expressed in B82 cells rv18pS286.

Kvl.3

MK3: In oocytes, mouse Kvl.3/MK3 unitary conductance rv13pS33 (cf. to human lymphocyte n-type K+ channel (13 pS)). RCK3: Chord conductance (see note 1) rv9.6pS22. Mouse Kvl.3 stably expressed in L929 cells rv 14 pS286.

Kvl.4

RCK4: RCK4 chord conductance (see note 1) rv4.7pS22.

Kvl.5

RMK2/isolate Kvl: Channel activity occurs as bursts of fast openings and closings with frequent openings to subconductance states. The main conductance state has a unitary conductance of 7.9 pS. Unitary events activate and exhibit holding potentialdependent inactivation44 . Human Kvl.S stably expressed in MEL cells rv8 pS286.

Kvl.6

HBK2/RCK2: HBK2: 8.7 pSi RCK2: 9.1 pS48.

Notes: 1. For derivation of chord conductance, see Glossary; values listed here all assume a reversal potential of -100 mV. 2. See also unitary conductance comparisons across Kv channels in Chandy and Gutman (1994, see Related sources and reviews, 48-56).

Voltage sensitivity IIntrinsic' voltage sensitivity 48-42-01: A defining function of Kv series of channels is their intrinsic voltage sensitivity, mainly (but not exclusively, see footnote in Table 23) associated with movement of charges within the S4 transmembrane domain upon depolarization, triggering protein conformational changes that open the channel (introduced in VLC key facts, entry 41; see also Sequence motifs, 48-24).

Segment S4 conveys the voltage dependence of the channels as well as the gating charges t which move across the membrane when the channel is activated277,361-363. The equivalent of 12-14 electronic charges are estimated to be transferred across the membrane during the activation of a single Shaker channel364 (also as cited in ref.365). Within Kvo. subunit tetramers, this

corresponds to approximately three charges per subunit. Steady-state measurements on Kvl and Shaker channels obtained by site-directed

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l_e_n_t_ry_48

---'_

mutagenesist show that stepwise reductions of positive charge within the S4 region correlate with a progressive decrease in the channels' overall gating valence t (gating charge)366 and hence alter tertiary structure367 and voltage dependence of gating368 . Charges that are 'buried' within transmembrane domains will interact more effectively with the transmembrane electric field than those at the surface365 . Note: This field provides only limited coverage of structure-function studies of voltage sensing in Drosophila Shaker channels. For supplementary information, see ref. 283 and those listed in Related sources and reviews, 48-56.

The tArg/Lys-X-X-Arg/Lys' and tleucine zipper' motifs 48-42-02: Although the overall predicted 'domain arrangement' of the proteins encoded by Shaker/Shab/Shaw/Shal gene locit are similar, each subfamily possesses a variable number of repeats of the' Arg/Lys-X-X-Arg/Lys motif' in the S4 putative transmembrane domain (see Sequence motifs, 48-24). A protein sequence motif reminiscent of the leucine zipper t is located immediately adjacent to the S4 transmembrane domain (the S4-S5 loop) of Shaker-, Shab- and Shal-related K+ channels321,369. Leucine zipper motifs (4 to 5 leucines repeated every seventh amino acid residue) are also found in S4 domains of voltage-gated Ca 2 + and Na+ channels and in M4 transmembrane domains of some ligand-gated channels - see also Voltage

sensitivity under INR K/Na IfhQ , 34-42.

Structural implications accompanying S4 movement from lipid to aqueous media 48-42-03: Using Fourier transform infrared (FTIR) spectroscopyt in aqueous solution, Drosophila Shaker S4 peptide in trifluoroethanol adopts an ahelical t conformation370 (in good agreement with the results of 2D NMRt studies of S4 peptide based on rat brain sodium channels - see VLC Na, entry 55). A predominantly a-helical structure is also observed when the S4 peptide is present in aqueous lysophosphatidylcholine micelles t in dimyristoyl phosphatidylcholine acid dimyristoyl phosphatidylglycerol lipid bilayers37o . In contrast to this, the S4 peptide in aqueous solution is in a random coil conformation. The coil-to-helix transition observed for the S4 peptide (upon its transfer from aqueous solution to lipid membrane) suggests the segment has a high degree of conformational flexibility and can undergo large changes in its structure in response to its environment370 (Le. as proposed to occur during voltage activation). Comparative note: The transition may also have some significance for gating mechanisms of ligandgated channels that retain an S4-like motif but are voltage-insensitive (e.g. the CNG channels, see entries 21 and 22).

Reporters for S4 movement prior to and during gating 48-42-04: Single cysteine substitutions have been introduced into the Shaker S4 segment, followed by voltage activation of the variants (in oocytes) while adding the membrane-impermeable cysteine-modifying reagent PCMBS (para-chloromercuribenzenesulphonate, 100 ~M)371. In these studies, PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C, suggesting that the exposure of

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en_t_ry_4_8_

84 during gating terminates at L366. By the criterion of cysteine modification, 54 domain movement occurs below the resting potential of the cell (i.e. when the channel would be non-conducting)371. In separate studies, internal and external accessibility of 54 residues in open and closed cysteine-substituted Shaker K+ channels to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethylammonium (MT8ET) indicated that the distribution of buried (voltage-sensing) residues changed (move outward) when channels open372. Furthermore, by combining site-specific fluorescent labelling of the Shaker channel protein with voltage clamping, the conformational change occurring during voltage-gating has been measured in real time373 . By this approach, channel activation was predicted to involve movement of at least seven amino acids of 54 from a 'buried' position, correlated with the displacement of the gating charge. Other work374 has concluded that movement of the 54 domain's N-terminal half (but not the C-terminal end) underlies gating charge, and that this portion of the 54 segment appears to move across the entire transmembrane voltage difference in association with channel activation374 . Note: Opening of a Shaker K+ channel is associated with a displacement of 13.6 electron charge units. Chargeneutralizing mutations of the first four positive charges in the Shaker 54 segment lead to large decreases (approximately 4 electron charge units each) in the gating charge. However, the gating charge of Shaker Ll10 (possessing 10 altered non-basic residues in 54) is identical to the wild-type channel374.

A mutant confirming 'separation' of voltage-sensing and ion-conduction functions 48-42-05: Mutant, non-conducting Shaker channels can still undergo the closed-open conformation in response to voltage changes: A mutation in the pore region of Shaker (W434F) completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the non-conductive mutant are identical in their kinetic and steady-state properties to those in conductive channels375 . Extensive mutational analyses of the segment encoding domain 54 in different voltage-gated K+channels have indicated that basic t residues at distinct positions in 54 encounter different structural interactions or charge environments that determine their role in gating. Table 23 summarizes properties of several Shaker channel mutants that have helped identify specified electrostatic interactions that may be important in the mechanism of voltage-dependent activation.

'On' and 'off' gating currents (see glossary)

48-42-11: Gating current t lon' transients for Kv-type and native potassium channels are fast «SO Jls) and have amplitudes up to several tens of picoamps. Upon repolarization to -100 mV following small depolarizations, loff' gating currents are observed, which rapidly «1 ms) reverse most of the 'on' charge displacement. However this fast recovery of gating charge is markedly reduced upon increasing the amplitude of the depolarizing pulse, and the temporary charge immobilization is complete within a few milliseconds at positive membrane potentials363 (cf. Voltage sensitivity under VLC Na, 55-42). This phenomenon occurs for both non-inactivating

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_ _ _ry_4_8 en t

l....--

_

Table 23. Electrostatic interactions in the Shaker S4 voltage sensor as predicted by structure-function analysis (From 48-42-05) Double mutation

Observed or inferred property

K374Q (S4 'neutralization' mutation - see note 1)

48-42-06: Blocks maturation of the protein, possibly interfering with folding into the native conformation.

R377Q (S4 'neutralization' mutation)

48-42-07: Blocks maturation of the protein (as above).

E293Q (specific 'second-site' rescue mutation in S2)

48-42-08: 'Specifically and efficiently' rescues K374Q. Suggests that K374 and £293 form a strong, local, electrostatic interaction that stabilizes the structure of the channel365 .

D316N (specific 'second-site' rescue mutation in S3)

48-42-09: 'Specifically and efficiently' rescues K374Q. Suggests that K374 and D316 form a strong, local, electrostatic interaction (as above}365 (see also note 2).

R368Q (S4 'neutralization' mutation)

48-42-10: Greatly decreases the valence t of the second component (q2) of charge movement and the proportion of the total charge it carries. R368Q (and R377K) decrease the voltage dependence of the whole-cell current and alter voltage-dependent gating at the single-channelleveI376 . Compared with the wild-type channel, they increase the latency to first opening, destabilize the open state, and alter the equilibria of voltage-dependent transitions, so that some of the charge movement occurs after the first opening376 .

Notes: 1. Although several charge neutralization mutants in S4 could not be functionally expressed, construction of multimeric Kvl.l/RCKI cDNAs has enabled functional expression of all charge neutralizations in the S4 segment227. 2. The S4 segment is not solely responsible for gating charge movement in Shaker K+ channels377. In channels containing neutralization mutations, four positions contribute significantly to the gating charge: £293, an acidic residue in S2 (see above) and three basic residues in the S4 segment: R365, R368 (see above) and R371. and inactivating K+ channels (compare to gating currents measured from Na+ channels, where a maximal two-thirds of the total gating charge mobilizes, and this immobilization is correlated with inactivation)305,378.

Sialidation affects voltage dependence of activation 48-42-12: In native cells, K+ channels form large, octameric sialoglycoproteins t (for details see VLC K Kv-beta, entry 47). Normal post-translational

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addition of negatively charged sialic acids (i.e. glycosylation) can be prevented by transfection of Kv channel cDNAs into Chinese hamster ovary cell lines deficient in glycosylation (Lec mutants)379. Kvl.l channels in Lec mutant lines show a dependence of activation (V 1/ 2 ) that is shifted to more positive voltages with slower activation kinetics compared with controls. A similar positive shift can be recorded in Kvl.ltransfected control cells following treatment of with sialidase t or by raising extracellular Ca2+ (without effect on the Lec mutants). These and other results have been taken to indicate that the presence of negatively charged sialic acid influences the local electric field detected by the Kv 1.1 voltage sensor.

Effects of proline substitutions in S4 segments 48-42-13: The steep voltage dependence of Kvl.l (RCKl) channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage dependent. Introduction of a proline residue into the Kvl.l S4 domain (L30SP, at a position corresponding to that in which a Pro is found in voltage-dependent sodium and calcium channels) at anyone of four domains in a concatenated tetrameric channel construct leads to currents with a Ishallower' activation curve380. Additionally, a fast component of deactivation (from strongly positive potentials) is detectable in these mutants. Notably, L30SP substitution in two domains is only tolerated if the domains were non-adjacent38o .

PHARMACOLOGY

Blockers Reported sensitivities of Kv channels to 'classical' blockers (caveats) 48-43-01: A large number of studies have reported properties of peptide toxin, ionic and pharmacological blockers of Kv1 subfamily channels in heterologous expression systems. In attempting to provide an overview of this literature, information types from different studies (pertaining to specified subunits or subunit combinations) have been placed under several italicized subheaders for each blocker or inhibitor (see footnotes to tables). These headers list general information prior to semiquantitative data - some lack of 'concordance' between independent studies make detailed quantitative comparisons difficult. Comparative data (indexed by Kv subunit) is limited to a single compilation table (Table 26). Several 'uncontrolled' variables influencing potency t and selectivity t of block may exist between studies in native and different heterologous expression systems (e.g. heteromultimer formations, association with Kvj3 subunits, variability in expression levels or complex mechanisms of block).

Steps towards rational drug design for K+ channel targets 48-43-02: Problems inherent in rational drug design for ion channel targets have been addressed in a meeting review38l . It is generally acknowledged

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1_ _

---'_

that K+ channels are 'difficult' drug targets, but that drug modulation of K+ channel gating (i.e. including facilitation of opening) has important therapeutic applications. Progress to date has largely depended on understanding in detail molecular interactions between K+ channel-selective peptide toxin blockers (or modulators) and their receptors in the P-region. To this end, a number of pioneering studies (e.g. ref. 23o,382-384) have used assignment of 'pairwise' amino acid interactions between blocker and target defining the spatial arrangement of ion channel residues. Scanning mutagenesis t approaches can identify sets of inhibitor residues critical for making energetic contacts with the channel; using thermodynamic mutant cycle analysis t channel residues relative to the known (rigid) toxin structure can be inferred (for further examples of this approach, see Table 24 and reviews on K+ channel-blocking peptide families 385 and K+ channel inhibitors386). Further properties of ionic and pharmacological blockers citing Kv1 subfamily channels are listed in Table 25.

Perspectives on 'specificities' of K+ channel-blocking toxins 48-43-03: As emphasized by Miller385, individual toxins falling into the charybdotoxin-, noxiustoxin-, kaliotoxin- and tytiustoxin-type subfamilies of K+ channel blockers cannot be relied upon to 'dissect' or selectively inhibit the numerous K+ currents found in native neurones. Natural venoms may typically comprise complex mixtures of "-150 distinct peptides, and only a small proportion (0.01-0.50/0) of these mixtures may have K+ channelblocking activity (the major proportion being represented by use-dependent t Na+ channel toxins which stabilize Na+ channel open states - see Blockers under VLC Na, 55-43). Epileptogenic activity induced by peptide toxins such as MCDP and DTx (see Table 24) may be due to block of K+ channels that are active at the resting membrane potential and control neuronal excitability387. K+ channel blockers are therefore likely to 'co-operate' with the Na+ channel toxins to initiate and maintain depolarization respectively, leading to, for example, disruption of neuronal activity and muscular tetany. Notably, the 'strategy' various venomous organisms use to immobilize their prey is remarkably similar despite the extreme biochemical diversity in venoms. An interesting example has been documented388 in the form of the fish-hunting marine snail Conus purpurascens, which combines neuromuscular block and excitotoxic shock to immobilize its prey rapidly. In this case, 'excitotoxic shock' is induced by the peptide kappa-conotoxin PVIIA (which inhibits Shaker channels) while delta-conotoxin PVIA delays Na+ channel inactivation388 . 48-43-14: Comparisons of toxin sequences that block Kv channels, highlighting the 'n-KTx' systematic nomenclature suggested by Miller, are shown in Fig. 11.

Summary of blocker data indexed by subunit/gene 48-43-30: Discrepancies in 'absolute' reported concentrations required for 500/0 block exist between studies, and this may reflect a dependence on the heterologous expression system employed. For example, compare (i) the higher potency of 4-aminopyridine sensitivity in mammalian cells such as

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Table 24. Properties of peptide toxin blockers citing Kvl subfamily channels (see also Table 25, indexing blocker properties by Kv subunit) (From 48-43-02) Toxin/peptide inhibitor

Description, examples and references

Agitoxins I, 2, 3

48-43-04: Purified peptides agitoxins I, 2 and 3 derived from LeiuIUs quinquestriatus var. hebraeus venom are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) and various Kv channels389. Thermodynamic mutant cycle analysis t has been used to map Shaker channel residues relative to the solved390 Agitoxin 2 structure. The K27M (inhibitor) - Y44SF (channel) interaction depends on the K+ ion concentration. These and other approaches384 including agitoxin footprinting391 predict a shallow K+ channel vestibule formed by the pore loops, with the selectivity filter located at the centre of the vestibule approx. sA from the extracellular solution384 .

Mutant cycle analysis

Agitoxin footprinting

Charybdotoxin (CTx)

Toxin-channel mapping

Transfer of toxin sensitivity

Pre-synaptic effects

II

48-43-05: Description: A peptide isolated from the

venom of the scorpion Leiurus quinquestriatus, originally reported as a specific blocker (sic.) of Kca channels (see entry 27). The large amount of information obtained on interactions of CTx with K+ channels has been reviewed385?392. Structural models of charybdotoxin (and other toxins) bound to Shaker and Kv channels have been developed through iterative structure-function analysis of toxin-channel interactions (e.g. refs 216?230?382?383?393-395) generally using the compact and rigid toxin molecular structural co-ordinates as templates for complementarity. This model has been subject to further testing by predicting throughspace electrostatic interactions between specific pairs of channel-toxin residues396 . Sites/mechanism: 'Transfer of scorpion toxin sensitivities from the 'highly sensitive' Kv 1.3 to the insensitive Kv2.1 potassium channel by exchange of a stretch of amino acids between SS and S6 ('the SS-S6 linker') helped confirm the P-region as a toxin receptor17 (see Blockers under VLG K Kv3-Shaw, 50-43). Native: rKvI.3: CTx-induced facilitation of transmission may be partly explained its effects on Kv channels in pre-synaptic terminals of hippocampal inhibitory neurones rather than BKca channels (see ILG K Ca, entry 27). In the presence of TEA+, application of CTx greatly increases the

lL...--e_n_t_ry_4_8

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Table 24. Continued Toxin/peptide inhibitor

Selectivity of CTx

Pore residues correlating with CTx sensitivity o:-Dendrotoxin (DTx)

Significance in first identifying native K+ channel complexes in brain

Inhibition of slowly inactivating neuronal currents

Description, examples and references amplitude of IPSCs t. In comparison, the specific BKca blocker iberiotoxin fails to augment IPSCs, whereas kaliotoxin and margatoxin (blocking Kv1.3»1.2) mimicks the facilitating effect of CTx137. Specificity: In a direct comparative study286, CTx blocked rKvl.2 and mKvl.3 channels, but had no effect on currents through mKv1.1, hKv1.5 and mKv3.1b channels. Kv1.1 and Kv1.5 have a phenylalanine (f"-J190A3) at a position on the outer mouth of the pore whereas Kv1.3 has a glycine (f"-J60 A3) at this position and was very sensitive to block by CTx. Kv1.2 has a glutamine (of intermediate size at f"-J144A3) at this position; this has been interpreted as permitting CTx to reach its binding site with some steric hindrance resulting in a weaker CTx block than that of Kv1.3. Note: The CTx resistance of mKv 1.1 is in conflict with earlier reports 22 in which ICso (rKv1.1, CTx) was determined as 22 nM. 48-43-06: Description: a-Dendrotoxin is a 59 aa

residue basic peptide from the snake Dendroaspis angusticeps (green mamba) venom. Used extensively to co-immunoprecipitate native K+ channel octomers (40:4,8) from mammalian brain (generally including Kvl.2, for other subunit compositions, see VLC K Kv-beta, entry 47 and text below). The use of dendrotoxins in K+ channel biology has been reviewed397. Sites/mechanism/specificity: DTx blocks current through mKv1.1 and rKv1.2 channels (see above) with high affinity (half-blocking concentration of f"-J20nM) and through mKv1.3 channels with lower affinity (half-blocking concentration of f"-J250 nM). In this comparative study286, there was no effect on current through hKv1.5 and mKv3.1b at 100nM. In Kv 1.1, three residues critical for DTx binding (A352P, E353S and Y379H) are located in the S5-S6 100p398. Native: Dendrotoxins act mainly on neuronal K+ channels. In general, DTx most effectively inhibits slowly inactivating neuronal K+ current. A-type Kv channels are blocked by higher DTx concentrations or are insensitive399 .

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Table 24. Continued Toxin/peptide inhibitor

Description, examples and references

DMB protein complex DMB protein, a native brain membrane K+ channel complex, has been purified and named on the basis of possessing multiple toxin binding sites for QTx (dendrotoxin), MCDP (mast cell degranulating peptide) and ,B-~Tx (,B-bungarotoxin)67 (see also VLC K Kv-beta, entry 47). MCDP and DTx may block K+ channels active at the resting membrane potential and that control neuronal excitability, possibly underlying their known epileptogenic Epileptogenic activities387. Some DTx-sensitive clones are activities insensitive to ,B-BTx, although this is in contrast to native currents in motor nerve terminals 40o . 'DTx-sensitive' homomultimeric channels (e.g. Kvl.l, Kvl.2, Kvl.6, all exhibiting 'delayedrectifier type' currents in oocytes) generally require Correlation with 4-AP only 50-200 JlM 4-AP to induce block (see 4-Ap, Table 25). sensitivities Kaliotoxin (KTx)

'KTx docking' models of the Kvl.3 vestibule

Margatoxin (MgTx)

l1li

48-43-07: Description: KTx, a Androctonus

mauretanicus mauretanicus (scorpion) venom peptide, was originally described as specific Ca2+-channel blocker (sic.). Potency/specificity: In a single comparative study286, half-blocking concentration of KTx for Kvl.l was rv40nM.. Kvl.3 was almost 2 orders of magnitude more sensitive to block by KTx than was Kvl.l, with a half-blocking concentration of rvO.65nM. Kvl.2, Kvl.5 and Kv3.1 were insensitive to KTx. Sites/mechanism: KTx residue Lys27 interacts with residues in the K+ channel signature sequence (GYGD) in Kvl.3; these and other results from KTx-docking studies have predicted the signature sequence extends into a shallow trough at the centre of a wider external vestibule230,401 (for illustration, see Fig. 10). 48-43-08: Description: 39 residue scorpion toxin.

Sites/mechanism: The solution structure of MgTx402 shows it to be similar to the related toxins charybdotoxin and iberiotoxin; additional residues insert in a manner that extends the ,B-sheet by one residue. Notes: MgTx has been noted to inhibit immune responses in vivo403 .

l_e_n_t_ry_48

----J_

Table 24. Continued Toxin/peptide inhibitor

Description, examples and references

Mast cell degranulating peptide (MCDP)

48-43-09: Description: MCDP (22 aa residues, isolated from bee venom) blocks voltage-gated K+ channels and has potent convulsant activity. Native: MCDP affects both fast-inactivating (native A-type) and slow-inactivating (delayed rectifier) K+ channels. See also DTx (this table). Specificity/potency: In a comparative studr86, mKvl.l and rKvl.2 are half-blocked by "J450nM MCDP; four Kv channels (mKvl.3, hKvl.4, hKvl.5 and mKv3.l) were resistant. Variable Kd values for mKvl.l (e.g. 10-fold lower in ref. 22 ) have may be attributable to toxin puritr86.

Maurotoxin

48-43-10: Chemical synthesis and characterization of maurotoxin, present in the venom of the Tunisian chactoid scorpion Scorpio maurus has been described404 . Synthetic maurotoxin blocks Kvl.l, Kvl.2 and Kvl.3 currents with half-maximal blockage (ICso) at 37,0.8 and l50nM, respectively.

Noxiustoxin (NTx)

48-43-11: Description: A 39 amino acid polypeptide

isolated from the Mexican scorpion Centruroides noxius (see also Pil, Pi2, Pi3, this table). Native: Blocks native (lipid bilayer solubilized) BKca channels and squid axon K+channels. Specificity: Kvl.3 and Kvl.2 are highly NTxsensitive (Kd "J2 nM)286. Pandinotoxins Pi I, Pi2 and Pi3

48-43-12: Three novel 35-residue peptides, Pil, Pi2 (proline at aa 7) and Pi3 (Glu at aa 7), purified from venom of the scorpion Pandinus imperator reversibly block ShakerB channels from the outside with 1: 1 stoichiometry (Pil IC so ''JlOnM405 or Kd "J32 nM in zero external [K+ ]406; Pi2 Kd "J8.2 OM; Pi3 Kd "J140OM)407. Pi2 and Pi3 share approx. 500/0 identity to noxiustoxin (this table); all peptides can displace the binding of [12S I]noxiustoxin to brain synaptosome membranes. Note: An independent group 408 has purified and characterized pandinotoxin (PiTx)-Ka, PiTx-K,B, and PiTx-Kr from venom of Pandinus.

Tytiustoxin or tityustoxin (TsTx)

48-43-13: Tityustoxin-K-alpha interacts with the o:-dendrotoxin-binding site on Kvl.2 K+ channel by binding to the same or closely related sites409.

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en_t_ry_4_8_

Figure 10. Schematic model showing the sites of interation between KTx and the external vestibule of Kv1.3. (Reproduced with permission from Aiyar et a1. (1995) Neuron 15: 1169-81.) (From 48-43-07) CHO to results obtained in whole-cell Xenopus oocytes241,452, and (ii) the slower inactivation of some channels in whole oocytes versus isolated patches (cited in ref.286). The reasons for these discrepancies remain unclear, although they may also reflect an altered accessory subunit constitution between different expression systems (see VLC K Kv-beta, entry 47). Table 26 summarizes Kv1 subfamily blocker data listed by Kv subunit/gene and indicates some reported discrepancies.

Reported variabilities in toxin sensitivities with level of mRNA expression in oocytes 48-43-31: Kv1.2: Injection of 'low' Kv1.2 cRNA concentrations (1""-J0.2ng) into

oocytes have been reported to form Kv1.2 channels sensitive to dendrotoxin I at an IC so of approx. 2 nM344 . In this study, 'high' cRNA concentrations (1""-J20ng) generated Kv1.2 channels which were largely insensitive to dendrotoxin I (IC so approx. 200 nM). At 'low' cRNA concentrations, the expressed Kv1.2 channels were also blocked by other polypeptide toxins such as MCDP (ICso 20 nM), charybdotoxin (IC so 50 nM), and ,B-bungarotoxin (ICso 50 nM), by binding to distinct but allosterically related sites on the channel protein344 . Notably, the channels formed following injection of 20ng cRNA/oocyte were 'totally resistant' to 100nM MCDP and 'hardly altered' by charybdotoxin and ,B-bungarotoxin (1 JlM)344 (see also variabilities in inactivation behaviour with level of mRNA expression reported in the

II

i_e_n_t_ry_4_8

_

Table 25. Properties of ionic and pharmacological blockers citing Kvl subfamily channels (From 48-43-02) Ionic/pharmacological blocker

Description, examples and references

4-(Alkylamino)-1,4dihydroquinolines

48-43-15: Synthesis and structure-activity relationshipst of a series of 4-(alkylamino)-I,4dihydroquinolines have been described410 as potential anti-inflammatory agents and novel inhibitors of voltage-activated n-type (rvKv1.3) channels on human T cells (IC 5o values from 10-5 to 10- 7 M. The naphthyl analogue 7c exhibits > 1DO-fold selectivity for inhibition of 125 [ I]charybdotoxin binding to n-type channels compared with inhibition of [3H]dofetilide binding to cardiac K+ channels 410 .

4-Aminopyridine (4- AP) 48-43-16: Description: 'Classical' K+ channel blocker. For further information, see this field in other K+ channel entries, this volume. Sites/mechanisms: Block by 4-AP of several cloned Kv1 subfamily channels increases the apparent rate Open channel block of inactivation, suggesting open channel block, with the blocker trapped in the closed channel286 . Effects of 4-AP in modulating slow Ie-type' Interference with inactivation of stably expressed Kv1.5 channels (in 'C-type' inactivation part by inhibiting gating currentst) have been described411 (see also Inactivation, 48-37). The characterization of two functionally distinct Blocker subsites Isubsites' for binding of internal blockers412 may explain why some blockers interfere with C-type inactivation through an allosterict effect, whereas others do not. Experiments performed with chimaeric channels suggest 4-AP-binding sites can be formed from the association of the N-terminal end of S5 and C-terminal end of S6, which are both thought to lie in the inner vestibule413 . In Kvl.4, 4-AP block is potentiated by removing the fast inactivation gate of the channel414 . Potentiation of 4-AP Furthermore, short-pulse trains that activate rKv1.4 block by removal of without inactivation induced more block by 4-AP N-type inactivation than a long pulse that activated and then inactivated gates (see Inactivation, the channel. Binding site accessibility is controlled 48-37) by the channel gating apparatus (i.e. suggesting that both activation and inactivation gates limit the binding of 4-AP to the channel) and binding site affinity modulated by membrane voltage414 . Native: See study on 4-AP block on native lymphocyte Kv channels415 .

II

_'----

e_n_try_4_8_

Table 25. Continued Ionic/pharmacological blocker

Description, examples and references

Specificity: 'Dendrotoxin-sensitive' homomultimer channels (e.g. Kvl.l, Kvl.2, Kvl.6, all exhibiting 'delayed rectifier-type' currents in oocytes) generally require 50-300 JlM 4-AP to induce block (see Q-DTx, Table 24). Potency: In comparative studies (e.g. ref, 416) 4-AP half-blocking concentrations ranged between rv200 and rv600 JlM for Kvl.l, Kvl.2, Kvl.3 and Kvl.5. Comparative note: mKv3.1 (entry 50) was rvl0 times Relative resistance of more sensitive to block by 4-AP, with a half-blocking A-type channels (for concentration of rv30 JlM. Generally, rapidly exceptions, see entries inactivating channels require millimolar concentrations of 4-AP for complete block. 50 and 51) Anandamide 48-43-17: Kv1.2: Anandamide was identified in porcine brain as an endogenous cannabinoid receptor ligand and is a possible counterpart to the psychoactive component of marijuana (8(9)- tetrahydrocannabinol or 6(9~THC). It has been reported417 that anandamide directly inhibits Shaker-related Kv1.2 channels (IC so , 2.7 JlM)417. 8(9)-THC: also inhibits Kvl.2 channels with comparable potency (IC so 2.4 JlM), as well as several Nacyl-ethanolamides with cannabinoid receptorbinding activity. 48-43-18: Capsaicin, derived from capsicum, is used Capsaicin to define nociceptive t sensory neurones and provides models for deep hyperalgesia. Kvl.l, Kvl.2, Kvl.3, Kvl.5and Kv3.1 are blocked by capsaicin (half-blocking concentration range from 23 JlM (for hKvl.5) to 158 JlM (for mKv3.1b)286. Note: Capsaicin has been shown to block voltage-gated K+ currents in native rabbit Schwann cells, dorsal root ganglion cells, T cells (type .I K+ currents, see Kv3.1), and vertebrate axons (for refs, see286). 48-43-19: CP-339,818 (l-benzyl-4-pentylimino-l, CP-339,818 competitively inhibits 4-dihydroquinoline) and two analogues (CP-393,223 [l 2S I]CTx from binding and CP-394,322) potently block Kvl.3 from the extracellular side in T lymphocytes 90 (IC so rv200 OM to the external and at 1: 1 stoichiometry). Block is use-dependent, vestibule of Kvl.3 with a preference for the C-type inactivated state of the channel (see Inactivation, 48-37). CP-339,818 is also a potent blocker of Kvl.4 but not Kvl.l, Kvl.2, Kvl.5, Kvl.6, Kv3.1-4, or Kv4.2. Notably, CP339,818 suppresses T cell activation in vitr0 90 (see Developmental regulation, 48-11). Relative sensitivity of delayed rectifiers

II

1'--_e_n_t_ry_4_8

---'_

Table 25. Continued Ionic/pharmacological blocker

Description, examples and references

Clofilium

48-43-20: For hKvl.5 stably expressed in CHO cells, steady-state half-inhibition concentration for the class ill antiarrhythmic compound clofilium in inside-out patches is 140 ± 80 nM (cf. the value of 840 ± 390 nM determined in outside-out patches). Clofilium accelerates apparent current inactivation but does not influence the kinetics of current activation or deactivation, with rate of onset of channel block voltage-independent and rate of recovery from block slower at hyperpolarized potentials. Elevation of [K+]o accelerates recovery, suggesting an 'activation trap' mechanism, Le. where clofilium is trapped near the pore418 . Some discrepancy in cited inhibitory concentrations exist, which report block at concentrations higher than the therapeutic dose (e.g. clofilium blocking hKvl.3 at 60 JlM 29 and hKvl.5 at 50 JlM 143 ) See also Blockers under VLG K eag/elk/erg, 46-43.

Acceleration of apparent inactivation

Activation trap

Docosahexaenoic acid (DHA)

48-43-21: External blockade of Kvl.5 by the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been reported419 . DHA accelerates of the apparent inactivation and decreases peak current in a manner similar to that produced by the class ill antiarrhythmic tedisamil.

Diacylglycerol (DAG)

48-43-22: Following activation of phospholipase C t , the common second messenger t intracellular I, 2-dioctanoyl-sn-glycerol (C8:0) (DOG) 'blocks' Kv1.3, Kv1.6 and Drosophila Shaker channels 420, appearing macroscopically as a large acceleration of inactivation rate (doubling the apparent inactivation rate at 162nM DOG). The action of DOG is independent of PKC activation, and appears to act by blocking channel open state rather than modulating gating (DOG and TEA+ ions interact at overlapping but non-identical sites when blocking Kvl.3)42o. Note: Longer carbon chain length DAGs (10 and 12 carbons) are less effective in producing this response.

Diltiazem (non-selective)

48-43-23: The non-selective Ca2 + and CNG channel blocker diltiazem (see Blockers under ILG CAT cGMp, 22-43) also inhibits various Kvl subfamily channels at micromolar concentrations 286 .

II

_L.-

en_t_ry_4_8_

Table 25. Continued Ionic/pharmacological blocker

Description, examples and references

Flecainide (non-selective)

48-43-24: Kvl.l, Kvl.2, Kvl.3, Kvl.5 and Kv3.1 are blocked by flecainide, a class Ic antiarrhythmic drug that inhibits IK but not ITO components in heart (half-blocking concentrations ranging from 53 ~M for mKvl.3 to 217~M for rKvl.2)286. Note: Since these concentrations are significantly higher than the therapeutic dose, it may indicate that homomultimers of these Kv channels are not the therapeutic targets of flecainide.

Nifedipine (non-selective)

48-43-25: Nifedipine blocks voltage-gated calcium channels at nanomolar concentrations (see Blockers under VLG Ca, 42-43); at micromolar concentrations, however, nifedipine also blocks types n (Kvl.3) and 1 (Kv3.1) voltage-gated K+ channels in lymphocytes. Current through various Kv channels is also inhibited by nifedipine at concentrations ranging from 5 ~M (for mKvl.3) to 131 ~M (for mKv3.1b)286.

Resiniferatoxin (capsaicin analogue)

48-43-26: The plant diterpene ester resiniferatoxin is an analogue of capsaicin and binds with high affinity to rat dorsal root ganglion and spinal cord. Resiniferatoxin blocks Kvl.l, Kvl.2, Kvl.3, Kvl.5 and Kv3.1 with half-blocking concentrations ranging from 3 ~M (for Kvl.3) to 46 ~M (for Kv3.1)286.

Terfenadine

48-43-27: Native cardiac atrial myocyte I Kur sensitive to 4-aminopyridine (this table) has similarities to current through Kvl.5o:. At therapeutic concentrations, the antihistamine terfenadine produces a time-dependent non-selective block in Kvl.5a current that is consistent with blockade from the cytoplasmic side of the channel421 . Note: Terfenadine also blocks cardiac current IK,r (entry 46) and IK,s (entry 54).

Tetraethylammonium ions (TEA or TEA+)

48-43-28: Description: 'Classical' K+ channel blocker. TEA+ ions were first used to selectively inhibit the potassium conductance in squid axons over 40 years ago. Sites/mechanism: Mapping of external and internal TEA-binding sites on Kv channels have been discussed in detail within several reviews (see Chandy and Gutman, 1994, and citations in Related sources and reviews, 48-56). External TEA interacts with a tyrosine at the C-terminal end of the

TEA+ site mapping

II

l_e_n_t_ry_4_8

---'_

Table 25. Continued Ionic/pharmacological blocker

Description, examples and references

P-region356 . Kv channels with tyrosines at this position, (e.g. mKv1.1 and mKv3.1) are half-blocked Interactions of TEA+ by rvO.3 mM external TEA286 . TEA-resistant with specified residues channels have a valine (rKv1.2) and an arginine (rKv1.5) in this position. Other published results 13 have stated lower sensitivity of human Kv1.1 to external TEA (Kd of 20 mM), i.e. unlike its mouse and rat homologues. Notably mKv1.3 (possessing a histidine in the homologous position to tyrosine, IC so rv10mM external TEA+ in L929, A4 fibroblasts and isolated oocyte patches33,286. Other whole-cell studies using two-electrode voltage clamp methods 22 report 50 mM external TEA+ as an ICso of rKv1.3. The Kv1.3 histidine residue (above) is also involved in slow 'C-type inactivation' (see Interference with Inactivation, 48-37); thus external TEA+ slows C-type inactivation down Kv1.3's apparent rate of inactivation (Kv1.3 blocked by TEA cannot inactivate332, and the titration of the histidine appears to change the rate of inactivation)34o. Other ions

48-43-29: S5-S6 segment mutations alter patterns of open channel block produced by intracellular Mg2 + (in the 'millimolar range'), Ba2 +359 and TEA+ (see above). 'Strong' inward rectifier K+ channels are several orders more sensitive to internal blockage by Mg2 + ions (i.e. in the 'micromolar- range' - for details, see INR K subunits, entry 33).

same study under Blockers, 48-43). For other variabilities in Kv current properties associated with expression level, see Current type, 48-34.

Channel modulation Listing of reported modulators for Kvl subfamily channels 48-44-01: A number of physiological and pharmacological agents that have been cited as influencing the electrophysiological characteristics of Kv1 subfamily channels appear in Table 27. While many of these may appear to have non-specific mechanisms of action, the listing includes several factors/ agents of potential physiological or pathophysiological significance, including modulatory effects by intracellular and extracellular ions, metabolites, signalling intermediates and redox modulators (compare with

similar listings in other Kv-series entries).

II

_____________________ en_t_ry_4_8.-

_ _ _u__

~1

Common name

0 -

Charybdotoxin-type: CTx lq2 IbTx LbTx

5

10

15

20

--1!L Turn .1L. 25

30

35

subfamily 1 ZFTNVSCTTSKE-eWSVCQRLHNTSRG-KCMNKKCRCYS ZFTQEsCTASNQ-CWSICKRLHNTNRG-KCMNKKCRCYS ZFTDVoCSVSKE-CWSVCKDLFGVDRG-KCMGKKCRCYQ

u-KTx Name

VFIDVS£SVSKE~WAP£KAAVGTDRG-K£MGKKCKCY?

1.1 1.2 1.3 1.4

Noxiustoxin-typ8: subfamily 2 NTx T I I NVKCT-SPKQCSKPCKELYGSSAGAKCMNGKCKCYNN MgTx T I I NVKCT-SPKQCLPPCKAQFGQSAGAKCMNGKCKCYP CITx1 I T I NVK£T-SPOO£LRP£KDRFGQHAGGK£ I NGK£K£YP

2.1 2.2 2.3

Kaliotoxin-type: subfamily 3 KITx GVEI NVKCSGSP-QCLKPCKDA-GMRFG-KCMNRKCHCTP? AgTx2 GVP I NVSCTGSP-QC I KPCKDA-GMRFG-KCMNRKCHCTPK GVP I NVPCTGSP-oe I KPCKDA-GMRFG-KCMNRKCHCTPK AgTx3 AgTx1 GVP I NVKCTGSP-QCLKPCKDA-GMRFG-KC I NGKCHCTPK VR I PVS£KHSG-o£LKP£KDA-GMRFG-K£MNGK£~TPK KITx2

3.1 3.2 3.3 3.4 3.5

Tytiustoxin-type: subfamily 4? TyKa VF I NAK£RGSPE-£LPK£KEA I GKAAG-K£MNGKCKCYP

4.1

Figure 11. Comparison of toxin sequences that block Kv channels, highlighting the a-KTx systematic nomenclature suggested by Mille?85. (Reproduced with permission from Miller (1995) Neuron 15: 5-10.) (From 48-43-14)

Ligands An ultra-high affinity, reversible radioligand for brain Kv channels 48-47-01: Kvl.2; Kvl.3: Monoiodotyrosine margatoxin ([ 125 I]_MgTx) has been described as an 'extraordinarily high-affinity ligand' for voltage-gated potassium channels in mammalian brain439. The [125 1]_MgTx K d of 0.1 pM to purified rat brain synaptic plasma membrane vesicle targets under equilibrium binding conditions has been confirmed by kinetic experiments (Kd 0.07 pM), competition assays employing native margatoxin (MgTx, K· 1"J0.15 pM), and receptor saturation studies (Kd 0.18 pM). Affinity labelling of the binding site in rat brain synaptic plasma membranes employing [125 1]_ MgTx and the bifunctional cross-linking reagent disnccinimidyl snberate induces specific and covalent incorporation of MgTx into a glycoprotein of Mr 74000, a value which reduces to 63 000 upon deglycosylation (compare Molecular weight (purified), 48-22). Antibody studies have suggested that at least Kvl.2 and Kvl.3 are integral constituents of the rat brain MgTx receptor complex. I"J

1

I"J

Receptor/transducer interactions Note: As further described below, some receptor/transducer interactions may be inferred from protein phosphorylation sensitivity (see Protein phosphorylation, 48-32) or from studies of channel modulation by receptor agonists in native cells (see this field in entries marked [native)}.

II

_e_n_t_ry_48

_

tPotential' versus tdemonstrated' couplings of Kv channels to receptor/transducers 48-49-01: This field is limited to studies which have reported heterologous coexpression of specific receptor subtypes with specified Kv channel types. Because of the large size of the Kv subunit family and the number of potential couplings to receptor/second messenger systems, there are a considerable number of predicted receptor/transducer/effector combinations operating within native cells. Some couplings can be inferred from the types of enzymatic effector protein(s) that a given receptor subtype can activate (e.g. phospholipase ct, adenylate cyclaser, guanylate cyclase t, etc.) although these are not equivalent to demonstrations of coupling. Many enzyme components may be part of a single receptor response, and studies often do not discriminate between (i) direct effects of an activated enzyme on a channel protein and (ii) an intermediate role for the same enzyme activating another enzyme within a signal amplification cascade (e.g. a tyrosine kinase isoform being activated by a protein kinase C subtype). Possession of substrate recognition motifs t within a channel's primary sequence (or even in vitro phosphorylation data) is not absolute proof of enzyme-channel interaction in vivo (although it might be evidence in support of it).

tReconstitution' of receptor/transducer/effector pathways 48-49-02: Relatively few studies have been published attempting to 'mimic' channel regulation as observed in native cells with 'entirely' cloned components. 'Partial' reconstitution of native properties may rely on ubiquitous expression of genes encoding other 'critical' components (e.g. those encoding G proteins and enzymes such as kinases or phosphatases). In isolation, 'heterologous reconstitution' data may be misleading without 'full' knowledge of the signal transduction pathway and some control over heteromultimer formation (especially those constituting the effector channel). With these caveats, however, heterologous reconstitution provides a useful route for determining stmcture-function relationships affecting receptor-linked channel modulation properties, particularly when these properties can be performed on native and heterologous cell preparations in parallel (see also next paragraph).

tPunctional consequences' of Kv channel modulation in situ 48-49-03: Receptor/transducer control of Kv (or other) channel activities can be interpreted in terms of the physiological effects which 'modulated' channel opening or closing would have in situ. Thus, the indirect effects of receptor agonists on properties such as voltage dependence of gating, kinetics of activation and current amplitude may be 'interpretable' in terms of effects on cellular function. 'Functional effects' may vary with cell type, but may include alterations in secretory behaviour, rates of action potential firing, action potential shape/duration or efficiency of coupling to muscular contraction or gene transcription events (for specific examples see Developmental regulation (field 11), Phenotypic expression (field 14), Protein phosphorylation (field 32), Channel modulation (field 44) and Receptor/transducer interactions (field 49) of most entries). In general, receptor-coupled modulation which opens K+ channels will hyperpolarize cells, inducing an 'inhibitory' phenotype (e.g. with respect to secretion or neurotransmission in excitable cells). Conversely, suppression of K+ channel current will assist depolarization of cells, inducing

II

II

Table 26. Summary of Kvl subfamily blocker data listed by subunit (From 48-43-30)

TEA (mM)

CTx (nM)

DTx (nM)

MCDP(nM)

4-AP (mM)

Other blockers/specific examples/additional notes (see also Tables 24 and 25)

mKvl.l

0.4 0.3

>1500

21

490

1.1 0.29

rKvl.l

0.6 0.8

22

12

45

1.0 0.16

hKvl.l

20

>100

ND

ND

1.1

rKvl.l/RBKl: TEA (or Et4N+) ion, IC so r-v0.5mM. Using concatenated DNAs to construct Kvl.l (RBKl)-based tetrameric channels containing four, two, or no TEA-sensitive subunits, it has been shown that bound TEA interacts simultaneously with all four subunits 422 . ,B-Bungarotoxin (r-v200 nM)i apamin-insensitive (r-v 1 J,1M).

rKvl.2

>560 129 10

17 6 1.7

24 4 2.8

440 180

0.6 0.8 0.2

hKvl.2

>50

10

ND

ND

0.8

>2000

0.20 0.4

rKvl.2/RCK2: TEA (and more potently, tetrapentylammonium) at the intracellular surface decreases channel open time and increases the duration of closed intervals. Extracellular TEA causes an apparent reduction in single-channel amplitude. Slower block at high-affinity internal site than at low-affinity external site. TEA is a voltage-dependent open channel blocker (see Table 25). The internal TEA-binding site is r-v25% into the membrane from the cytoplasmic margin. External TEA also requires an open channel, but block has less voltage sensitivity. External and internal TEA sites define the inner and outer margins of the aqueous pore 423 . (b

mKvl.3

11

2.6 0.5-20

250

rKvl.3/RCK3/MK3: Oocytes expressing MK3 are sensitive to a wide spectrum of pharmacological agents including (in increasing order of potency)

='

f"1"

~ ~

00

mKv1.4

rKv1.4

>100

>40

>200

>2000

hKv1.4

>50

ND

ND

ND

rKv1.5

>40

>200

>200

>600

hKv1.5

330 >40

>1000

>1000

>10000

0.27 >0.1

rKv1.6

4 7 1.7

1 >3000

20 25

10 200

1.5 0.3

hKv1.6

7

1

20

10

1.5

hKv1.3

II

13

>5003,12

503 11 12 >40 13 1414 30 15 >1600

rKv1.3

>10003

0.8

ND

ND

>1000

>200

>2000

1.5

0.19 0.3 ND

13 1.2 0.8 0.7 0.4

TEA, 4-AP, quinine, verapamil and CTx. Type n lymphocyte channels (",native Kv1.3) show the same order of sensitivity33. RCK3 and MK3 are not blocked by nanomolar concentrations of DTx and MCDp22,67. rKv1.4/RCK4: Transient currents can be recorded in the presence of non-inactivating currents by using TEA at 10-100mM (e.g. ref. 22,255 (ef. RCK1, above).

hKv1.5/hPCN1: Outward current is inhibited by 4-AP dependent on current activation and is enhanced by repetitive stimulation (in 50 JlM 4-AP ",30-38 % block; in 100 JlM 4-AP 54-62 % block; Ki ",<0.10mM. Kv1.5/hPCN1: Relatively insensitive to barium ion (e.g. 10 JlM Ba2 + produces approx. 53% block. Insensitive to external Ca 2 + (1-10mM) or Cd2 + (100 JlM). 1-V relation is unaffected by external Mg2 + (10mM)28. hKv1.6/HBK2/rKv1.6/RCK2: ID so (500/0 inhibition of peak current at 20 m V test potential) for 4-AP (1.5mM), TEA (7mM), DTx (20nM), MCDP (10nM), CTx (10M) f1-BTx (»200nM)48.

Values are taken from the summaries by Chandy and Gutman (1994) which also contains a comprehensive bibliography for the data sources. These largely overlap with those cited in this field. Values quoted are to two significant figures with comparable values differing by less than 100/0 shown as identical. Numbers on different lines indicate (reported) variability in 'absolute' values (see text).

("D

~

f"'1'"

~ ~

00

_L--

e_n_try_4_8_

Table 27. Kvl subfamily channel modulation (From 48-44-01) Modulator

Specificity (as cited), description and references

Arachidonate (released 48-44-02: hKv1.1~: Intracellular application of by Ca2+ -independent Ca2+-independent phospholipase A 2 (ciPLA2, 40 kDa isoform) has been reported to (i) concomitantly phospholipase A2 ) increase the rate of activation of the macroscopic Kv1.1 current in Sf9 cells (from T(act) =6.25 ±0.76ms to T(act,ciPLA2) = 2.78 ± 0.78 ms at 40 m V) and (ii) induce channel inactivation (from no observed See also 2n2+ inactivation to T(inact) = 103 ±6ms at 40mV)424. These effects (i) were dependent on the modulation of docosahexaenoic acid enrichment of Sf9 cell phospholipids in esterified arachidonic acid (for background, see ILG K AA inhibition of Kvl.2 [native], entry 32); (ii) were abolished by (this table) pre-treatment of the ciPLA2 by the inhibitor (E)-6(bromomethylene)-3-( 1-naphthalenyl)-2Htetrahydropyran-2-one; (iii) occurred prior to development of alterations in cellular permeability. Effects of ciPLA2 were indistinguishable from the effects of exogenously applied arachidonic acid (AA), which also specifically and reversibly increased the rates of (i) channel activation (from T(act) =5.73±0.88ms to T(act,AA) = 1.91 ±O.39ms at 40mV} and (ii) inactivation (from no observed inactivation to T(inact,AA) == 76.6 ± 1.4 fiS at 40mV)424. Ca2+ ions, extracellular 'Stabilization' of K+ channels by [Ca 2 +]o (comparative note only)

48-44-03: Drosophila Shaker: A 'critical requirement' for Ca2 + ions in the bathing medium has been shown during recording of recombinant Shaker-type K+ channels 425 . Upon removal of Ca 2+, Shaker K+ currents disappear and are replaced by a steady non-selective leak conductance. Control cells (devoid of Shaker channels) do not develop the leak conductance when Ca2+ was removed. This behaviour parallels responses seen in native neuronal membranes 426 and indicates that extracellular Ca2 + is required to stabilize the native conformation of voltage-gated K+ channels (see also

Sequence motifs, 48-24 and Protein phosphorylation, 48-32). Ca2+ ions, intracellular [Ca2+Jielevations following gating of intracellular stores

48-44-04: Kv 1.2: Oocyte-expressed Kv 1.2 channels have been reported to be 'blocked' by an increase in intracellular Ca2 + from inositol trisphosphate-sensitive pools344 (see ILG Ca InsP3, entry 19) and by the phorbol ester PMA that activates protein kinase C (see Protein

phosphorylation, 48-32).

l_e_n_t_ry_48

_

Table 27. Continued Modulator

Specificity (as cited), description and references

Diacylyglycerols (DAG, independent of PKC)

48-44-05: Comparative note only: Intracellular 1,

2-dioctanoyl-sn-glycerol (C8:0) (DOG) appears to block the open state of Kv1.3, Kv1.6 and Drosophila Shaker channels, rather than modulate gating, because DOG and the open channel blocker TEA interact when blocking channels 420 (for further

details, see Blockers, 48-43). Non-oxidative metabolites of ethanol (fatty acid ethyl esters) accelerating Kv1.1 activation kinetics

48-44-06: Kv1.1: Major metabolites of ethanol in neural tissues (fatty acid ethyl esters) markedly accelerate the kinetics of the voltage-induced activation of Kv 1.1 427. External application of ethyl oleate (20 JiM) to Sf9 cells expressing Kv1.1 channel results in decreased rise times of the macroscopic current (e.g. from 51.7 ± 13.1 to 12.8 ± 3.0 ms at 0 mV for 10-900/0 rise times) and a 10mV hyperpolarizing shift (at 0 m V) in the voltage dependence of channel activation. These results have been discussed in terms of pathophysiologic consequences of ethanol abuse in excitable tissues 427.

H+ ions, extracellular

48-44-07: Kv1.2; Kv1.4: Lowering external pH suppresses Kv1.2 and Kv1.4 currents in a concentration-dependent manner428, with inhibition of Kv1.2 alone being voltage dependent. Using chimaeric Kv1.2/1.4 channels the amino acids responsible for H+ modulation were suggested to lie within the first half of the 85-86 linker region428 .

Titration of surface 48-44-08: Kv 1.5: Parallel studies429 on the effects of negative charges by H+ [H+]o on Kv 1.5 expressed in oocytes revealed (i) (see also Sr2 + ion and reductions in the maximal conductance (Gmax ) N-dodecyl-guanidine consistent with H+ ions entering and 'plugging' the modulation, this table) channel pore and (ii) a shift of the activation curve in a depolarizing direction. These effects were explained in terms of a 'fixed surface charge model', where the 'titration of surface negative charges by H+ was sensed by channel gating machinery', changing the voltage dependence of channel activation429. K+ ions, extracellular

(see also K+ modulation of native inward rectifier channels under Channel modulation in INR K [native], 32-44)

48-44-09: rKv1.4: In addition to to 'local' modulation by redox potential (this table) and receptor-coupled phosphomodulation (see Protein phosphorylation, 48-32), activity of Kv 1.4 channels is highly sensitive to external K+ concentratrion43o . Note: Local [K+]o may increase during prolonged neuronal activity, thus [K+]o-sensitive K+ channels may form part of a use-dependent adaptive mechanism for controlling

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_8_

Table 27. Continued Modulator

Specificity (as cited), description and references excitability in axons and terminals (see also Subcellular locations, 48-16 and Channel modiulation under INR K [native], 32-44).

Nystatin

48-44-10: Nystatin (frequently used in perforated patch t recordings to preserve intracellular components) inhibits Kv1.3 in CHO cells (ICso approx. 3 JlM, a concentration 'significantly below' those used to induce perforation)431.

Sr2+ ions, extracellular 48-44-11: A principal modulatory effect of extracellular strontium ions (Sr2+; 7-50mM) on oocyte-expressed Kv1.1 Kv1.5, Kv1.6, Kv2.1 or Kv3.4 is a shift of the GKV curve along the potential axis. This effect has been interpreted as 'screening' of fixed surface charges. Estimated charge densities show 'good correlation' with the total net charge of Surface potentials extracellularly located amino acid residues of the linearly related to channel segment linking S5 and the pore. Notably, estimated surface potentials are linearly related to activation midpoint potentials of different the activation midpoint potential, suggesting a functional role for variable surface charges432 . channels Zn2 + ions, extracellular

Mutants in the 'pre-pore' region affecting activation and Zn2 + modulation properties

48-44-12: rKvl.l; hKvl.4; hKv1.5: Elevated extracellular Zn2 + ions (2-1000 JlM) can induce a shift in the activation curves (in the depolarizing direction) for rKvl.1, hKv1.4 or hKv1.5 channels433 . Additionally, Zn2-t- shifts the steady-state inactivation curve for hKv1.4 (also in the depolarizing direction). As a consequence of Zn2+modulation, activation kinetics of the K+ currents are slower (an effect likely to delay repolarization of the neuronal action potential). The modulatory action of Zn2+ appears to show some 'subtype selectivity' and may predict a common 'Zn2 +-binding domain' which could interact with the S4 voltage sensor in Zn2 +-sensitive K+ channel subtypes433 (for variability between isoforms, see Domain functions, 48-29). See also notes 1 and 2. For Kvl.5, mutants A455K, H461K and E454Q in the 'pre-pore' domain (between S5 and H5) have been shown to affect gating, activation kinetics and Zn2+ modulation434 . The midpoints of activation (V 1/ 2 ) of all three mutants are shifted in the depolarizing direction compared to wild-type Kv1.5 channels (V 1/ 2 , wild-type = 4.5 ± 1.8 mV; V 1/ 2 , E454Q= 19.5 ±3.1 mV; V 1/ 2 , A455K= 19.6± 1.9mV;

1L-_e_n_t_ry_4_8

_

Table 27. Continued Modulator

Specificity (as cited), description and references V 1/ 2 , H46lK== l8.9±2.3mV). E454Q mutants also show a relative slowing of activation kinetics relative to the wild-type Kvl.5 (tl/2, wildtype==5.33±0.7ms; tl/2, E454Q==11.75±2.6ms). All three mutant channels exhibit decreased Zn2+ sensitivity, with E454Q showing a 10-fold lower affinity. These and other data have been taken to support a model for gating modulation in which the 'pre-pore domain' may interact with extracellular ions and the S4 voltage sensor434 .

Zn2+ modulation of docosahexaenoic acid inhibition of Kvl.2

48-44-13: The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), which is is highly enriched in neuronal membranes produces a potent inhibition of Kvl.2 (1.8 ± 0.1 JlM) and Kv3.la (690±60nM) at +40mV) when applied to the extracellular face of the channels. Micromolar concentrations of [Zn2 +]0 non-competitively t antagonizes DHA inhibition of Kvl.2 channels (with little effect on DHA inhibition of Kv3.la)435.

N-Dodecyl-guanidine 48-44-14: Kvl.4: n-Dodecyl-guanidine (C-12-G, Modulation of external 1-20 J-LM, see notes 3 and 4) induces a surface potential concentration-dependent positive shift in rKvl.4 voltage dependence of activation, inactivation, rate of decay during depolarization, and rate of recovery from inactivation for current when the subunit is expressed in oocytes 436 . C-12-G is likely to act at an extracellular site (i.e. modulatory effects are observed following its addition to bath solutions but not when applied to the cytoplasm of oocytes). Effects of C-12-G are antagonized by elevated [Mg2 +]o or by external guanidine ions (see note 3) but can be augmented by lowering the ionic strength t of external solutions. From these and other results 436, C-12-G appeared to exert its actions by causing a positive shift in the external surface potential around rKvl.4, without altering (i) the ion permeation process or (ii) voltage-independent transition steps. Intracellular redox state (oxidative and reductive agents affecting channel gating)

48-44-15: Kvl.4 expressed in oocytes can be regulated by the reducing agent glutathione, exerting its modulatory effect predominantly at a cysteine redox site in the inactivation bal1308 (for

further details, see Channel modulation under VLG K Kv-beta, 46-44; see also next paragraph).

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_S-------J

Table 27. Continued Modulator

Specificity (as cited), description and references 48-44-16: The cysteine-specific oxidizing agents 2, 2'-dithiobis-5-nitropyridine (DTBNP, 50 JlM) and chloramine-T (CL-T, 500 JlM) remove inactivation of Kvl.4 expressed in HEK-293 cells. These effects can be reversed by the reducing agent dithiothreitol (DTT, 10mM). DTBNP and CL-T also slow Kvl.4 deactivationt while increasing the voltage sensitivity of deactivation. Thus, in general, oxidation tends to stabilize the open state of Kvl.4, both by removing inactivation and slowing deactivation437.

Oxygen free radical generators

48-44-17: Photoactivation of rose bengal, a generator of reactive oxygen species 'drastically inhibits' activity of Kvl.3, Kvl.4, and Kvl.5 (as well as Kv3.4, see entry 50 and Kir2.3/IRK3, see entry 33)438. In parallel experiments, Kvl.2, Kv2.1, Kv2.2 (entry 49), Kv4.1 (entry 51), Kirl.l, Kir2.1 (entry 33) and minK (entry 54) were'completely resistant' to this treatment. Another generator of reactive oxygen intermediates, tert-butyl hydroperoxide has been shown capable of removing fast inactivation processes of Kvl.4 (and Kv3.4) without apparent effect on other channels 438 .

Notes: 1. Zn2 + modulation of ion channel gating may be of significance in regions where Zn2 + is found in abundance, such as mossy fibre nerve terminals of the hippocampus (see Protein distribution, 48-15). 2. For studies of extracellular Zn2 + modulation in other ion channel families see ELG CAT ATP (entry 06), ELG CAT GLU AMPA/KAIN (entry 07), ELG CAT GLU NMDA (entry 08) and ELG Cl C;ABA A (entry 10). 3. C-12-G is an amphipathic t compound with a guanidine moietyt, which is positively charged at physiological pH, and a hydrophobict side-chain. 4. C-12-G also alters properties of native canine ventricular myocyte fA (in a similar manner to rKvl.4) but has 'negligible effects' on the voltage dependence of the slow delayed rectifier K+ channel in the same cell type (see IK,s, described under VLG K minK, entry 54).

an 'excitatory' response. In non-excitablet cells (i.e. cells that do not pass action potentials, e.g. endothelium and blood cells), a depolarizing response following closure of Kv channels may reduce the driving force for calcium entry. Characterization of co-expression for distinct receptor/transducer/effector components with lopposing' effects on membrane potential in specified cell types may thus help 'model' the contribution which specific channel subtypes make in homeostatic responses of native cells.

"---e_n_t_ry_4_8

------'_

Table 28. Co-expression of specified receptor and Kvl channel isoforms in heterologous cells (From 48-49-04) Kvl.l isoforms co- 48-49-05: mKv1.1/MBK1: Following co-expression of MBK1 channels with mouse 5-HT 1C receptors in expressed with Xenopus oocytes, application of serotonin results in a 5-HT1c receptors suppression of the I K amplitude over rv20 min 44o . Suppression can also be effected by activation of G proteins and Ca 2+ injection. These modulatory effects are blocked by the protein kinase inhibitor H-7 but appear independent of protein kinase A and C. Suppression may be due to the actions of a Ca2+ /calmodulin-activated phosphatase, with recovery from suppression being due to the action of a protein kinase 44o . Kvl.2 isoforms co-expressed with endothelin receptors (subtype ETA), Muscarinic M 1

Muscarinic M 3

!32 adrenergic receptors

Kvl.3 isoforms co-expressed with 5-HT2 receptors

48-49-06: Following co-injection of Kv1.2 and ETA receptor cRNAs in oocytes, endothelin (0.1-100nM) suppresses the amplitude of Kv1.2 current in a dose-dependent manner441 . 48-49-07: rKv1.2/RAK: Activation of M 1 muscarinic acetylcholine receptors in Xenopus oocytes potently and acutely suppresses co-expressed Kv1.2 current amplitude through a pathway involving [Ca 2+h elevation and phospholipase C. 48-49-08: In oocytes or COS cells co-expressing human muscarinic receptor M3 and hKv 1.2, acetylcholine (ACh, 100 JlM) decreases the whole-cell Kv channel current by approx. 72 % over 20 rnin 442, an effect mimicked by phorbol esters. Muscarinic receptor M3 activation suppresses hKv1.5 in a manner similar to hKv1.2 channels 442 . 48-49-09: Demonstration of both positive and negative regulation of Kv1.2 (RAK) by two independent G protein-linked pathways has appeared443 resulting in (i) a suppressive effect of activating muscarinic acetylcholine receptors (mAChR) and (ii) a 'strongly enhancing' effect of activating !32-adrenergic receptors (!32AR), the latter effect depending on phosphorylation at the single PKA consensus phosphorylation site located near the N-terminus of the channel protein443 (see Protein phosphorylation, 48-32). 48-49-10: hKv1.3/HLK3: Application of 5-HT to Xenopus oocytes co-expressing a cloned 5-HT2 receptor with HLK3 induces a long-lasting inhibition of the voltage-gated K+ current265 . For Idual

_L..-

e_n_try_4_8_

Table 28. Continued

regulation' of the n-type K+ current by PKC and PKA in T lymphocytes, see Protein phosphorylation, 48-32.

II

5-HTIC receptors

48-49-11: mKvl.3: In oocytes co-expressing the mouse 5-HT IC receptor and mouse Kvl.3 channel, addition of 100llM 5-HT causes a complete and sustained suppression of Kvl.3 currents in approximately 20min268 . The 5-HT-mediated suppression of Kvl.3 currents proceeds via activation of a pertussis toxin-sensitive G protein (and a subsequent rise in intracellular Ca2 +, but Ca2 + does not directly block the channel)268. Notably, this study concluded that despite the ability of phorbol esters to 'independently' suppress Kvl.3 currents, PKC activation did not appear to be part of the pathway specifically linking 5-HT IC receptor to Kvl.3 channels. Removal of the first 146 amino acids from the N-terminal region of mKvl.3 does not alter the time course of 5-HT-mediated suppression of Kvl.3 current (this region contains a putative tyrosine kinase and PKA phosphorylation sites, see Protein phosphorylation, 48-32 and Fig. 4 under Encoding, 4819). Furthermore, treatment of oocytes with calmodulin or phosphatase inhibitors does not alter 5-HT-mediated modulation268 . Note: Kv3.l channels (see VLG K Kv3-Shaw, entry 50) are not susceptible to suppression by 5-HT in analogous studies.

Fas receptor

48-49-12: Activation of the Fas receptor is associated with induction of apoptosis, tyrosine phosphorylation and inhibition of Kvl.3 in Jurkat T lymphocytes (see Protein phosphorylation, 48-32).

Kvl.5 isoforms co-expressed with PDGF receptors, FGF receptors, thrombin receptors, 5-HTIC receptors

48-49-13: Kvl.5: In oocytes, activation of receptors that activate phospholipase C (e.g. those for platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), human thrombin or 5-hydroxytryptamine (rat 5-HT I c) mediate a decline in the Kvl.5 current amplitude, with a half-time of about 20 min. These reductions in K+ current amplitude occur with little change in the kinetics or voltage sensitivity of activation274 . Mutant FGF receptors, which do not activate phospholipase C-,1 but retain other functions, are unable to modulate the Kvl.5 currents. Simultaneous injection of inositol trisphosphate (InsP3, see ILG Ca InsPJ, entry 19) and superfusion of phorbol l2-myristate l3-acetate can reproduce the modulation of the Kvl.5 current274 .

l_e_n_t_ry_4_8

_

Heterologous co-expression of 'specified' receptor and Kvl channel proteins 48-49-04: Known receptor/G protein transducer combinations with reference to their known effector pathway(s) are listed in Resource A - G protein-linked receptors, entry 62. 'Subsets' of these are further described in field 49 of appropriate entries. Table 28 lists examples of channel modulatory patterns for specified Kv isoforms which have been studied by transient t or stable t expression of channel cDNAs in cells with characterized receptor/second messenger systems (see general notes and caveats above).

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 48-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction eiJ layout of entries, entry 02).

Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in Introduction eiJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouring t analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers), author /reference or nomenclature. Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly reported and related sequences. Kv nomenclature

Species, Database locus original isolate name (bold)

Accession

Sequence/ discussion

hKvl.l

Human, eDNA gb: HUMKCHA

gb: L02750

Kamb, Proc Natl Acad Sci USA (1989) 86: 4372-6. Mathew, Soc Neurosci Abstr (1989) 15: 540. Ramaswami, Mol Cell Neurosci (1990) 1: 214-23. Christie, Science (1989) 244: 221-4.

HuK(I)

rKvl.l

Rat, eDNA RBKI

gb: RATPTP

gb: M26161

II

_

entry 48

1......----

Kv

nomenclature

_

Species, Database locus original isolate name (bold)

Accession

Sequence/ discussion

gb: X12589

Baumann, EMBO T(1988) 7: 2457-63. Tempel, Nature (1988) 332: 837-9.

rKv1.1

Rat, cDNA RCKI

gb: RATCKIA

mKv1.1

Mouse, cDNA MBKI

gb: MUSPCPl j gb: Y00305 j gb: MUSKCHPIA: gb: M36456 gb: MMPCPl

mKv1.1

Mouse, genomic gb: MUSMKIA MKI

gb: M30439

hKv1.2

Human, cDNA HuK(IV)

gb: L02752

cKv1.2

Canine, colonic gb: not found circular smooth muscle CMSKI

mKv1.2

Mouse, genomic gb: MUSMK2A MK2

rKv1.2

Rat, cDNA BK2

rKv1.2

Rat, cDNA NGKI

rKv1.2

Rat, cDNA RAK

gb: RATRAK

gb: M74449

rKv1.2

Rat, cDNA RCK5

gb: RNRCKS

gb: X16003

Kv1.2

Toad, cDNA XSha2

gb: XELPOTCH

gb: M35664

hKv1.3

Human, genomicgb: HUMKCHN HGK5

gb: M38217

hKv1.3

Human, cDNA HLK3

gb: HUMKCHC

gb: RATBK2

Chandy, Science (1990) 247: 973-5.

Kamb, Proc Natl Acad Sci USA (1989) 86: 4372-6. Mathew, Soc Neurosci Abstr (1989) IS: 540. Ramaswami, Mol Cell N eurosci (1990) I: 214-23. gb: not found Hart, Proc Natl Acad Sci USA (1993) 90: 9659-63. gb: M30440 gb: J04731

see BK2

gb: HUMKC:HAN gb: M85217

Chandy, Science (1990) 247: 973-5. McKinnon, I BioI Chern (1989) 264: 8230-6. Yokoyama, FEBS Lett (1989) 259: 3742. Paulmichl, Proc Natl Acad Sci USA (1991) 88: 7892-5. Stiihmer, EMBO 1(1989) 8: 3235-44. Ribera, Neuron (1990) 5: 691701. Cai, DNA Cell BioI (1992) II: 163-72. Attali, TBioI Chern (1992) 267: 8650-7.

I

-

entry 48

Species, Database locus original isolate name (bold)

Accession

Sequence/ discussion

hKv1.3

Human, genomicgb: HUMPCD hPCN3

gb: M55515

Philipson, Proc Natl Acad Sci USA (1991) 88:

mKvl.3

Mouse, genomic gb: MUSMK3A MK3

gb: M30441

Kv

nomenclature

53-7. Chandy, Science (1990)

247: 973-5. Grissmer, Proc Natl Acad Sci USA (1990) 87:

rKv1.3 rKv1.3 rKv1.3

Rat, genomic Isolate KV3

gb: RATKV3AA

Rat, cDNA RCK3

gb: RATKV3AB

Rat, genomic RGK5

gb: M31744

9411-15. Swanson, Neuron (1990)

gb: X16001

4: 929-39. Stiihmer, EMBO

gb: RATRGK5

gb: M30312

T(1989)

8: 3235-44. Douglass, T Immunol (1990)

144: 4841-50. hKv1.4 hKv1.4

Human, cDNA HKl Human, cDNA hPCN2

gb: HUMVENHKl gb: M60450

Tamkun, FASEB

gb: HUMPCC

gb: M55514

T(1991)

5: 331-7. Philipson, Proc Natl Acad Sci USA (1991) 88:

53-7. Philipson, Nucleic Acids Res (1990) 18:

hKv1.4

Human, cDNA HuK(II)

gb: HUMKCHB

gb: L02751

7160. Kamb, Proc Natl Acad Sci USA (1989) 86:

4372-6. Mathew, Soc Neurosci Abstr

(1989) 15: 540. Ramaswami, Mol Cell Neurosci (1990)

rKv1.4 rKv1.4

Rat, cDNA RCK4 Rat, cDNA RHKl

gb: RNRCK4

gb: X16002

1: 214-23. Stiihmer, EMBO

gb: RATKCHAN

gb: M32867

T(1989)

8: 3235-44. Tseng-crank, FEBS Lett

(1990) 268: 638. bKv1.5

Bovine, cDNA and genomic

gb:

gb:

Garciaguzman, FEBS Lett

(1994) 354: 173-6.

II

_L-

e_n_try_4_8_

Kv nomenclature

Species, Database locus original isolate name (bold)

Accession

hKv1.5

Human, genomicgb: HUMPOTCH gb: M83254 HCKI

hKv1.5

Human, cDNA gb: HUMVENK2

gb:

HK2

hKv1.5

Human, cDNA hPCNI

gb: HUMPCA

gb:

mKv1.5

Mouse, cardiac cDNA

gb:notfound

gb:

rKv1.5

Rat, genomic Isolate KVl

gb: RATKV1AA

gb: gb:

RKv1.5

Oryctolagus gb: RABPCA cuniculus (strain ORF: 598 aa Japanese White Rabbit) heart cDNA

rKv1.6

gb:

Curran, Genomics (1992) 12: 72937. M60451 Tamkun, FASEB T(1991) 5: 331-7. M55513 Philipson, Proc Natl Acad Sci USA (1991) 88: 53-7. not found Attali, TBiol Chem (1993) 268: 24283-9. M27158; Swanson, M37145 Neuron (1990) 4: 929-39. D45025 Sasaki, FEBS Lett (1995) 372: 20-4.

gb: HSHBK2

X17622

Rat, genomic Isolate KV2

gb: RATKV2AA

Rat, cDNA

gb: RRRCK2

gb: M27159; gb: gb: M37146 gb: X17621

Rat, cDNA HBK2

rKv1.6 rKv1.6

Sequence/ discussion

Grupe, EMBO T (1990) 9: 174956. Swanson, Neuron (1990) 4: 929-39.

RCK2

Kv1 subfamilyrelated

Rabbit (partial length from RT-PCRt)

gb: RABVGKCA togb: M81350 toDesir, Am T Physiol (1992) RABVGKCE M81354 262: FI51-7. inclusive inclusive

Drosophila Shaker

Shaker locus

(example only)

gb: M17211

Papazian, Science (1987) 237: 749-53.

Gene mapping locus designation 48-54-01: Human chromosomal locus names assigned by the Human Gene Mapping Workshopt (HGMW) are as follows: Kvl.l - KCNAl j Kvl.2 KCNA2 j Kvl.3 - KCNA3 j Kvl.4 - KCNA4 j Kvl.5 - KCNA5 j Kvl.6 KCNA6 j Kvl.7 - KCNA7. Kvl.8 - KCNA8 (see Chromosomallocation, 4818). By convention of the International Commitee on Mouse Genetic Nomenclature444 mouse Kv loci are designated in lower casej e.g. Kcna1, Kcna2, Kcna3 etc. Notes: 1. Italicized designations are generally used on printed maps but are usually not preserved in online resources such as OMIM. 2. Kvl.5 (KCNA5) was termed 'KCNAI' in ref. 42.

II

1'--_e_n_t_ry_48

---I_

Miscellaneous information Cloning of invertebrate and lower prokaryotic homologues of Shaker (examples) 48-55-01: A number of reports have appeared examining homologues t of voltage-gated K+ channels in a wide diversity of organisms such as Escherichia COlle445 (see below), Streptomyces lividans 446 , Saccharomyces cerevisiae447 (budding yeast), Arabidopsis448 (e.g. KAT 1, see entry 46); primitive metazoans (e.g. jellyfish), Aplysia (see entry 52), nematode worms (e.g. C. elegans), parasitic trematode worms 449 (see below), squid450 etc. While largely beyond the scope of these entries, evolutionarily distant homologues of Kv channels are of great interest in deducing lineages of ion channel gene/molecular evolution, in particular the representation of protein motifs that are highly conserved thoughout all organisms. For example, the kch sequence predicts a protein 417 residues long with 'extensive similarity' to eukaryotic potassium channel proteins445 . Further analysis predicts six apparent transmembrane regions with a 'potassiumselective' P (or HS) I pore' region motif between SS and S6 (compare field 40, this entry). kch gene sequences are conserved across a series of 38 wild-type reference (ECOR) strains, and while examples of 'importation' of eukaryotic genes into bacteria are known, there is no direct evidence that kch has been 'imported'. A multigene family of putative Kv channels has also been described in the ciliate protist Paramecium tetraurelia, which is an important model for metazoan nerve and muscle electrical excitability451. Ancient diploblastic metazoans (e.g. jellyfish and other coelenterates) have been shown to possess multiple Shaker subfamily members (e.g. jShakl and jShak2, from the hydrozoanfjellyfish Polyorchis penicillatus, in phylum Cnidaria451 ,453 which express transient outward currents in oocytes). Given that there is lack of direct evidence that the Escherichia K+ channel gene was 'imported' from genomes of higher organisms, its existence supports the view that the Shaker K+ channel subfamily was functionally established prior to the first major radiation of metazoans.

Related sources and reviews See also review references cited in entries 47 to 51. 48-56-01: Major sources used in this entry include general reviews including Shaker-related K+ -channels and accessory subunits299,454-456; retrospective review on voltage-dependent potassium channels457; interspecies conservation of K+ channel genes3,8; earlier (pre-1994) reviewing aspects of Shaker-related vertebrate K+ channel functions 67,458-47o - see also VLG key facts, entry 41; earlier (pre-1994) reviews relating structural domains to functional properties of cloned K+ channels260,301,302,309,310,353,354,356,362,393,471-475; early mutational studies for Shaker mutations eliminating fA in Drosophila 476; cloning of Drosophila Shaker51,477-479; early single-channel analyses in myotubes and neurones480; isolation of additional Drosophila K+ channel genes encoding Shab, Shaw and Sha1164; interpretation of amino acid sequences of voltagedependent K+ -channels481 .

III

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_8__

48-56-02: Kinetic mechanisms of voltage-gated channels (including Shaker channels)482,483; the Shaker 84 segment and mechanisms of voltage gating (including Shaker)283,362,368,484,485; K+ channel pore structure/function486 and Brown et al. (1993); molecular determinants of ion conduction and inactivation in K+ channels (1995 review)487; proposal of canaliculi for ion and gating charge traversal supported by cysteine scanning mutagenesis and toxin-channel interaction site mappin{ll; later (post-1994) reviews: molecular/functional diversity of K+ channels (1995)488; overview of protein phosphorylation of Kv channels256; structural motifs underlying Kv channel function 489,490; subunit constitutions of native K+ channels 491 ; toxins purified from animal venoms as specific ligands to distinguish K+ channels 68,69; Kv channels in lymphocyte function (in part)92; voltage and cyclic nucleotide-gated potassium channels in kidney492; myocardial K+ channels (1996 review)493 (see also this field in other entries).

Book references: Brown, A.M., Drewe, J.A., Hartmann, H.A., Taglialatela, M., Debiasi, M., Soman, K. and Kirsch, G.E. (1993) The potassium pore and its regulation. In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 74-80. Annals of the New York Academy of Sciences, vol. 707, New York. Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand- and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton. Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA. Lewin, B. (1994) Genes v: Oxford University Press, Oxford and New York. Li, M., Isacoff, E., Jan, Y.N. and Jan, L.Y. (1993) Assembly of potassium channels. In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 51-9. Annals of the New York Academy of Sciences, vol. 707, New York. Saito, M., Zhao, M.L. and Wu, C.-F. (1993) In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 392-5. Annals of the New York Academy of Sciences, vol. 707, New York. Spooner, P.M., Brown, A.M., Catterall, W.A., Kaczorowski, G.J. and Strauss, G.J. (eds) Ion Channels in the Cardiovascular System. Futura, New York. Stefani, E., Toro, L., Perozo, E. and Bezanilla, F. (1994) Gating currents of cloned Shaker K+ channels. In Handbook of Membrane Channels (ed. C. Perrachia), pp. 199-210. Academic Press, San Diego.

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VLG K Kv2-Shab Edward C. Conley

Vertebrate K+ channel subunits related to Drosophila Shab (KvQ subunits encoded by gene subfamily Kv2) Entry 49

See note on coverage at the head of VLG K Kv1-Shak, entry 48. General properties of Kv channel subunits expressed in heterologous cells are summarized in the fields of the entry 48.

NOMENCLATURES

Abstract/general description 49-01-01: Vertebrate K+ channel subunits whose primary amino acid sequences

are most closely related to those of Drosophila Shah gene are grouped in the voltage-gated K+ channel subfamily 2 (Kv2, this entry). In comparison with the Kvl subfamily, the Kv2 subfamily is small, its prototype members members including Kv2.1 (in rat, often referred to by its trivial/clone name DRK1, originally isolated by expression cloning) and Kv2.2 (in rat, originally designated as the ~ircumvillate papilla ~elayed Eectifying ~+ channel or cdrk). Various species homologues of these genes have been described {see Table 1 under Gene family, 49-05} with representatives from lower vertebrates (e.g. Xenopus) and invertebrates (e.g. Aplysia) have been particularly valuable for defining genotype-phenotype relationships for the Shab-related channels {see below}. The relationship of the 'non-expressible' cDNA clone IK8 to the Kv2 family is uncertain, but limited phylogenetic analysis has placed it on the gene family branch leading to Shab {for details/references on IK8, see VLC K Kvx, entry 52}. 49-01-02: As indicated by their trivial names in use (e.g. DRKl) Kv2 subfamily

channels have been taken for 'archetypal' gelayed !ectifier-forming K+channels, particularly as the K+ current in Drosophila neurones and muscle (encoded by Shab) has many similarities to the 'classical' HodgkinHuxley delayed rectifier. Comparative analysis of heterologously expressed versus native cell currents have shown a high degree of similarity irrespective of evolutionary position, e.g. from invertebrates (e.g. flies), lower vertebrates (e.g. frog), and higher vertebrate cell types. Striking observations exist 'matching' Kv2 subfamily mRNA expression correlated with in situ recordings in excitable tissues of developing Xenopus embryos {see Developmental regulation, 49-11, mRNA distribution, 49-13, and note on Kv{3 subunits under Protein interactions, 49-31}. 49-01-03: Kv2 subfamily polypeptide isoforms exhibit discrete temporal patterning during neuronal development. Extensive studies using molecular probes for Kv2 (particularly in in rat and Xenopus) have suggested distinct physiological roles for Kv2 polypeptide isoforms during development. For example, Kv2.2 transcripts are detectable in eggs; following fertilization, 'segregation' of Kvl and Kv2 subtype expression occurs (e.g. during development of different lineages of spinal neurones). 49-01-04: Further underlining a fundamental role in neuronal differentiation,

striking changes in patterns of Kv2.1 protein abundance and distribution

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_

(without effects on mRNA levels) occur following treatment of cells with certain growth factors. For example, redistribution of Kv2.1 to the growth cones of PC12 phaeochromocytoma cells has been reported following treatment with nerve growth factor t (NGF) (see Developmental regulation, 49-11). Additionally, the neuropeptide thyrotropin-releasing hormone (TRH) has been shown to affect transcription of the Kv2.1 (and Kvl.S) K+ channel genes in clonal pituitary cells (ibid.). 49-01-05: A number of systematic (comparative) in situ mRNA hybridization and protein immunolocalization studies have been published (for summaries, see the fields mRNA distribution, 49-13 and Protein distribution, 49-15). On the basis of these studies, Kv2 subfamily members have largely undetermined roles in vertebrate heart and skeletal muscle, while the significance of differential expression patterns in the nervous system are not well understood. In Drosophila, however, the Shab locus has been determined to be the exclusive gene underlying delayed rectifier currents in both muscle and neurones. In muscles, Shab removes 'virtually all' of the whole cell delayed rectifier current (IK ) without altering the transient A-current (encoded by the Shaker gene, see VLC K Kv1-Shak, entry 48). In neurones, the Shab mutation also removes most of IK , without altering the transient component (A-current, encoded by the Shal gene - see VLC K Kv4-Shal, entry 51). Selective cell-type expression of Shab splice variants appears to correlate with different electrophysiological phenotypes in identified lobster neurones (see mRNA distribution, 49-13). Within other invertebrates, Aplysia Kv2.1 has been shown to have a role in action potential broadening associated with peptide-secretory events from the bag cell neurones of Aplysia (see Phenotypic expression, 49-14). 49-01-06: Kv2.1 proteins show interesting patterns of cell- and subcellular localization. For example, rKv2.1 'monospecific' antibody immunocytochemistry has determined that rKv2.1/DRKl protein is restricted to neurones, where staining is present on proximal dendrites and cell bodies but not on axons or glial cells; for Kv2.2 immunoreactivity occurs in small cells which resemble interneurones (i.e. at axonal locations). There is increasing evidence for differential expression of Kv2.1 polypeptide length variants during brain development, and subcellular trafficking of channels has been observed (e.g. see effects of NCF, paragraph 49-01-04). There is some evidence that such plasma membrane 'targeting' of Kv2.1 during differentiation is dependent upon an C-terminal protein domains. Subunits of approximately 38kDa have been co-immunoprecipitated with Kv2.1 from native membranes; the relationship of these subunits to known Kv,B subunits is outlined under Protein interactions, 49-31. 49-01-07: Although the existence of alternative splicing of primary transcripts is implied through the differential transcript size classes observed on Northern blots (see Table 3, this entry), other explanations can account for these (see Cene organization, 49-20). A primary transcript from the single Shab gene in the lobster Panulirus interruptus does undergo alternate splicing to produce multiple transcripts with discrete cell-type localizations. Amino

_ entry 49

- - - - - - acid sequence alignments of the Shab-related isoforms rKv2.1 and rKv2.2, together with that of Drosophila Shab (e.g. Fig. 1, this entry) show open reading frame lengths of Kv2 subfamily members are relatively long compared to known members of the Kvl, Kv2 and Kv4 subfamilies (e.g. 857 aa, 132 kDa native Q subunit Mr for Kv2.1; compare the Encoding fields, 48-19, 50-19 and 51-19). The 'unusually long' C-terminus of Kv2 subfamily proteins (e.g. Kv2.1/443aa; Kv2.2/384aa); may underlie a role in subcellular targeting (see above). Cross-referenced examples of protein domain conservation/divergence and protein domain functions (as determined by structure-function analysis) appear in Tables 5 and 6, respectively. 49-01-08: Protein phosphorylation dependent on protein kinase A has been described as 'essential' for function/modulation of Kv2.1; mammalian Shabrelated isoforms retain a consensus site for phosphorylation by protein kinase C in the 84/85 intracellular loop (for background, see Protein phosphorylation, 49-32 and under VLG K Kv-Shak, 48-32). In vivo, phospholabelling of Kv2.1 occurs at an early stage in biosynthesis, presumably on the endoplasmic reticulum, as indicated from increased sizes of Kv2.1 expressed in COS-l cells that can be accounted for by protein phosphorylation alone. 49-01-09: Vertebrate Shab-related channels have been generally described as supporting 'slowly activating, slowly inactivating' sustained outward currents. In Drosophila, the Shab gene was often described (along with the Shaw gene) as encoding the 'delayed rectifier subtypes' in comparison to the 'transient (A-current) subtypes' encoded by Shaker and Shal. Despite marked primary sequence divergence, Kv2.1 and Kv2.2 currents show 'similar' (but not identical) properties. 'Typical' current responses of Kv2.1 and Kv2.2 appear under Current type, 49-34. Although not necessarily relevant to Shab channels in vivo, simple 'classification' of Shab-related channels as 'delayed rectifiers' does not take into account possible sensitivity of inactivation properties on 'accessory' subunits (see VLG K Kv-beta, entry 47) or effects of protein terminal deletions on channel properties (see Inactivation, 49-37). 49-01-10: Kv2.1 wild-type channels (or chimaeras containing segments of Kv2.1) have been used extensively in studies of K+ permeation/selectivity and blocker-binding sites. Ironically, this is partly due to Kv2.1's relative insensitivity to certain 'classical' blockers (and in consequence its ability to act as an 'acceptor' for 'donated' high-affinity binding sites/residues within chimaeric or mutant channels. Table 7 (see Selectivity, 49-40) attempts to summarize some conclusions/proposals from studies that have used Kv2.1 channels or their derivatives. Two peptides isolated from the venom of a Chilean tarantula, hanatoxin t (HaTxt) and hanatoxinz (HaTxz), unrelated in primary sequence to other K+ channel-selective toxins have been described as selective blockers of Kv2 subfamily channels. Structurefunction analyses have helped to define critical residues comprising binding sites for 4-aminopyridine, tetraethylammonium ions, H+, Zn2+ and external/internal Ba2+ ions. Notably, there are presently few examples of

entry49

_

I" - - - - - - - - - - - - - - - - '

heterologous coupling of Kv2 subfamily members to G protein-linked receptors, yet there is precedence for receptor-linked signalling affecting channel activities (see Receptor/transducer interactions, 49-49).

Category (sortcode) 49-02-01: VLG K Kv2-Shab, i.e. Q subunits of vertebrate voltage-gated potassium channels encoded by Kv gene subfamily 2 whose primary (amino acid) sequences show highest identity to those of Drosophila Shab (see Gene family, 49-05). Homomultimeric t channels formed from Kv2 Q subunits are normally named in accordance with the eDNA name (see Channel designation, 49-03). Assigned names of subfamily Kv2 gene (chromosomal) loci are listed under Gene mapping locus designation, 49-54.

Information sorting/retrieval aided by designated gene family nomenclatures 49-02-02: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: Kv2 (for reports or reviews on channel properties applicable to heterologouslyt expressed recombinantt subunits encoded by cDNAst or genes t ). For advantages of UBIs and guidelines on their implementation, see Resource T- Search criteria.

Channel designation 49-03-01: Vertebrate voltage-dependent K+ channel genes/cDNAs/channels related to Drosophila Shab are presently designated as Kv2.1 and Kv2.2 by the published nomenclature 1,2. Strictly, Kv genes t (i.e. chromosomal sequences) should be referred to by their gene locus designation (see Gene mapping locus designation, 49-54) although in practice 'Kv numbers' are used to conveniently 'specify' genes, mRNAs, cDNAs, cRNAs and protein subunit variants arising from or associated with expression of a given Kv gene product or locus.

Current designation 49-04-01: For currents conducted by homomultimerict channel assemblies in heterologous cells, this could follow the shorthand form I Kvn .n , where n.n is the Kv subfamily number of the gene encoding the monomer, although this type of designation is rarely used.

Gene family See background to the extended Kv family in this field under VLG K Kvl-Shak, 48-05.

49-05-01: Vertebrate Shab-related K+ channel subfamily 2 members are designated as Kv2.n by the agreed nomenclature 1,2. To indicate their

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Table 1. Kv nomenclature and original names in use for Shab-related vertebrate voltage-dependent K+ channel cDNAs/subunits (subfamily 2). cDNA Iclone' names (isolates) in common use are indicated in bold. See also table footnotes and VLC key facts, entry 41. (From 49-05-01) Kv designation

Human isoforms

Kv2.1 hKv2.1 hDRKI 3 ,4 Names in use and refs (notes 1 and 2)

Rat isoforms

Mouse isoforms

Other species

rKv2.1 DRK1 5

mKv2.1 mShab 6

Other Drosophila Shab 7,8 Xenopus Kv2.1

Xshab9 9 Aplysia Kv2.1

Aplysia Shab 10 Kv2.2 Names in use and refs

hKv2.2

rKv2.2 CDRK or cdrk (see note 3)

mKv2.2

Other Xenopus Kv2.2

Xshab12 9

Notes: 1. If the original nomenclature is used to identify a data source in this entry, the Kv nomenclature 1,2 also appears. Original names are described as isolates within the entries, e.g. 'the isolates mShab and DRK1'. 2. Generally, only supplementary or contrasting category information for channel'equivalents' are listed in entries. 3. CDRK is derived from ~ircumvillate papilla !lelayed !ectifying ~+ channel (for further description, see Cloning resource, 49-10 and Isolation probe, 49-12). 4. Note the relationship of the cDNA clone IK8, which limited phylogenetic analysis has placed on the gene family branch leading to Shab (for details/ references on IK8, see VLC K Kvx, entry 52).

relatedness to the Sh superfamilyt of genes, some authors have used the designation ShII to designate mammalian homologues of Shab (Table 1). A listing of vertebrate Shab-related cDNAs encoding voltage-gated K+ channel subunits, with 'systematic' and 'isolate' (clone) names are given in Table 1.

EXPRESSION For references comparing expression determinants applicable to all Kv channel subfamilies, see the EXPRESSION section under VLC K Kv1-Shak, entry 48.

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Cell-type expression index Cell-type expression patterns implied using molecular probes specific for Kv2 family members are described under Cloning resource, 49-10, Isolation probe, 49-12, mRNA distribution, 49-13 and Protein distribution, 49-15.

Identified Shah or 'Shah-like' currents in native cells (examples) 49-08-01: 1. The delayed rectifier K+ current in Drosophila neurones and muscle has many similarities to the 'classical' Hodgkin-Huxley delayed rectifier. Mutant analysis has determined the channels underlying this current to be encoded by Shab 12 (see Phenotypic expression, 49-14). 2. Electrophysiological and pharmacological characteristics of the 'type II channel' studied in rabbit Schwann cells cultured from neonatal sciatic nerve and from the lumbar/sacral spinal roots are similar to that displayed by the mammalian Shab subfamily 13 (for brief description, see Cell-type expression index under VLC K DR [native], 45-14). 3. A Shab subfamily channel has been noted in rat cochlea 14 . 4. On the basis of in situ hybridization experiments, Kv2.2 channels play early roles in excitable tissues of developing Xenopus embryos, signals which correlate with in situ recordings (for further details see Developmental regulation, 49-11 and mRNA distribution, 49-13).

Channel density Apparent local 'clustering' of Kv2.1 protein as evidenced by immunolocalization studies 49-09-01: Kv2.1: Several descriptions of 'discrete' immunoreactivity patterns observed for Kv2.1 (described under Subcellular locations, 49-16) are indicative of high densities of Ilocalized' protein expression; this pattern is not observed for Kv2.2 staining (ibid.).

Importance of increased Kv current densities accompanying Xenopus neuronal differentiation 49-09-02: A developmental increase in density of delayed rectifier potassium current (IKv ) in embryonic Xenopus spinal neurones shortens action potential durations and limits calcium influx governing neuronal differentiation (for a brief review and further references, see ref.15; for likely mechanisms of K+ current increase, see Developmental regulation, 49-11).

Cloning resource Sources/positive controls for Kv2 cDNAs 49-10-01: rKv2.1/DRKl: RNA transcriptst generated from a 3.3-4.2kb sizeselected rat brain cDNA library5. mKv2.1/mShab: Kv2.1 transcripts can be detected in atrial tumour myocytes derived from transgenic mice (AT-l cells) at 1 day in culture 16 (for further details on AT-1 cells, see Cloning resource under VLC K eag/elk/erg, 46-10). rKv2.2/CDRKl: cDNA library prepared from circumvallate papillae (taste receptor cells) of rat tongue for partiallength sequences, followed by rescreening of a rat brain cDNA library with a 438bp fragment of the cdrk sequence (N-terminal) at high stringencytll.

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Developmental regulation Kv2.1 polypeptide isoforms exhibit discrete temporal patterning during neuronal development 49-11-01: Temporal expression patterns of Kv2.1 (drk1) mRNA has been studied in the developing rat brain17. The three size classes of Kv2.1 mRNA (4.4, 9.0, 11.5kb) show different patterns of expression between embryonic and post-natal developmental stages. In the embryonic brain, the 4.4kb Kv2.1 transcript is predominant, while in the adult brain the 11.5 kb Kv2.1 transcript is predominant. These changes in transcript profile can be correlated with changes in expression of Kv2.1 polypeptide isoforms (detected by immunoprecipitation t reactions using antibodies selective for the Kv2.1 C-terminal)17. These results suggest distinct physiological roles for the Kv2.1 polypeptide isoforms encoded by each transcript size class during development.

Segregation of Kvl and Kv2 mRNAs during development of Xenopus spinal neurone subtypes 49-11-02: Xenopus Kv2.1 and Kv2.2 (cloned in ref.9) induce expression of delayed rectifier potassium currents, which can determine action potential waveform in all Xenopus embryonic primary spinal neurones 9. Transcripts from both Kv2 genes are present in developing embryos; notably, however, only Kv2.2 mRNA is detectable in embryonic spinal neurones, localized to the ventral spinal cord neurones. By comparison, Kv1.1 transcripts are found in dorsal spinal cord neurones 18 (see also VLC K Kvl-Shak, entry 48). Note: Currents supported by 'heterologous' expression of Xenopus Kv2.1 and Xenopus Kv2.2 in Xenopus oocytes (as published in ref.9) are shown in Fig. 2 under Current type, 49-34.

Kv2.2 may underlie endogenous delayed rectifiers sometimes seen in Xenopus oocytes 49-11-03: Xenopus Kv2.2: ISH: Notably, Xenopus Kv2.2 mRNA is detectable in eggs from some female frogs and the properties of activation of Kv2.2 resemble the delayed rectifier current expressed endogenously in some oocyte clutches. Additionally, there are striking 'redistributions' of Kv2.2 transcripts in the course of early Xenopus embryo development from early gastrula (stage 9, 7 hours post-fertilization, where Kv2.2 is diffusely distributed in the dorsal lip of the blastopore), late gastrula (stage 12, 14 hours, where Kv2.2 localizes to the dorsal ectoderm and presumptive neural tissue) to the neurula (stage 19; 20 hours, where Kv2.2 is localized along the entire neural tube)9.

Single-cell RT-PCR establishing candidate gene expression associated with native current development 49-11-04: Xenopus Kv2.2: A developmental increase in density of delayed rectifier potassium current (IKv ) in embryonic Xenopus spinal neurones shortens action potential durations and limits calcium influx governing neuronal differentiation (for a brief review and further references, see ref. 15). Maturation of IKv depends on de novo mRNA synthesis19; in later

e_n_t_ry_49

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studies it was determined that Xenopus Kv2.2 (Shab) and Kvl.l (Shaker) transcripts are expressed 'heterogeneously' during the same period over which an 'homogeneous' development of I Kv develops in diverse types of Xenopus spinal neurones (maintained both in vivo and in culture)15. Kvl.l and 2.2 are candidates for generation of IKv (see note 1); independent correlation of gene expression with current properties shows that where Kv2.2 transcripts cannot be detected (see note 2) they have a characteristic voltage dependence of activation of native (mature) IKv 15 • Notes: 1. Kvl.l mRNA is detectable (see note 2) in a maximum ~ 30% of cells (while IKv is immature); Kv2.2 mRNA appears later in ~ 60% of mature neurones). 2. This study15 used a novel internal control depending on co-detection of a ubiquitously expressed 'housekeeping' gene (EF-la) along with Kv channel mRNA signals; this device controlled for (i) successful aspiration (sampling) of single-cellular mRNA and (ii) permitted reliable scoring of cells in which Kv gene transcripts were 'not detected'.

NGF changes Kv2.1 protein abundance/distribution without effects on mRNA levels 49-11-05: Redistribution of Kv2.1 to the growth cones of PC12 phaeochro-

mocytoma cells following treatment with nerve growth factor t (NGF) suggests a role for this channel in neuronal differentiation (for details, see Subcellular locations, 49-16). Notably, RNAase protection assays have indicated that increases in Kv2.1 protein that follow NGF treatment (see note) occur without an increase in steady-state levels of Kv2.1 mRNA following NGF treatment20 . Note: As detected by immunoblot, Kv2.1 polypeptide elevation is observable 'within 12 hours', while elevated levels are maintained for 'at least 6 days' during continuous NGF treatment20.

TRH enhances excitability by inhibiting K+ channel gene expression in GH3 pituitary cells 49-11-06: The neuropeptide thyrotropin-releasing hormone (TRH) decreases

transcription of the Kv2.1 (and Kvl.5) K+ channel genes in clonal pituitary cells. The primary effect on mRNA can be observed at the protein level within a 12 hour period as judged by immunoblottini1, and this can be correlated with a decrease in voltage-gated K+ currents. These results indicate how neuropeptide regulation of K+ channel gene expression can produce long-term changes in neuronal action potential activity and synaptic transmission21 . Such 'prolonged' changes are additional to the familiar 'rapid' functions of neurotransmitters (e.g. altering action potentials by 'rapidly' modulating K+ channel gating in neuronal, endocrine, and cardiac cells).

Regulation of Kv polypeptide abundance throughout in vitro versus in vivo development 49-11-07: rKv2.1; rKv2.2: A comparative study relating differential spatio-

temporal expression of Kv2.1, Kv2.2 (plus Kvl.4, Kvl.5 and Kv4.2) polypeptides in developing hippocampal neurones in situ versus in vitro has appeared22 (for brief description, see 'Monotonic development of Kv protein expression patterns in developing hippocampus' under Developmental regulation of VLC K Kv1-Shak, 48-11). Differences between levels of

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Table 2. Reported tissue distributions of Kv2 subfamily mRNAs (From 49-13-01) Kv2.1

49-13-02: rKv2.l/DRKl: Northerns: Aorta: + + +. Atrium: + +. Brain: + + + +. Liver: -. Skeletal muscle: -. Ventricle: +. With probes for the 3' UTR t the expression level of DRKI was reported to be higher in heart and skeletal muscle than in brain or any other tissue23 (see also Transcript size, 49-17). ISH: DRKI mRNA is 'homogeneously' distributed in brain11,23 with marginally higher expression levels in the piriform cortex, the olfactory tubercule, hippocampal regions CAl through CA3, the dentate gyrus, and the medial habenular nucleus. DRKI is expressed at low levels in cerebellum and lower levels in the brainstem23 . 49-13-03: rKv2.1: Using a quantitative RNAase protection t assay comparing the abundance of 15 different potassium channel mRNAs in rat cardiac atrial and ventricular muscle24, Kv2.1 was one of only five (Kv1.2, Kv1.4, Kv1.S, Kv2.1 and Kv4.2) that were judged to be expressed at 'significant levels' (see also mRNA distribution under VLC K Kv4-Shal, 51-13). A comparative expression assay of five K+ channel subunit mRNAs (Kv1.2, Kv1.4, Kv1.S, Kv2.1 and Kv4.2) also analysed by RNAase protection assay in the rat ventricle has appeared25 (see also mRNA distribution under VLC K Kv1-Shak, 48-13).

49-13-04: Ferret Kv2.1/Kv2.2: ISH: Fluorescent-labelled oligonucleotide probes for Kv2.1 and Kv2.2 have been used to perform in situ hybridization studies on enzymatically isolated myocytes from subregions of ferret heart (including the sinoatrial node (SAN), right and left atria, right and left ventricles, and interatrial and interventricular septa)26. In general, this technique can detect transcripts for Kv2.1 (at moderate abundance) and Kv2.2 in working myocytes (for further details, see mRNA distribution under VLC K Kv1-Shak, 48-13). Kv2.2

49-13-05: rKv2.2/CDRK1: Northerns: Greatest density of CDRK1 mRNA occurs in the olfactory bulb, followed by the cerebral cortex, hippocampus and cerebellum 11 . In peripheral tissues, CDRK and DRKI (Kv2.1) mRNAs appear to be 'reciprocally distributed'll as estimated by densitometric scans of Northern blots, with relative expression levels as follows (CDRK1/DRK1): cardiac atrium (0/1.1); cardiac ventricle (0.1/1.7); skeletal muscle (0/1.3); olfactory epithelium (0.2/1.0); retina (0.1/2.6); kidney (0.2/1.9); tongue epithelium (1.3/0.4); circumvallate papillae (1.2/0.2); liver (0/0)11. ISH: Localized distribution patterns suggests the channel mediates functions that are concentrated in distinct subregions: 'Prominent' signals in the olfactory bulb (localized to the granule cell layer) and the cerebellum (granule cells)ll. Strong signals are also evident in the hippocampus throughout the CAI-CA4 region (pyramidal cells), as well as in the dentate gyrus (granule cells). High levels of CDRK1

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Table 2. Continued mRNA are also detected in the amygdala, piriform and enterorhinal cortices. 'Moderate' CDRKI mRNA densities are detectable in the olfactory tubercle, layers of the cerebral cortex, and brainstem. CDRKI probes detect a low level of expression in the remaining regions of brain. No CDRK mRNA signals can be detected in the molecular layer of cerebellum 11 .

expression for all subtypes studied except Kv2.1 indicate additional mechanisms operating in situ that are absent in vitro that play important roles in determining polypeptide abundance22 .

Isolation probe 49-12-01: rKv2.1/DRKl: Originally obtained5 using sequence-independent (expression cloning t ) methods involving injection of Xenopus oocytes with pools of cRNA t transcripts generated from a cDNA library (see Cloning resource, 49-10). rKv2.2/CDRK: A 438 bp PCR-generated fragment from the cdrk sequence, originally obtained from a rat circumvillate papillae (tongue epithelium) cDNA libraryll. This probe did not retrieve any full-length sequences from the original library, but did retrieve a full-length sequence (ORF 2406 nt, 802 aa) from a rat brain cDNA library screened at high stringency t.

mRNA distribution 49-13-01: A summary of reported tissue distributions for Kv2 subfamily mRNAs appears in Table 2; see also evidence for 'segregation' (i.e. differential expression) of Kv2.1 and Kv2.2 into spinal cord neuronal subtypes during Xenopus development under Developmental regulation, 49-11.

Differential splicing of the Shah gene product in identified lobster neurones 49-13-06: ISH: Expression patterns of alternatively spliced t products of the single Shab gene in identified neurones of the lobster pyloric network have been determined by in situ hybridization techniques27. Selective cell-type expression of these splice variants appears to correlate with different electrophysiological phenotypes (e.g. in identified neurones of the pyloric network of the stomatogastric ganglion). The Shab gene is 'consistently expressed' at a low abundance in the ventricular dilator cell, but at a high abundance in the pyloric dilator cell; no expression can be detected in the the lateral pyloric or inferior cardiac cells. Interestingly, Shab gene expression in the anterior burster cell is inconsistent, and reportedly varies from animal to animal; similarly, the 'electrophysiologically heterogeneous' group of eight pyloric constrictor cells also displays differential expression27.

_'---

e_n_try __ 49_

Phenotypic expression For likely roles of Kv2 subfamily channels in early embryonic/neuronal development, see Developmental regulation, 49-11.

The major delayed rectifier is encoded by Shah in Drosophila neurones and muscle 49-14-01: In Drosophila, the Shab locus has been determined to be the exclusive gene underlying delayed rectifier currents in both muscle and neurones 12. In muscles, Shab removes 'virtually all' of the whole-cell delayed rectifier current (I K ) without altering the transient A-current (encoded by the Shaker gene, see VLG K Kv1-Shak, entry 48). In neurones, the Shab mutation also removes most of IK , without altering the transient component (A-current, encoded by the Shal gene - see VLG K Kv4-Shal, entry 51). In addition to the major delayed rectifier current associated with Shab gene products, the Shaw gene contributes a relatively small 'leak' current to most neurones and muscle cells. Thus A-currents are encoded by distinct genes in muscle (Shaker) and neuronal cell bodies (Shal), the predominant I K in both muscle and neurones is encoded by the same gene (Shab)12. Comparative note: Selective cell-type expression of Shab splice variants appears to correlate with different electrophysiological phenotypes in identified lobster neurones28 (for examples, see mRNA distribution, 4913). General note: Among the many mammalian K+ channel cDNAs that express IK-like currents in oocytes, those from Kv2.1 (DRKl)-specific templates produce current that 'most closely resembles' neuronal I K in voltage-dependence, kinetics of activation, pharmacological characteristics, and lack of inactivation over tens of milliseconds 5 ,29.

Aplysia Kv2.1 contributes to action potential broadening in peptidergic neurones 49-14-02: Changes in action potential shape (Le. height and width) are associated with peptide-secretory events from the bag cell neurones of Aplysia. These 'electrically homogeneous' neurones control reproductive behaviour, in that they are normally silent, but in response to brief synaptic stimulation, they generate a train of action potentials (extending to approx. 30 min) resulting in multiple neuropeptide secretion (including neuropeptide Y and egg-laying hormone (for refs, see ref. 10). Currents detected following expression of a cDNA encoding Aplysia Shab (aKv2.1, whose mRNA can be detected in bag cell neurones) match the voltage dependence and kinetics of IK2, one of the two delayed rectifiers in bag cell neurones. In common with I K2 , Aplysia Shab current inactivates within several hundred milliseconds, and occurs by a process whose properties do not match those previously described for C-type and N-type mechanisms 1o. Computational modelling of native versus heterologous expression data predicts that Aplysia Shab current contributes to the normal enhancement (broadening) of action potentials that occurs in the bag cell neurones at the onset of neuropeptide secretion10 (as above). Notes: 1. The action potential broadening phenotype (above) contributes to the progressive potentiation of neuropeptide release; this mechanism involves multiple distinct phases and

l_e_n_t_ry_49

_

is known to involve complex second messenger modulation of both outward K+ and inward Ca2+ channels (as reviewed in ref.30). 2. See also paragraph 4955-01 under Miscellaneous information, 49-55.

Protein distribution Kv2.1 proteins are expressed in neurones but not in glial gells or

axons 49-15-01: rKv2.1/DRK1: 'Monospecific' antibodies for the DRKI polypeptide have been used to stain sections of adult rat brain cortex. These studies have revealed that rKv2.1/DRKl protein is restricted to neurones, where staining is present on proximal dendrites and cell bodies but not on axons or glial cells 29.

Contrasting immunolocalizations for Kv2 subfamily proteins 49-15-02: In a direct comparative study31, Kv2.1 (DRK1) immunoreactivity in the cerebral cortex was localized to pyramidal cells, whereas Kv2.2 (CDRK, the 'circumvillate papilla delayed rectifier' - see Cene family, 49-05) immunoreactivity occurs in small cells which resemble interneurones (i.e. at axonal locations )(see also general patterns ofimmunoreactivities described under Subcellular locations, 49-16). For evidence relating expression patterns of Kv2.1 polypeptide variants throughout rat brain development, see Developmental regulation, 49-11. Polyclonal antibodies against Kv2.1 have been used as part of a comparative study describing differential expression of five Kv channels (Kvl.2, Kvl.4, Kvl.5, Kv2.1 and Kv4.2) in adult rat heart32 (see Protein distribution under VLC K Kv1-Shak, 48-15).

Subcellular locations C-terminal-dependent plasma membrane targeting of Kv2.1 differentiation 49-16-01: rKv2.1/DRK1: Kv2.1 has been immunolocalized to high-density plaques'exclusively' on the cell body and the proximal portions of dendrites t of central neurones. In the PC12 phaeochromocytoma cell model, undifferentiated cells localize endogenous Kv2.1 to the cell-cell adherent junction and cell-substratum interfaces. Strikingly, PC12 cells that have been differentiated with nerve growth factor t (NGF) redistribute Kv2.1 to the growth cones t of extending neurites t2o,33. Levels of Kv2.1 polypeptide in PC12 membranes increase 4-fold as part of the NGF response. When PC12 cells are transfected with a mutated version of Kv2.1 lacking the C-terminal 318 amino acids, the truncated channel does not localize to the cell-cell adherent junction, but is 'non-specifically' distributed throughout the plasma membrane2o,33. Comparative notes: 1. The NGF response of PC12 is a well-defined model for subcellular trafficking of K+ channels during differentiation; within the Kv1 (Shaker-related) subfamily, precedents exist for involvement of Kv{3 subunits in trafficking and membrane insertion of Kvo: subunits (for details, see Kv{32 under VLC K Kv-beta, entry 47; see also notes on Kv{3 subunits under Protein interactions, 49-31). 2. A significant co-localization of fodrin t and actin t has also been observed in this PC12 model. 3. Kvl.5 protein (see VLC K Kv-Shak, entry 48) exhibits a

_L-

e_n_try __ 49_

markedly different localization to Kv2.1 in PC12 cells. 4. Compare evidence for C-terminal ~subcellular targeting' domains in alternatively spliced products of the Kv3.2 gene in Subcellular locations under VLG K Kv3Shaw, 50-16.

'Discrete' versus 'diffuse' localizations of Kv2.1 and Kv2.2 49-16-02: Direct comparison of immunohistochemical staining using antibodies selective for (Kv2.1) DRK1 and Kv2.2 (CDRK, the 'circumvillate papilla delayed rectifier') (see Gene family, 49-05) has revealed striking subcellular localization patterns31,34. Whereas DRK1 tends to show discrete immunoreactivity in cell bodies (soma) and proximal dendrites (dendritic processes), CDRK appears diffusely distributed in cell bodies but is also localized to fibres in some brain areas 31,34 (see also Protein distribution, 4915). In an independent studr9, the 'specific immunoreactivity' for Kv2.1 was detectable in cortical neurones (see Protein distribution, 49-15) and appeared to be cell surface associated, which supports the possibility that DRK1 protein plays a role in surface K+ conductive pathways in the plasma membrane of cortical neurones.

A Kv2.1 cytoplasmic domain associated with polarized expression and clustering 49-16-03: Expression of wild-type Kv2.1 and two Kv2.1 carboxy-terminal truncation mutants (~C318 and ~CI87) in a model polarized t kidney epithelial cell-type (MDCK) display different subcellular localizations 77: (i) Both wild-type Kv2.1 and ~C187 localize to the lateral membrane in high does not localize, but expresses uniformly density clusters while (ii) ~C318 on both apical and lateral membranes. However, when the influenza virus hemagglutinin protein antigen is fused to the segment of Kv2.1 that differentiates the two truncation mutants (aa 5~~6-666), expression in MDCK cells shows polarized expression and high-density clustering of the chimaera (Le. in a manner similar to that observed for wild-type Kv2.1). Notably, polarized expression and clustering of Kv2.1 correlates with detergent solubility, suggesting that interaction with the detergent-insoluble cytoskeleton t may be required for the correct subcellular localization of Kv2.1 subunits 77.

Cell surface 'clusters' of Kv2.1 protein expression in COS cells 49-16-04: Immunofluorescent localization of Kv2.1 in COS cells shows intense surface labelling with no intracellular pools of 'retained' protein apparent35 . Immunogold electron microscopy confirms that the expressed Kv2.1 polypeptide is found on the cell surface in small clusters or patches of 10-15 gold particles35 (see also Protein phosphorylation, 49-32 and Miscellaneous information, 49-55).

Kv subtypes localized to specific subcellular domains within individual retinal neurones 49-16-05: Comparative note: As part of a large study identifying and

localizing five Kv subunits in the mouse retina, Kv2.1 (together with Kv1.2 and Kv1.3) showed variable subcellular distribution depending upon cellular context (in contrast to consistent localizations of Kv1.4 (to axonal portions)

lL...--_ _ _ry_4_9

_

en t

Table 3. mRNA transcript sizes derived from Kv2 subfamily K+ channel genes (From 49-17-01) Kv2.1

49-17-02: rKv2.1/DRKl: Two transcripts, r-v4 kb and r-vl0 kb hybridize to DRKI probes37. With probes for the 3' UTRt a prominent band (cardiac) or weak band (adult skeletal muscle) at 4.3 kb is visible in RNA from neonatal and adult tissues. In brain, several bands between 4 and 6 kb (lower intensity) can be detected23 . A minor band for DRKI at 8.9kb has been reported 11 . 49-17-03: rKv2.1/DRKl: In an independent study, Northern t analysis revealed three size classes (4.4, 9.0, 11.5 kb) of Kv2.1 K+ channel transcripts are detectable in rat brainj the proportion and expression patterns of these change through rat brain development (for details, see ref.17 and Developmental regulation, 49-11).

Kv2.2

49-17-04: rKv2.2/CDRKl: Prominent transcripts in poly(A)+ mRNA from rat brain (olfactory bulb) at 13.5 kb and 6.2 kbj weaker bands at 8.0kb and 3.7kb (see also mRNA distribution, 49-13).

and Kv4.2 (to somatodendritic portions)36. Overall these results suggested that 'each K+ conductance responds uniquely to local voltage and second messenger signals'. For further information on Kv1 and Kv4 subcellular targeting, see this field under VLC K Kv1-Shak, 48-16 and VLC K Kv4Shal, 51-16.

Transcript size 49-17-01: Kv2.1; Kv2.2: No extensive study of transcriptional control/ mapping of mammalian Kv2 family genes was published at the time of entry compilation. Although the existence of alternative splicing of primary transcripts is implied through the differential transcript size classes observed on Northern blots, other explanations can account for these (see Cene organization, 47-20). Listings of mRNA transcript sizes reported for Kv2 subfamily genes appear in Table 3.

SEQUENCE ANALYSES For references comparing sequence analyses applicable to all of the Kv channel subfamilies, see the SEQUENCE ANALYSES section under VLC K Kv1-Shak, entry 48.

Chromosomal location 49-18-01: Table 4 summarizes chromosome locations of Shab-related K+ channel genes in the human and mouse genomes. For further background to these studies and their evolutionary implications, see Chromosomal location under VLC K Kv1-Shak, 48-18.

_

entry 49

1---

_

Table 4. Chromosome locations reported for Kv2 subfamily genes (From 49-18-01)

Consensus/summary

Further details

49-18-02: hKv2.l: Human chromosome 20q4, and Kv2.1 later by by fluorescence in situ hybridization of hKv2.I, KCNB1, Chr 20qI3.2 genomic PI clones to 20qI3.238 consistent with that mKv2.I, Kcnb1, Chr 2 predicted by mouse/man syntenyt. A polymorphic (no conflicts, no (GA) microsatellite repeat was identified in one of paralogyt) the PI clones. Assignment to 20qI3.2 eliminated KCNB1 as a candidate for the gene associated with benign familial neonatal convulsions (BFNC), which had been previously localized to 20qI3.3; KCNIB maps more than 30 cM proximal to the BFNC locus38 . 49-18-03: mKv2.l: Mouse chromosome 2, apparently unlinked to any other K+ channel gene39. The Kcnb1 gene maps close to the mutation wst (wasted) on the distal region of mouse chromosome 2, which is associated with neurological disease (see also this field under VLC K Kv1-Shak, 49-18). Kv2.2

49-18-04: hKv2.2: Not found.

hKv2.2, KCNB2, 49-18-05: mKv2.4: Mouse chromosome 1, at a not found proximal position very close to the centromeret39. mKv2.2, Kcnb2, Chr 1 See also IChromosomal mapping in the mouse of (no conflicts, no paralogyt) eight K+ channel genes representing the four Shaker-like subfamilies Shaker, Shab, Shaw and Shal,40. Note: 1. This column lists species equivalents (see also Table 8 under VLC K Kv1Shak, 48-18) notes on phenotypes (if known) and other cross-references. For sources of consolidated marker sets for all ion channel genes, see resources such as Online Mendelian Inheritance in Man (OMIM).

Encoding Sequence alignment of Kv2 subfamilJl members 49-19-01: An amino acid sequence alignment of the Shab-related isoforms rKv2.I and rKv2.2, together with that of Drosophila Shab is shown in Fig. 1. The open reading frame length of 857 aa for mouse, rat and human isoforms of Kv2.1 (see legend to Fig. 1) was originally reported as 853 aa5 but was reassigned following analysis of longer 5' untranslated regions 23 . The longer versions (i.e. containing four extra N-terminal amino acid residues (Met-ProAla-Gly) express I delayed rectifier type' currents indistinguishable from the original isolate discovered by expression cloning5 . The open reading frame lengths of Kv2 subfamily members are relatively long compared to known

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= t"1"

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150

rKv2.1 (ORK1 ) rKv2.2(CORK)

MPAG -AEICAPPG -VGQLQGGQAAGQQQQQQQATQQQQHSKQQLQQQQQQQQQLQLICQHQQQQQDI LYQQHNEAIAIARGLQAATPADI GDNQPYYDTSGNWWERAMGAGGAGAYGGIGIGSLPAAGGAAYHLGPANPAGLVSRHLOYGOGGHLAGPSAGLP

rKv2.1(ORK1 ) rKv2.2(CDRK)

EPME I 300 LNRKT--- -L- - - - - -W- I - - -T- - - - -KI - - - - -N- - - - ---- -- --AGAVGSGAGAGAGAGASVTGSGSGAGTGTGTGAGSGSGSGAAGKEVRYAPFPVASPTHSIPTTSQQIVGGVGGVGVGGASSQSI SGGVPTHSQSNTTGALQRTH-- -M-- I - -P- -FM-AQ- - -VNS- -SI - - - -VR- - -- - ---E- --H

Shab

Shab

rKv2.1(ORK1 ) rKv2.2(CDRK)

Shab

rKv2.1(DRK1 ) rKv2.2(CORK)

Shab

rKv2.1(DRK1 ) rKv2.2(CDRIC)

Shab

rKv2. 1(ORIC1 ) rlCv2.2(CORIC)

Shab

rlCv2.1(ORK1 ) rlCv2.2(CORK)

Shab

rlCv2. 1(ORK1 ) rKv2. 2( CORK)

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~TKHGSRSTS~lPP

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lNFYRTGRlHMMEEMCAL~FSQELDY\lGIDE

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Figure 1. Amino acid sequences of the Shab-related isoforms rKv2.1 and rKv2.2, together with that of Drosophila Shabo Open reading frame lengths in the Kv2 subfamily include hKv2.1/hDRK1: 857 aa (not shown, see note in paragraph 49-19-01); rKv2.1/ DRK1: 857 aa; mKv2.1/mShab: 857 aa (not shown); rKv2.2/CDRK1: 802 aa. (Alignment kindly provided by George Gutman, University of California at Irvine.) (From 49-19-01)

II

~

_'----

e_n_try_4_9_ _

members of the KvI, Kv2 and Kv4 subfamilies (compare the Encoding fields, 4819, 50-19 and 51-19). Other notable features of the Kv2 subfamily sequences are outlined in Sequence motifs, 49-24, Protein phosphorylation, 49-32 and Domain conservation, 49-28. See also general features of Kv channel primary sequences denoted in entry 48. Note that individual nucleotide and/or amino acid sequences may be retrieved for local analysis using the accession numbers listed under Database listings, 48-53.

Gene organization Variable mRNA sizes of Kv2 gene products 49-20-01: Presently known vertebrate Shab-related proteins possess relatively conserved N-termini but relatively long and variable C-termini (see Domain arrangement, 49-27). At the time of compilation, no extensive account was found describing variation due to alternate splicing at the 3' end of the coding regions of mammalian Kv2 genes, although this remains one possibility (other formal possibilities would be alternative 5' transcript starts, or alternative polyadenylation sites). (See Transcript size, 49-17; compare this field under VLC K Kv3-Shaw, 50-20 and VLC K Kv4-Shal, 51-20).

The single lobster Shah gene product does undergo alternative splicing 49-20-02: A primary transcript from the single Shab gene in the lobster Panulirus interruptus undergoes alternate splicing to produce multiple transcripts28 . Selective expression of these splice variants correlates within different electrophysiological phenotypes in identified neurones of the pyloric network in the stomatogastric ganglion (see mRNA distribution, 49-13).

Protein molecular weight (purified) Protein molecular size determinations following immunoblotting 49-22-01: rKv2.1/DRKl: Anti-DRKI antibodies have been reported to react with a native 130 kDa polypeptide on immunoblots of adult rat brain membranes29 . In separate experiments, anti-Kv2.1 antibodies recognize a single polypeptide population of 132 kDa polypeptide in phaeochromocytoma (PCI2) cell membranes (as distinct from a heterogeneous population which is detected with the same antibody in adult rat brain)2o. In vitro translation of the Kv2.1 (DRKl) cRNA yields a 'core' 95kDa polypeptide, similar to that predicted from a calculated Mr based on the open reading frame and consistent with a protein that has not been N-glycosylated t (see Protein molecular weight (calc.) 49-23 and Sequence motifs, 49-24).

Protein molecular weight (calc.) 49-23-01: rKv2.1/DRKl: 95.3kDa (95294Da) on the basis of the earlier5 853 aa protein sequence (now 857 aa - see Encoding, 49-19). rKv2.1/DRKl: 90.6 kDa (90645 Da). Note: DRKI is 'unusually large' for a Kv series polypeptide (see Domain arrangement, 49-27).

l_e_n_t_ry_49

----'_

Sequence motifs 49-24-01: rKv2.1/DRK1; rKv2.2/CDRK1: N-Gly: There are four consensus sequences for N-glycosylation but only one is likely to be functional, in the second S3/S4 extracellular loop (rKv2.1:aa 279, rKv2.1:aa 287). Note: The mammalian Shab sequences (but not the Drosophila Shab sequence) lack a consensus N-glycosylation site in the SI/S2 extracellular loop as commonly observed in the Shaker-related channels (for further comparisons, see Domain conservation, 49-28).

Southerns 49-25-01: Kv2 subfamily variants described thus far exist as single-copy genes t. Low-stringency t Southern blot t genomic analysis produces crosshybridizing bands which may represent hitherto unidentified members of the Kv2 subfamily ll.

STRUCTURE AND FUNCTIONS For references pertinent to all of the Kv channel subfamilies, see the STR UCTURE AND FUNCTIONS section under VLC K Kv1-Shak, entry 48.

Domain arrangement tUnusually long' C-termini of Kv2 subfamily proteins 49-27-01: rKv2.1/DRK1: The subunit domain arrangement of Shab-related K+ channels is generally similar to that illustrated in the protein domain topography model [PDTM] (Fig. 6) under VLC K Kv1-Shak, entry 48. Note, however the Kv2.1 (DRKl) ORFt encodes an N-terminal domain of 182aa, a I core' region of 232 aa and a C-terminal domain of 443 aa which is relatively long for the Kv subfamily members. For the dependence of subcellular targeting of Kv2.1 on the presence of the C-terminal, see Subcellular locations, 49-16. rKv2.2/CDRK1: In comparison, isolate CDRKI comprises an N-terminal domain (190 aa), core region (228 aa) and C-terminal domain (384 aa).

Domain conservation 49-28-01: Kv2.1/Kv2.2: Mammalian Shab-related isoforms show a striking conservation of the hydrophobic core region (discussed under VLC K Kv1Shak, entry 48) but the SI/S2 extracellular loop is represented by a short conserved segment (in the Shaker-related channels, this loop is highly variable in both length and sequence). Other comparative features are shown in Table 5.

Domain functions (predicted) 49-29-01: Examples of protein domain functions or 'phenotypes' (mostly derived or predicted by structure-function analysis) for Kv2 subfamily

III

_'--

e_n_try_4_9___

Table 5. Patterns of domain conservation/divergence in Kv2 subfamily K+ channel proteins (From 49-28-01) Feature/region

Comparison

N-terminus

An approx. 200 aa segment in the N-terminal region adjacent to Sl is 'highly conserved' in known mammalian isoforms. Drosophila Shab has an 'extra' 240aa N-terminal segment which displays some repetitive elements (poly-Glu, Q; poly Gly-Ala, GA - see Fig. 1 under Encoding, 49-19).

Hydrophobic core

Highly conserved (see paragraph 49-28-01).

81/82 extracellular loop

Short conserved segment (compare Shaker-related channels, see paragraph 49-28-01). No N-glycosylation motif present, but an N-gly motif is present in Drosophila Shab (see paragraph 49-24-01).

83/84 extracellular loop

Shorter and more highly conserved in Shab-related sequences than Shaker-related sequences. Motif for N-glycosylation is present in all presently known Kv2 subfamily sequences.

C-terminus

C-termini of Shab-related proteins are the longest of all Kv-family proteins (see Domain arrangement, 49-27). Shab-related C-termini show very little sequence conservation (see Gene organization, 49-20).

Other motifs

Described under Sequence motifs, 49-24 and Protein phosphorylation, 49-32.

members are given in Table 6 (cross-referenced to fields of the entry where further information appears).

Predicted protein topography 49-30-01: Kv2 subfamily channel subunits would be expected to form oligomeric complexes similar to those described for the Shaker-related subfamily (see VLG K Kv1-Shak, entry 48).

Protein interactions Putative domains mediating alternative subcellular targeting 49-31-01: The C-terminal domain of Kv2.1 has been implicated in mechanisms of subcellular targeting and protein localization in a nerve growth factor t (NGF)-differentiated PC12 phaeochromocytoma cell model (see Subcellular locations, 49-16 and compare evidence for C-terminal tsubcellular targeting' domains in alternatively spliced products of the Kv3.2 gene under Subcellular locations of VLG K Kv3-Shaw, 50-16).

II

lL....--_ _ _ry_4_9 en t

_

Table 6. Cross-referenced listing of domain functions predicted or inferred from structure-function/mutational/chimaeric analyses on Kv2 subfamily members (From 49-29-01) Function/motif/phenotype

See field cross-reference

ILocalized' protein expression

Channel density, 49-09; Subcellular locations, 49-16 Developmental regulation, 49-11 Subcellular locations, 49-16; Protein interactions, 49-31 Protein interactions, 49-31

Kv2.1 polypeptide variants C-terminal targeting

f3 subunit associations; oligomeric assembly mechanisms Phosphomodulation sites N-glycosylation motifs Inactivation mechanisms, 'minimal' channels, P-type inactivation Selectivity determinants/pore structure Inorganic and organic ion blocker binding sites Volatile anaesthetic modulation

Encoding, 49-19; Protein phosphorylation, 49-32 Sequence motifs, 49-24 Inactivation, 49-37

Selectivity, 49-40; Table 7 Blockers, 49-43 Channel modulation, 49-44

Preliminary evidence for novel Kv(3 subunits interacting with Kv2 family members 49-31-02: rKv2.1/DRKl: 'Monospecific' antibodies for the DRKI polypeptide identify a 38 kDa neuronal peptide which tightly associates with the channel protein29 . The 38 kDa protein is immunologically distinct from DRKI polypeptide, and the two proteins exist in a stoichiometric relationship as evidenced from relative amounts detected in co-precipitation experiments. The proteins are 'tightly associated' by non-covalent links, and the mobility of the DRKI peptide does not change upon reduction of disulphide bonds. The DRKl-associated 38 kDa protein has similar properties to f3 subunits associated with channels sensitive to the epileptogenic neurotoxins dendrotoxin and f3-bungarotoxin (see refs 41 ,42 and the fields Protein molecular weight and Protein phosphorylation under VLC K Kv1-Shak 48-22 and 48-32, respectively). Notably, however, interaction of the Kv-beta subunits Kvf31 and Kvf32 with members of the Kv2 (Shab-related) and Kv3 (Shaw-related) Q subunit subfamilies cannot be detected by immunoblot analysis (for refs, see VLC K Kv-beta, entry 47). The 38 kDa polypeptide co-immunoprecipitating with Kv2.1/DRKl subunits (above) most likely represents a distinct (i.e. novel) Kvf3 subunit.

Overview of Kv2 subunit assembly interactions (see also entry 49) 49-31-03: An N-terminal deletion mutant of Kv2.1 has been shown to form functional homomultimeric channels43; moreover, co-expression of this

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_9_

deletant with a similar N-terminal deletant from the Shaker subfamily (hKvl.4) can form functional hybrid channels (despite members of different K+channel subfamilies normally not co-assembling - see Protein interactions under VLC K Kv1-Shak, 48-31). These and other results 43 have been used to propose that the N-terminal of the Kv channels subserves two functions: (i) it provides a domain necessary for heteromultimeric (but not homomultimeric) channel assemblies within a subfamily and (ii) it provides a domain that can prevent co-assemblies between subfamilies. Notes: 1. Earlier studies44 of Drosophila Shab had established that Shaker, Shal, Shab and Shaw expressed independent K+ current systems as a consequence of such 'molecular barriers to heteropolymerization'. 2. Other studies using Kv2.1 subunits to delineate K+ channel assembly domains (e.g. refs 45- 47) are described under VLC K Kv1-Shak, 48-31.

Protein phosphorylation PKA-dependent phosphorylation appears lessential' for function/modulation of Kv2.1 49-32-01: rKv2.1/DRK1: The DRKI C-terminus contains two consensus sequences for cAMP-dependent phosphorylation (aa positions 440 and 492). Application of 8-Br-cAMP to oocytes expressing the ~NDRKI channel (with the first 139 amino acids of the N-terminus deleted) induces a voltage-independent elevation of current amplitude48 . This effect is not observed for wild-type (full-length) DRKI channels or endogenous current. Augmentation of current ~NDRKI can be blocked by pre-injection of oocytes with Walsh peptide (an inhibitor of protein kinase A). Walsh peptide also reduces both ~NDRKI and wild-type DRKI currents. Significantly, substitution of the serine residues by alanine at one or both of the two consensus PKA phosphorylation sites (above) on the C-terminus produced non-functional channels48 . rKv2.2/CDRKl: Two of ~NDRKI consensus cAMP-phosphorylation sites are present in rKv2.2 (aa 448 and 500) at homologous positions to those in rKv2.1.

Conserved PKC motifs in Kv2 subfarrlily proteins 49-32-02: Kv2.1/Kv2.2: Mammalian Shab-related isoforms retain a consensus site for phosphorylation by protein kinase C in the 54/55 intracellular loop

(for background, see Protein phosphorylation under VLC K Kv-Shak, 48-32).

In vivo phospholabelling of Kv2.1 occurs at an early stage in biosynthesis 49-32-03: COS-l cells transiently expressing rKv2.1 at high levels express a 108kDa polypeptide species which is larger than the size of the 'core' polypeptide synthesized in vitro (95 kDa, see Protein molecular weight (purified) 49-22). The increased size of Kv2.1 in COS-l cells appears to be due to a posttranslational modification that occurs early (tl/2 == 5 min) in biosynthesis (presumably on the endoplasmic reticulum). Strikingly, the increased size can be accounted for by protein phosphorylation alone (based on patterns of in vivo 32 P-Iabelling and sensitivity to alkaline phosphatase digestions)35. See also Subcellular locations, 49-16 and Miscellaneous information, 49-55.

1'--_e_n_t_ry_4_9

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ELECTROPHYSIOLOGY

Activation General features of Kv2 family currents 49-33-01: rKv2.l/DRKl: In the oocyte expression system, Kv2.l currents generally activate relatively slowly (requiring > 100 ms for full activation). Kv2.l channels open at test potentials more positive than - 20 mY and display sigmoidal activation which is voltage dependent (time to halfmaximal activation approx. 20-100 ms). See also effect of protein termini deletions on activation (cross-referenced under Domain functions, 49-29). 49-33-02: rKv2.2/CDRKl: Similar to DRKl, i.e. relatively slow-activating following voltage steps (see Current type, 49-34 and compare Shaker-related channels, entry 48); half-maximal activation '"'-'21 ± 2 ms after depolarization to Omy11 . See also rKv2.1 data as part of tKv channel surface potential related to activation midpoint potential' cited under Activation of VLC K Kv3-Shaw, 50-33.

Current type Despite marked primary sequence divergence, Kv2.1 and Kv2.2 currents are similar 49-34-01: Vertebrate Shab-related channels have been generally described as supporting 'slowly activating, slowly inactivating' sustained outward currents 5,11,49. In Drosophila, the Shab gene was often described (along with the Shaw gene) as encoding the 'delayed rectifier subtypes' (e.g. ref. 49) in comparison to the 'transient (A-current) subtypes' encoded by Shaker and Shal. Figure 2 summarizes Itypical' current responses of Xenopus Kv2.l and Xenopus Kv2.2 following 'heterologous' expression in Xenopus oocytes. Minor differences between these currents and those observed with mammalian Kv2 homologues in the Xenopus expression system are described in the legend to Fig. 2. Note: In observing ttypical' current characteristics, note that any Q subunit homomeric or heteromeric channel complex may be susceptible to functional modulation (see also paragraph 49-31-02).

Current-voltage relation Rectification behaviour induced by millimolar intracellular Mg2+ and Na+ ions 49-35-01: Kv2.l/DRKl: In intact oocytes, current-voltage relationships of Kv2.l (DRKl) display inward-going rectification at potentials> +100my51 . This rectification can be (i) abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions and (ii) restored by introducing Mg2 + or Na+ ions into bath solutions. The inward rectification induced is not directly comparable to that observed in the strongly rectifying inward rectifier (Kir) channels (see INR K [subunits], entry 33) since 'highmillimolar' concentrations of intracellular ions are required for appreciable

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e_n_try_4_9_

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u - 3 -J---:;"-~~-~==;===;===='l -120 -100 -80

-60

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-20

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20

Membrane Potential (mV)

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-0.5 -1--...--...---..-------,..-----,--50 -25 0 25 SO 75 11

Voltage (mV)

Figure 2. Xenopus Kv2.1 and Xenopus Kv2.2 following expression in Xenopus

oocytes. For records (a) and (b), currents were elicited following injection of cRNAs t in response to 60 ms voltage steps to potentials ranging from -50 to +100mV from a holding potential of -BOmV. (c) Comparison of kinetics of current activation by superimposition of records (a) and (b), illustrating that the Kv2.1 cRNA-induced current (dotted line) rises faster than the Kv2.2 cRNA-induced current (solid line). (d) Tail current analyses for Xenopus Kv2.1 channels indicating that they are K+ selective (i.e. varyinf external [K+ J shifts the reversal potential as predicted by the Nernst

l'---e_n_t_ry_4_9

_

block of Kv2.1 (e.g. half-maximum blocking concentrations 4.8 ± 2.5 mM for Mg2 + and 26±4mM for Na+ at +50mV). For Kv2.1, increasing [K+]o reduces the degree of rectification of intact cells51 . Note: hKv2.1/h-DRKl: It has been noted that the instantaneous t current-voltage relationship of hKv2.1 in oocytes rectifies outwardly to a higher degree than predicted by the Goldman-Hodgkin-Katz t equation5o .

Inactivation 49-37-01: rKv2.1/DRKl: Following its original cloning and heterologous expression in oocytes, DRKI was arbitrarily defined as a 'delayed rectifier' as it 'did not inactivate within 500 ms'. Although not necessarily relevant to Shab channels in vivo, this simple type of 'classification' did not anticipate later findings on sensitivity of inactivation properties on accessory subunits (see VLG K Kv-beta, entry 47) or effects of protein terminal deletions on channel properties (see next papargraph and the cross-referenced list under Domain functions, 49-29).

Kv2.1 N- and C-terminal domain functions and 'minimal' channels 49-37-02: rKv2.1/DRKl: Deletions t in either the cytoplasmic N-terminal segment (182aa) or the C-terminus (443aa) of the DRKI protein affect activation and inactivation properties of the expressed channels in oocytes 52 . Relatively small deletions at the N-terminus (e.g. 16 aa) or a large deletion at the C-terminus (e.g. 318 aa) result in minor shifts of the voltage dependence of

equation for a K+ -selective channel). (e) Current amplitudes in the presence of variable concentrations of TEA (see Blockers, 49-43). Xenopus Kv2.1 and Kv2.2 currents are less sensitive to TEA than the Kv2 mammalian homologues (compare millimolar TEA required to block 500/0 of Xenopus channels Kv2.1 (40mM) and Xenopus Kv2.2 (25mM) compared to 5-10mM for rat and human Kv2 isoforms). (I) Normalized conductance versus voltage relationships for Xenopus Kvl.l, Kvl.2, Kv2.1, Kv2.2 cRNAinduced currents. Conductance, G, was calculated for a given voltage command, ~ and its corresponding steady-state current response, I, from the formula G == I/(V - EK ), where the equilibrium potential for K+(E K ) was -116mV (for background, see Glossary and VLG key facts, entry 41). Half-maximal activation (V 1/ 2 ) was achieved at values of +25.0 ± 0.1 m V (mean ± SD, n == 7) for Xenopus Kv2.1 currents and at +30.0±5.5mV (mean ± SD; n == 3) for Xenopus Kv2.2 currents. Record (f) illustrates how Xenopus Kvl currents achieve Gmax at less positive membrane potentials than do the Xenopus Kv2 currents. Xenopus Kv2.1 and Kv2.2 data 'deviate consistently' from the Boltzmann isotherm as described for human Kv2.1 channels expressed in oocytes50 (see Current-voltage relation, 49-35). (For details of recordings and current fitting see Burger and Ribera (1996) J Neurosci 16: 1412-21, from which this figure was used with permission.) (From 49-34-01)

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activation and inactivation in the hyperpolarizing direction. Larger deletions at the N-terminus (e.g. 101 or 139 aa) remove inactivation and markedly slow activation and deactivation (Le. the steady-state activation curvet shifts approx. ",20-30 mV in a depolarizing direction). Unexpectedly, kinetics are largely restored in truncated DRK1 channels that have most of both N- and C-termini removed (e.g. the 396 aa mutant channel ~N~C, which has 139 aa deleted from its N-terminus and 318 aa deleted from its C-terminus). These studies52 concluded that the N- and C-termini are not essential for voltage-dependent activation, inactivation and deactivation, although they can have profound modulatory effects upon these properties. These experiments also defined a 'minimal DRKI channel' only marginally longer than that encompassed by the 'core' Sl-S6 domains (see Amino acid composition under VLC K Kvl-Shak, 48-26). The 396aa mutant channel cDNA ~N~C thus encoded 'sufficient protein' for the formation of channels with kinetics of a delayed rectifier52 . Supplementary note: Since wild-type Kv2.1/ DRK1 is a slowly inactivating channel (see Current type, 49-34), these results did not necessarily contradict the inactivation 'ball domain' hypothesist proposed for fast-inactivating channels (see next paragraph).

Inactivation determined by a single site in K+ pores tP-type-inactivation' 49-37-03: Kv2.1: Mutation of position 369 within the P-region of slowinactivating, wild-type Kv2.1 can produce fast inactivation with properties distinct from both N-typet or C-typet inactivation53 (see also Inactivation field under VLC K Kvl-Shak, 48-37).

Selectivity A series of postulates underlying the proposal of a structural motif for the Kv2.1 channel pore appears in ref. 54. In press update: These results should

be compared to those of the first crystal structure for a K+-selective channel (ref. 79 ).

Kv2.1 has been used extensively in studies of K+ channel selectivity 49-40-01: In general, all Shab-related isolates show high selectivity of K+ over

Na+ ions (Le. varying the external K+ concentration shifts reversal potential with a slope of ",48 mV per 10-fold change in external K+ concentration). Many mutagenesis studies (several involving Kv2.1 wild-type channels or chimaeras containing segments of Kv2.1) have identified a region of the S5-S6 loop of voltage-gated K+ channels (P-region) responsible for permeation/selectivity properties and other functions such as TEA+ block (for details, see Selectivity under VLC K Kvl-Shak, 48-40 and Blockers, 48-43). The detailed nature of these studies (and reliance on specific residue numbering systems) precludes a simple comparative listing for structure-function determinants. Table 7, however, attempts to summarize some conclusions from studies that have used Kv2.1 channels or their derivatives to answer general questions relating to selectivity.

II

1__e_n_t_ry_49

_

Table 7. References detailing properties of Kv2 subfamily selectivity functions (From 49-40-01). In press update: see also crystal structure of the protype K+ -selective channel described in ref, 79. Kv

Brief description of use/application and references

Kv2.1

Use of Kv2.1 chimaeras in establishing character of pore-lining residues

49-40-02: rKv2.l/DRKl: Point reversions t have been made in chimaeric DRKl-based channels (which differ by 9 aa from DRKl) to 're-establish' Kv2.l pore properties55 . These studies helped establish that K+ -selective pores have (i) an inner, deep region where the permeation properties of ions are regulated by interactions with non-polar amino acid residues and (ii) an outer region where ions may be regulated by charged residues 55 .

Kv2.1/Kv3.1

CHM

A leucine residue acting as a tK+ -to-Rb+ conductance

switch'in deep pores 49-40-03: The chimaera CHM (formed by replacing the pore region of Kv2.l with that of Kv3.l) displays ion conduction properties which resemble Kv3.l. A point mutation in CHM (L374V), restores the channel to a Kv2.l phenotype56 (see note below) and switches the K+ /Rb+ conductance profiles. 'Quantitative restoration' of the Kv2.l K+ /Rb+ profiles actually requires simultaneous point mutations at three non-adjacent residues, suggesting the possibility of interactions between residues within the pore56 . This study concluded that (i) Leu374 is responsible for differences in K+ and Rb+ conductance between Kv2.l and Kv3.l and (ii) the location of Leu374 (close to residues critical for block by internal TEA) are consistent with it forming part of a cationbinding site deep within the pore56 . Note: In CHM, K+ conductance (GK ) is three times greater than that of Kv2.l and GRb/GK == 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.l and Kv3.l). 49-40-04: In both CHM and Kv2.l, channels with the substituted hydrophobic residues Valor lIe at position 374 express Rb+-preferring pores while channels with the substituted polar residues Thr or Ser express K+-preferring pores57 (see also internal TEA blockade under Blockers, 49-43).

Kv2.1

Overlap of external selectivity determinants and external TEA + -binding site in Kv2.1 49-40-05: Orientation of the 'critical residue' 369 was determined (with respect to the aqueous lumen of the pore) by replacing the non-polar lIe at 369 (in Kv2.l) with a basic His. The Ile369His substitution produced a Cs+-selective channel with Cs+ :K+ permeability ratio of 4 (compared to 0.1 in the wild type)58. On the basis of these and other

II

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Table 7. Continued Kv

Brief description of use/application and references

Kv2.1

experiments concluding that position 369 forms part of binding site for external TEA+ blockade (see Blockers, 49-43), it has been suggested that the imidazole side-chain of His369 is exposed to the aqueous lumen at a surface position near the external mouth of the pore 58 .

Kv2.1

Residues exposed at Kv2.1 channel outer and inner mouths

49-40-06: Cysteine substitution/scanning mutagenesis t experiments within the carboxyl half of the pore-forming region of Kv2.1 have indicated position 378 to be exposed at the outer mouth of the channel, while position 374 is exposed at the inner mouth, with both residues immersed in the aqueous phase59 . These findings and other data are consistent with the aa 383-369 region spanning the pore as a non-periodic stmcture. Independent Cys substitution experiments in Kv2.1 have indirectly indicated that the side-chain at position Cys393 (in transmembrane domain S6) is involved in (i) conformational changes during transitions between open and closed states and (ii) the control of ion permeation6o . By domain swopping t experiments61 , it was determined that both transmembrane domains S5 and S6 contribute to the inner mouth of the Kv2.1 pore; different residues regulate ion conduction and blockade by internal TEA and 4-AP (see Blockers, 49-43).

Voltage sensitivity Comparative analysis of S4 regions 49-42-01: Specific differences in the charge content of the 84 region between mammalian RCKI (Kvl.l) and the Drosophila Shabll have been determined as I sufficient' to account for the distinct gating valence t of each channel62. Residue differences in the S4 region alone cannot, however, account for each channel's characteristic voltage range of activation62. See also effects of channel protein termini deletions on voltage dependence of activation (cross-referenced in Domain functions, 49-29). PHARMACOLOGY

Blockers A new class of peptide inhibitors of Kv2 family channels 49-43-01: rKv2.1/DRKl: Two peptides isolated from the venom of a Chilean tarantula, hanatoxin 1 (HaTx1 ) and hanatoxin2 (HaTx2), unrelated in primary

l_e_n_t_ry_4_9

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sequence to other K+ channel-selective toxins have been described 78 as 'selective' blockers of Kv2 subfamily channels. Shaker-related, Shaw-related, and eag K+ channels are relatively insensitive to HaTx. Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx. Prior to the hanatoxins, no potent (or selective) blockers were available for Kv2 subfamily channels. Typically, 500/0 inhibitory concentrations (IC so ) for 'classical' K+ channel blockers include 4-aminopyridine (4-AP, 0.5 mM, see next paragraph); tetraethylammonium ions, TEA+ (10mM)5. Kv2.1 in oocytes apamin (~1 M) and CTx (~1 M). is insensitive to C0 2 + (~2 mM), Cd2 + (~0.2mM), 'High-millimolar' intracellular Mg2 +and Na+ has been reported to induce inward rectification {for brief description, see Current-voltage relation, 4935. rKv2.2/CDRK1: TEA ICso ",,7.9± 1.7mM (mean SEM, n == 5)11.

Definition of 4-aminopyridine (4-AP}-binding sites in the inner mouth of K+ channels 49-43-02: Critical regions for binding of 4-AP have been identified by segmental exchanges using chimaeric channels using the tlow-affinity block' phenotype of Kv2.1 (IC so 18 mM) replaced with thigh-affinity block' phenotype of Kv3.l (IC so 0.1 mM)63. 4-AP sensitivity was not transferred with the S5-56 linker (pore or P-region), but a chimaera encompassing the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A 'double chimaera' of the cytoplasmic halves of 55 and 56 'fully transfers' 4AP sensitivity. Note: Since 4-AP block is inhibited by tetrapentylammonium, it was concluded that determinants of 4-AP binding were associated with the S6 segment that forms the cytoplasmic vestibule of the pore (a site that appears to overlap a quaternary ammonium site)63.

Intracellular structure and channel gating differences accounting for 4-AP sensitivities 49-43-03: Whole-cell analysis in the oocyte expression system has determined

that 4-AP sensitivity of Kv2.1 is approximately 150-180 times less than that of Kv3.l 64 (see previous paragraph). Whereas the mechanism of 4-AP block in both channels appears qualitatively similar (reaching its blocking site via the cytoplasmic side of the channels), the ON-rate for block is strongly accelerated when channels open and 4-AP is trapped in closed channels64 . Kv3.l's high 4-AP sensitivity relative to Kv2.l is associated with both a slower OFF-rate (and therefore increased stability of the blocked state) as well as a faster ON-rate (and therefore increased access to the binding site)64. These results appear consistent with 4-AP having an intracellular binding site that is 'guarded' by the gating mechanism, which differs substantially between Kv2,l and Kv3.1. Note: The spindle tubule-disrupting agent cytochalasin-B can affect kinetics and sensitivity to internal TEA in Kv2.1 (DRK1) channels expressed in oocytes65 .

Transfer of a toxin receptor to (normally insensitive) Kv2.1 channels 49-43-04: Transfer of charybdotoxin sensitivity from the (highly sensitive) Kvl.3 (KV3) channel to the insensitive Kv2.l (DRKl) channel has been demonstrated by 'donation' of the Kv1.3 segment between domains S5 and

II

--------entry 49

S6 comprising the pore-forming region (Ithe S5-S6linker', see the PDTM, Fig. 6, under VLC K Kvl-Shak; see also Table 7 under Selectivity, 49-40)66. This study also identified specific residues in the S5-S61inker which confer toxin sensitivity to Kv2.1.

Overlap of external TEA + -binding site and selectivity determinants 49-43-05: In general, multiple site-directed mutagenesis studies (many involving Kv2.1 or chimaeras containing segments of Kv2.1) have identified a region of the S5-S6 loop of voltage-gated K+ channels (P-region) responsible for tetraethylammonium (TEA +) block and, in addition, permeation/selectivity properties (see Selectivity under VLC K Kvl-Shak, 48-40 and also this entry, 49-40). Block by external TEA+ is reduced about 20-fold in the Ile369His mutant of Kv2.1 (described in Table 7 under Selectivity, 49-40). Block by internal TEA+ is unaffected by this mutation. External protons (H+) and Zn2 + (which interact with the imidazole ring t of histidine side-chains) both block the Ile369His mutant Kv2.1 channel much more potently than the wild-type Kv2.1 channel58 (see notes 1 and 2). On the basis of these experiments, it has been suggested that the imidazole side-chain of His369 is exposed to the aqueous lumen at a surface position near the external mouth of the pore 58 . Notes: 1. In this study, Zn 2+ and H+ block was voltage-independent, and the proton blockade had a pKa of about 6.5, consistent with the pKa for histidine in solution. 2. The histidylspecific t reagent diethylpyrocarbonate t displayed greatly exaggerated blockade' of the Ile369His channel compared to wild-type Kv2.1. For related findings on the His369 residue also conferring Cs+ selectivity, see Table 7 under Selectivity, 49-40. I

Side-chains forming a K+ / Rb+ filter and a site for blockade by internal TEA 49-43-06: In both the CHM chimaera and Kv2.1 (for designation, see Table 7 under Selectivity, 49-40) Valor lIe substitutions at position 374 stabilize while Thr or Ser destabilize blockade by internal TEA+, which confirms the importance of hydrophobic interactions for blockade57. In these experiments, TEA+ blockade was dependent upon the charge carrier (Le. where K+ or Rb+ selectivity could be changed - see Table 7 under Selectivity, 49-40 - TEA+ was more effective in the presence of the ion having the larger conductance.

Block of Kv2.1 channels by external TEA depends on the permeating ion 49-43-07: Kv2.1 shows a competition mechanism of selectivity similar to that of calcium channels 67. In the presence of high [K+h and high [Na+]o current through Kv2.1 is 'almost exclusively' carried by K+. Under these conditions, external TEA+ (30 mM) blocks these currents by approx. 87%68. In the absence of both [K+h and [K+]o, large currents are carried by Na+. Under these conditions, external TEA+ (30 mM) has no effect on Na+ currents through the channel68 . As [K+h is increased, between 0 and 140mM, the percentage of current blocked by TEA+ progressively increases from 0 to 870/0. These and other data were taken to indicate that (i) in the presence of

l"---e_n_t_ry_4_9

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Na+ and absence of K+, TEA+ does not bind to the channel, and that (ii) addition of low concentrations of K+ facilitates TEA+ binding67,68.

Regulation of barium ion block of Kv2.1 by Va1374 49-43-08: Ba2 + blocks the Kv2.1 pore at both internal and external sites, which are clearly distinguishable. Internal Ba2 + ion (see notes 1 and 2) blocks wild-type open Kv2.1 (DRK1) channels with a Ko ~ 13 JlM69. Blockade appears to involve more than one site and shows some voltage dependence, increasing at more positive potentials. Val374Thr or Val374Ser (pore) mutants of Kv2.1 produce a significant decrease in the rate of dissociation of internal Ba2 + from the pore (although Val3 74Ile has little effect). For wild-type channels, external Ba2 + decreases the rate of activation of the K+ current (but in this case was unaffected by the Val374Thr substitution) consistent with Ba2+ being able to interact with closed Kv2.1 channels. External Ba2+ also induces a very low-affinity (Kd ~ 30 mM) and voltage-independent block of the open Kv2.1 channel. Notes: 1. Ba2 + has the same crystal radius as K+ but blocks rather than permeates the ion-conducting pore of K+ channels and has therefore been used as a probe of residues lining the pore of K+ channels (see also Blockers under INR K [native], 32-40 and INR K [subunits], 3340). 2. Effects of substitution at position 374 (with residues having polar hydroxyls) are consistent with position 374 being at 'a surface position critical for ion permeation', near the inner mouth of the pore69 .

Channel modulation Mechanisms of halothane and ketamine inhibition of Kv2.1 show significant differences 49-44-01: The two general volatile anaesthetics ketamine (25, 50 and 75 JlM) and halothane (10/0, 20/0 and 40/0) reduce peak current amplitudes of both wild-type Kv2.1 or a variant lacking the C-terminal 318 amino acids (Kv2.1~C318)7o. Both anaesthetics reduce Kv2.1 and Kv2.1~C318 in a dose-dependent and reversible manner while halothane (alone) accelerates the time constant of current inactivation and induces inhibition that is voltage dependent. Use dependence t of both ketamine and halothane inhibition can be observed for both Kv2.1 and Kv2.1~C318, an effect probably attributable to augmentation of C-type inactivation t (for background, see Inactivation under VLC K Kv1-Shak, 48-37). Notably, Kv2.1~C318 showed decreased sensitivity to both anaesthetics, although the mechanism for this is unclear 70 .

Receptor/transducer interactions Interactions with native receptors 49-49-01: No examples of heterologous co-expression of Kv2 subfamily members with G protein-linked receptors were found during entry compilation. Note, however, the studies involving interactions of Kv2 subfamily channels with nerve growth factor (NGF) and thyrotropinreleasing hormone (TRH) described under Developmental regulation, 49-11 and Subcellular locations, 49-16.

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Lack of direct effects of denatonium on Kv2.2 (expressed in tongue epithelia) 49-49-02: rKv2.2/CDRKl: The intensely bitter tastant t denatonium (extra-

has no effect on expressed CDRKI cellular concentration rvlO-lOO~M) channels 11 . In isolated taste cells, denatonium reduces voltage-activated K+ currents of the delayed rectifier type 71 probably through a receptormediated mechanism 72.

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 49-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction eiJ layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in the Introduction eiJ layout of entries, entry 02, at the front of the book). For example, sequences of cross-species variants or related gene familrt members can be readily accessed by one or two rounds of neighbouring analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers), author/reference or nomenclature (Table 8). Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly reported and related sequences.

Gene mapping locus designation 49-54-01: Human chromosomal locus names assigned by the Human Gene Mapping Workshopt (HGMW) are as follows: Kv2.1 - KCNBl; Kv2.2 KCNB2 (see Chromosomal location, 49-18). By convention of the International Committee on Mouse Genetic Nomenclature 73 mouse Kv loci are designated in lower case, e.g. Kcnb1, Kcnb2. Italicized designations are generally used on printed maps but are usually not preserved in online resources such as OMIM.

Miscellaneous information Novel methods employed for heterologous expression of Shah subfamily channels 49-55-01: Kv2.1 channels have featured in several novel methods for

heterologous expression of K+ channels including methods that rely on (i)

Table 8. Kv nomenclature

('l)

Species, DNA source

Original isolate, ORF

Database locus

Accession

Sequence/discussion

t:S

t"1"

~ ~

\0

hKv2.1

Human, cDNA and genomic DNA

hDRKI ORF: 854aa ORF: 858aa (calc. from Genbank entries)

HUMKV21CH

gb: L02840 gb: X68302

Ikeda, Pfliigers Arch-Bur T Physiol (1992) 422: 201-3. Allbrecht, Recept Channels (1993) 1: 99-110.

mKv2.1

Mouse, cDNA

MUSSHAB

gb: M64228

rKv2.1

Rat, cDNA

mShab ORF: 857aa DRKI ORF: 853aa

RSDRKIPC

gb: X16476

Pak, TNeurosci (1991) 11: 869-80. Frech, Nature (1989) 340: 642-5.

Xenopus Kv2.1

Xenopus tadpole brain cDNA library Aplysia nervous system cDNA libraries Rat, cDNA

Aplysia Kv2.1

rKv2.2

Xenopus Kv2.2

Drosophila Shah

II

Xenopus tadpole brain cDNA library Shab locus

Xshab9 876aa

gb: U20342

Burger, TNeurosci (1996) 16: 1412-21.

Aplysia Shah ORF: 905aa

gb: not found

Quattrocki, Neuron (1994) 12: 73-86.

gb: M77482

Hwang, Neuron (1992) 8: 473-81. Burger, TNeurosci (1996) 16: 1412-21.

CDRK ORF: 802aa Xshab12 898aa

RATCDRK

DROSHABA (example only, Shahll protein mRNA) FlyBase: FBgnOO03383 ORF: 925aa

gb: U20343

gb: M32659

Butler, Nucleic Acids Res (1990) 18: 2173-4. Wei, Science (1990) 248: 599-603.

_'--

-:--

9 ----J1 e_n_t_ry_4_

direct cytoplasmic microinjection of cRNA into single cells followed by electrophysiological recording3 ; (ii) transient expression using the COS-I cell line35, which produces Kv2.1 currents are 'virtually indistinguishable' from Xenopus oocytes (although differences in voltage-dependent properties can be observed). In order to to aid the expression of the Aplysia Shab clone in Xenopus oocytes10, complementary linker/adaptor sequences (CAGATCTGCCGCCACC and GCATGGTGGCGGCAGATCTGAGC~ containing an internal BgID site) were used to create a translation initiation signal (Kozak) motif 74 adjacent to the ATG start codon within the expression construct.

Related sources and reviews 49-56-01: Many of the review references listed under this field in the entry VLC K Kvl-Shak (48-56) are pertinent to the Shab family. Discussion on the 'sets' of K+ channels conserved from flies to humans, including Shab family members is included in ref. 75 •

Book reference Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand- and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton.

Feedback Error-corrections, enhancements and extensions 49-57-01: Please notify specific errors, omissions, updates and comments on

this entry by contributing to its e-mail feedback file (for details, see Resource

T- Search criteria). For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 49-32-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback eiJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

REFERENCES 1

2

3 4

5

6 7

8

Chandy, Nature (1991) 352: 26. Gutman, Semin Neurosci (1993) 5: 101-6. Ikeda, Pfliigers Arch-Eur TPhysiol (1992) 422: 201-3. Allbrecht, Recept Channels (1993) 1: 99-110. Frech, Nature (1989) 340: 642-5. Pak, TNeurosci (1991) 11: 869-80. Butler, Science (1989) 243: 943-7. Butler, Nucleic Acids Res (1990) 18: 2173-4.

I

entry 49

9 10 11 12 13

14 15

16 17 18 19

20 21 22 23

24 25

26 27 28 29

30 31 32

33 34

35 36 37

38 39

40

41

42 43

44 45

46 47 48

49

50 51 52 53 54

55 56

57

Burger, TNeurosci (1996) 16: 1412-21. Quattrocki, Neuron (1994) 12: 73-86. Hwang, Neuron (1992) 8: 473-81. Tsunoda, TNeurosci (1995) 15: 5209-21. Baker, TPhysiol (1996) 490: 79-95. Kelley, Am THum Genet (1993) 53: 1620. Gurantz, TNeurosci (1996) 16: 3287-95. Yang, Circ Res (1994) 75: 870-8. Trimmer, FEBS Lett (1993) 324: 205-10. Ribera, TNeurosci (1993) 13: 4988-96. Ribera, Neuron (1989) 2: 1055-62. Sharma, T Cell Biol (1993) 123: 1835-43. Takimoto, TNeurosci (1995) 15: 449-57. Maleticsavatic, TNeurosci (1995) 15: 3840-51. Drewe, TNeurosci (1992) 12: 538-48. Dixon, Circ Res (1994) 75: 252-60. Gidhjain, Circ Res (1996) 79: 669-75. Brahmajothi, Circ Res (1996) 78: 1083-9. Baro, Recept Channel (1994) 2: 193-205. Baro, TNeurosci (1996) 16: 1687-1701. Trimmer, Proc Natl Acad Sci USA (1991) 88: 10764-8. Conn, Mol Neurobiol (1989) 3: 237-73. Hwang, TNeurosci (1993) 13: 1569-76. Barry, Circ Res (1995) 77: 361-9. Scannevin, Soc Neurosci Abstr (1995) Abstract 121.2. Hwang, Neuroscience (1993) 55: 613-20. Shi, TBiol Chem (1994) 269: 23204-11. Klumpp, Vis Neurosci (1995) 12: 1177-90. Paulmichl, Nature (1992) 356: 238-41. Melis, Genomics (1995) 25: 285-7. Lock, Genomics (1994) 20: 354-62. Klocke, Genomics (1993) 18: 568-74. Rehm, FEBS Lett (1989) 249: 224-8. Rehm, Proc Natl Acad Sci USA (1988) 85: 4919-23. Lee, Biophys T (1994) 66: 667-73. Covarrubias, Neuron (1991) 7: 763-73. Li, Science (1992) 257: 1225-30. Shen, Neuron (1993) 11: 67-76. Deal, T Neurosci (1994) 14: 1666-76. Wilson, Pfliigers Arch-Eur TPhysiol (1994) 428: 186-93. Wei, Science (1990) 248: 599-603. Benndorf, TPhysiol (1994) 477: 1-14. Lopatin, TGen Physiol (1994) 103: 203-15. VanDongen, Neuron (1990) 5: 433-43. Debiasi, Pfliigers Arch-Eur TPhysiol (1993) 422: 354-63. Lipkind, Proc Natl Acad Sci USA (1995) 92: 9215-19. Kirsch, Neuron (1992) 8:'499-505. Kirsch, Biophys T (1992) 62: 136-44. Taglialatela, Pfliigers Arch-Eur TPhysiol (1993) 423: 104-12.

_'--

58 59

60 61 62 63

64 65

66 67 68

69 70

71 72 73

74 75 76 77

78

79

Debiasi, Biophys T (1993) 65: 1235-42. Pascual, Neuron (1995) 14: 1055-63. Zuhlke, Recept Channels (1994) 2: 237-48. Shieh, Biophys T(1994) 67: 2316-25. Logothetis, Neuron (1993) 10: 1121-9. Kirsch, Neuron (1993) 11: 503-12. Kirsch, T Gen Physiol (1993) 102: 797-816. Lopatin, Biophys T (1994) 66: AI09. Gross, Neuron (1994) 13: 961-6. Korn, Science (1995) 269: 410-12. Ikeda, TPhysiol (1995) 486: 267-72. Taglialatela, Mol Pharmacol (1993) 44: 180-90. Kulkarni, Anesthesiology (1996) 84: 900-9. Spielman, Brain Res (1989) 503: 326-9. Hwang, Proc Natl Acad Sci USA (1990) 87: 7395-99. Ried, Genomics (1993) 15: 405-11. Kozak, Nucl Acids Res (1987) 15: 8125-48. Salkoff, Trends Neurosci (1992) 15: 161-6. Roe, TBiol Chern (1996) 271: 32241-46., Scannevin, TCell Biol (1996) 135: 1619-32. Swartz, Neuron (1995) 15: 941-9. Doyle, Science (1998) 280: 69-77.

e_n_try_4_9_1

VLG K K v3-Shaw Edward C. Conley

Vertebrate K+ channels related to Drosophila Shaw (Kva subunits encoded by gene subfamily Kv3) Entry 50

See note on coverage at the head of VLG K Kv1-Shak, entry 48. General properties of Kv channel genes expressed in heterologous cells are summarized in the fields of entry 48.

NOMENCLATURES

Abstract/general description 50-01-01: Vertebrate K+ channel subunits whose primary amino acid sequences are most closely related to those of Drosophila Shaw are grouped in the voltage-gated K+ channel subfamily 3 (Kv3, this entry). In mammals, the Shaw-related gene subfamily consists of four independent genes (Kv3.1 to Kv3.4) encoding a total of (at least) 11 transcripts generated by alternative splicing (Kv3.1a, Kv3.1b, Kv3.2a, Kv3.2b, Kv3.2c, Kv3.2d, Kv3.3a, Kv3.3b, Kv3.4a, Kv3.4b, Kv3.4c - for details, see tabulations in Gene family, 50-05 and Gene organization, 50-20). To indicate their relatedness to the Sh superfamily of genes, some authors have used the designation ShIll to designate mammalian homologues of Shaw (see Gene family, 50-05). 50-01-02: The developmental expression of Kv3 subfamily members has been studied in much detail. Generally, Kv3.1b appears to be the predominant splice variant in adult brain, whereas Kv3.1a appears as the predominant species in embryonic and perinatal neurones 1 (see Developmental regulation, 50-11). Double-immunostaining techniques for Kv3.1b protein and the Ca 2 +-binding protein parvalbumin have demonstrated that Kv3.1 b is present in approx. 800/0 of the 'parvalbumin-positive subset' of GABAergic inhibitory interneurones in the rat hippocampus. Kv3.1 channels have been assigned a tentative role in fast action potential repolarization (i.e. channels containing Kv3.1b subunits may impart 'fast-spiking' characteristics in neurones that possess them. 50-01-03: Patch-clamp experiments have demonstrated that T lymphocytes can express at least three distinct types of voltage-dependent K+ channel (type n, type n' and type 1, the latter probably encoded by Kv3.1). The expression pattern of these channels is largely determined by the state of T cell differentiation and mitogenic activation. The channels appear to play essential roles in the physiology of lymphocytes, including the regulation of membrane potential, cell volume, calcium signalling and mitogenic activation. 50-01-04: As summarized in Table 2 under mRNA distribution, 50-13, much work has provided information about the overlapping distribution patterns of Kv3 subfamily members. Some studies have invoked overlapping Kv3 mRNA distributions (and some functional co-expression data) to suggest a common occurrence of Kv3 subfamily heteromultimers in the CNS (see also Inactivation, 50-37). Three of the four Shill genes studied (Kv3.1, Kv3.2 and Kv3.3) are expressed 'mainly in the CNS' albeit Kv3.4 transcripts are more abundant in skeletal muscle.

---------

entry SO

50-01-05: Forms of Kv3.1 are abundant in neurones that generate trains of high-frequency action potentials in response to synaptic inputs, for example in neurones of the auditory brainstem (capable of 'phase-locking' their action potentials on to sinusoid stimulus frequencies of up to several kilohertz) and hippocampal and cortical interneurones, which can generate trains of action potentials at several hundred hertz (see Phenotypic expression, 50-14). The properties of Kv3.1 provide rapidly firing neurones with a high 'safety factor' for impulse propagation (e.g. preventing lovershoot' of action potentials as a feature of its fast closure upon repolarization, and minimizing the relative refractory period that follows individual action potentials). Kv3 subunitcontaining channels have thus been suggested to play roles in shortening of action potential durations, setting frequencies of action potential trains (by controlling interspike resistance), modulating pre-synaptic neurotransmitter release and limiting post-synaptic 'spread' of depolarization induced by receptor-channel openings. Multiple immunolocalization studies have confirmed that Kv3.1b protein is associated with various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei. Dual pre- and post-synaptic (i.e. 'peri-synaptic') locations of Kv3.1 have suggested roles in shaping fast synaptic transmission in auditory pathways (see Subcellular locations, 50-16). 50-01-06: Disruption of the gene encoding mKv3.1 in mice results in altered slow- and fast-twitch muscular contractile properties - see Phenotypic expression, 50-14. Kv3.1-deficient mice perform 'significantly worse' in tests for co-ordinated motor skill, with Kv3.1-deficient muscles generating significantly smaller contractile forces during single twitches or tetanic conditions. Both fast and slow skeletal muscles of Kv3.1-/- mice display slower time-to-peak force and time-to-relax responses, properties which promote muscular tetanyt at lower stimulation frequencies. mKv3.1-/- mice also display significantly lower body weights than the control and a reduced acoustic startle response (although they otherwise exhibit similar spontaneous locomotor and exploratory activities and do not display increased susceptibility to seizures). 50-01-07: Alternatively spliced C-termini of Kv3 subfamily channels may determine their subcellular targeting (e.g. Kv3.2 channels, see Subcellular locations, 50-16). In support of this, immunocytochemical and confocal microsopic analyses of the polarized cell model MDCK transfected with various Kv3 eDNA expression constructs reveal a basolateral distribution pattern for the Kv2.3a variant, whereas Kv~3.2b, Kv3.2c and Kv3.2d products localize to the apical domain. Since the Kv3.2a to Kv3.2d products differ only in their C-terminal domains (see Gene organization, 50-20), this suggests the C-terminus possesses a subcellular membrane targeting domain. 50-01-08: Multiple Kv3 subfamily transcripts are represented in brain mRNA pools. Northern blotst of rat brain poly(A)+ RNA have thus identified multiple transcript sizes (listed in Table 4 under Transcript size, 50-17). Different transcript sizes may be accounted for by alternative transcriptional start sites, alternative polyadenylation sites or large untranslated regions.

l_e_n_t_ry_50

---'_

Alternative transcript sizes from each Kv3 subfamily gene could provide a mechanism for distinct subcellular localizations, specific regulation of translation or mRNA stability and turnover. 50-01-09: Experiments in cell lines suggest that Kv3.1 transcription is regulated by Ca2+ 1 cAMP and growth factor signal transduction cascades (see Gene organization, 50-20). Following deletion analyses, further functional elements of the Kv3.1 promoter have been delineated, including the essential promoter, multiple silencing and enhancing elements, a cAMP/calcium response element (eRE) and an AP-l-binding site (ibid.). 50-01-10: The vertebrate Shaw-related channel mRNAs undergo extensive alternative splicing events within their protein-coding regions (for example, generating potassium channel subunits with variant C-termini). The existence of alternative splicing in the Kv3 subfamily was initially identified when genomic and eDNA sequences encoding the isolate/clone KV4 diverged at the 'breakpoints' of genomic/eDNA co-linearity. Genomic Southern blots t confirmed the existence of alternative splicing from singlecopy Kv3.1 (and later Kv3.2) genes (see Southerns, 50-25). The linear organization, regulatory mechanisms and splicing patterns of the four independent Kv3.1 to Kv3.4 subfamily genes appear broadly similar, consistent with an evolutionary origin at some time involving duplication of an ancestral gene. Overviews of intron/exon usage in the Kv3 gene subfamily are described in annotations to Figs 3 to 6 under Gene organization, 50-20. 50-01-11: The leucine heptad repeat motif (similar to the leucine zipper t sequence described in Sequence motifs under VLG K Kv1-Shak, 48-24) is interrupted in the Shaw-related channels (the fourth Leu is substituted by Phe). This conserved substitution may contribute to the observed shifts in voltage activation thresholds (to more positive membrane potentials) in Kv3 subfamily channels compared to the Shaker-related K+ channels (see Phenotypic expression, 50-14 and Activation, 50-33). Other notable motifs found in Kv3 subfamily protein sequences are described in Table 6 under Sequence motifs, 50-24. 50-01-12: All Kv3 subfamily channels show a similar voltage-dependence of activation, but oocytes co-injected with Kv3 cRNAs encoding channels with markedly different inactivation rates (e.g. Kv3.1 plus Kv3.4) produce currents suggestive of heteromultimer formation (see Fig. 7 under Protein interactions, 50-31). Putative Kv3.1:Kv3.4 heteromultimers thus display fast-inactivating currents that are several-fold larger than those seen in oocytes injected with the same amount of Kv3.4 cRNA alone (ibid.). 50-01-13: Although several subfamily-specific assembly domains (i.e. those preventing heteromultimerization between Kv subfamily proteins and other Kv subfamilies) have been described, some apparent exceptions to 'subfamily-specific association' have been reported for Kv3 subfamily members (see Protein interactions, 50-31). 50-01-14: Modulation of Kv3 subfamily channel function by phosphorylation/ dephosphorylation (e.g. as part of neurotransmitter receptor signalling

II

_ L....----

entry SO _

cascades) is likely to be an important mechanism for controlling thresholds of pre-synaptic and post-synaptic excitability (see Phenotypic expression, 50-14). Mechanisms for modulation of Kv3 protein function by description of structural transitions or subunit assemblies/disassemblies are poorly developed, but extensive primary sequence analysis has predicted multiple candidate sites for kinase modification (Table 7 under Protein phosphorylation, 50-32). Strikingly, protein kinase C (PKC) treatment can specifically eliminate rapid (N-type) inactivation of hKv3.4, converting the channel to a non-inactivating delayed rectifier (ibid.) Furthermore, the hKv3.4 N-terminal domain is a substrate for 'direct' in vitro phosphorylation by PKC at two serine residues within the N-terminal inactivation gate domain. Suppression of heterologously expressed Kv3.1 and Kv3.2 by protein kinases C and A (respectively) has been reported (ibid.). Notably, Kv3.2 proteins can also be phosphorylated in situ by intrinsic PKA with a localization consistent with a role in regulation of thalamocortical synaptic efficacy and, in consequence, altered global states of awareness. 50-01-15: All heterologously expressed Kv3 subfamily channels show a similar voltage-dependence of activation, generally requiring membrane potentials more positive than -10mV to activate significant currents (i.e. more depolarized than typical in other Kv families). Such 'high-voltage activating' channels have been ascribed specific cellular functions (see this field, above and Phenotypic expression, 50-14). 50-01-16: Kv3.1 and Kv3.2 transcripts support 'very similar' currents in oocytes that can be generally described as slowly rising, delayed rectifier type outward currents that inactivate 'extremely slowly'. Conversely, Kv3.3 and Kv3.4 transcripts express A-type inactivating currents that can be broadly distinguished by (i) Kv3.4's macroscopic inactivation rates being an order of magnitude faster than Kv3.3; (ii) Kv3.4 current rise time being shorter than other Kv3 channels. These kinetic properties enable Kv3.3 channels to conduct in the steady state over a wider range of potentials than Kv3.4. In vivo modulation (e.g. redox modulation), various subunit associations and protein phosphorylation/dephosphorylation events may markedly influence these properties.

50-01-17: All Kv3 subfamily currents show a decline in conductance at membrane potentials more positive than -+-20 mV (albeit this is less evident in Kv3.1). Kv3.2a channels display a 'fast-flickering' at voltages more positive than +20mV, an effect that may be due to fast voltage-dependent open-channel block by intracellular components such as Na+ or Mg2 + ions (see Current-voltage relation, 50-35). Initial characterization of the rat Shaw-related (Raw) channels revealed an intense rectification that was dependent on the presence of Mg2 + in the bathing solution (ibid.). 50-01-18: On the basis of measured effects on rapid (N-type) inactivation following application of various peptides on homomeric Kv1.1 currents in CHO cells, it has been proposed that the hydrophobic N-terminal region of the hKv3.4 inactivation (ball domain) peptide blocks the channel pore while the adjacent positively charged region interacts with negative charges on the

l_e_n_t_ry_s_O

_

channel protein (see Inactivation, 50-37). Separate experiments in oocytes showed inactivated Kv3.4a channels were able to conduct (Le. 're-open') during repolarization of the membrane (ibid.). Experiments involving fastapplication of ball domain peptides have suggested this phenomenon may be due to voltage-dependent release of the inactivation gate. 50-01-19: Studies of Kv3.1 wild-type channels or chimaeras containing

segments of Kv3.1 were important in defining the HSt or P-domain1 function in K+ -selective channels. For example, in Kv3.I/NGK2, a 21 aa segment was shown to confer pore properties such as selectivity, singlechannel conductance and sensitivity to internal/external TEA+. Exchange of P-domains between Kv3.1 and Kvl.3 (both T cell channels but with widely divergent pore properties) have also been important for identifying single residues in the deep pore acting as a K+ -Rb+ conductance switch. 50-01-20: The conductance-voltage relationships of Kv3.4 channels have been shown to be influenced not only by the number of basic residues in segment S4 (see entry 48), but also the neighbouring amino acids (see Voltage sensitivity, 50-42). For transient Shaw-related subtypes (e.g. hKv3.4b/HKShmC) the parameter of 'time-to-peak' (the time for current to reach a maximum following a depolarization) decreases with increasing depolarization. The rate at which the current declines or inactivates during a pulse is also voltage dependent, increasing with increasing depolarization (ibid.). 50-01-21: Kv3 subfamily-selective pharmacology and receptor/transducer

coupling mechanisms are poorly developed, and the patterns of block using presently available compounds is very similar between subfamily members. Kv3 channels are relatively sensitive to both 4-aminopyridine (4-AP) and tetraethylammonium ions (see comparative data in Table 10 under Blockers, 5043). Conversely, Kv3 subfamily channels are relatively insensitive to toxins that block other Kv channels (Table 10). Various segmental exchanges and point mutations of non-conserved residues between high-affinity TEA+binding sites in Kv3.l and other Kv channels with distinct pore properties have been important for definition of 'critical' amino acid residues affecting block. 50-01-22: A number of common channel modulators have been shown to have functional effects on Kv3 subfamily channels. For example, exposure of the cytoplasmic side of inside-out membrane patches containing fast-inactivating Kv3.4 or Kv1.4 channels to mild oxidizing conditions (e.g. air exposure) results in removal of the inactivating process (see Channel modulation, 50-44). Inactivation can be restored by exposure to reducing agents (e.g. dithiothreitol or glutathione) or by reinsertion of the patch into cells ('patch-cramming'). These modulatory effects have been shown to be critically dependent upon oxido-reduction of a cysteine residue in the respective N-terminal 'ball' domains of these channels (ibid.). In separate studies, a role for co-expressed Kv3.3a and NADPH oxidase functioning as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines has been proposed. Inactivation processes are also

II

_""---

e_n_try_5_0_

susceptible to chemical modification agents such as N-bromoacetamide (ibid.).

Category (sortcode) 50-02-01: VLG K Kv3-Shaw, i.e. Q subunits of vertebrate voltage-gated potassium channels encoded by Kv gene subfamily 3 whose primary (amino acid) sequences show highest identity to those of Drosophila Shaw (see Gene family, 50-05). Homomultimeric t channels formed from Kv3 Q subunits are normally named in accordance with the eDNA name (see Channel designation, 51-03). Assigned names for subfamily Kv3 gene (chromosomal) loci are listed under Gene mapping locus designation, 50-54.

Information sorting/retrieval aided by designated gene family nomenclatures 50-02-02: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: Kv3.ns where n is a designated number in the systematic nomenclature and (where appropriate) s is a designated ~plice variant letter (e.g. Kv3.2d). Within this entry, paragraph 'running orders' (sort orders) are largely determined alphanumerically by systematic nomenclatures, i.e. denoting species and gene product prefix combined with any trivial or clone name(s) where these have been used in the source reference (e.g. rKv3.2d/Rawl:). Where properties are likely to apply to all or several subfamily proteins (i.e. irrespective of species or isolate) the 'species' term may be omitted. Entry updates covering novel isoforms will index information using these conventions established from systematic nomenclatures based on gene family relationships.

Channel designation Designation of Kv3 subfamily splice variants 50-03-01: In mammals, the Shaw-related gene subfamily consists of four independent genes encoding a total of (at least) 11 transcripts generated by alternative splicing t (for details, see Gene family, 50-05 and Gene organization, 50-20). The multiplicity of 'isoforms' encoded by the Kv3 subfamily has inevitably led to some inconsistency in naming (initial descriptions of identical variants sometimes having been given different names by different authors). Designations used in this entry to denote different variants (employing the systematic nomenclature2 ,3 are summarized in Table 1 (for general background, see Channel designation under VLG K Kv1-Shak, 48-03).

Potential confusion between systematic and certain original clone names 50-03-02: To ensure there is no confusion between the Shaw subfamily clone named KV4 (ref. 4, systematic name Kv3.1, this entry) all occurrences of the former are capitalized and/or are preceeded by the words 'isolate' or 'clone'. Some authors have used the distinctive clone name 'NGK2-KV4' to describe

l'---e_n_t_ry_s_O

_

variants of the Kv3.1 subtype (see also Table 1 and Gene organization, 50-20). Additional instances of potentially confusing nomenclatures (for isolate/ clones KV1, KV2 and KV3) are described in note 2 under Table 1 of VLG K Kv1-Shak, 48-05.

Cross-references to other designations in use 50-03-03: (i) To indicate their relatedness to the Sh superfamilyt of genes, some authors have used the designation ShIll to designate mammalian homologues of Shaw (see Gene family, 50-05). (ii) T lymphocyte channels (likely to be encoded by the Kv3.1 gene) have been designated type 1 (see Developmental regulation, 50-11 and Phenotypic expression, 50-14). (iii) On the basis of its hydrogen peroxide sensitivity, the Kv3.3a channel has been designated KH202 in ref. s (see Channel modulation, 50-44). Note: In cases where it is important to distinguish Kv gene loci from Kv gene products conventional gene locus designations can be used (see Gene mapping locus designation, 50-54).

Current designation 50-04-01: See Table 1 under Gene family, 50-05; for general background, see

this field under VLG K Kv1-Shak, 48-03. The native canine cardiac atrial current designated IK,ur,Id (or I-Kur,I-d) was defined as the 'ultrarapid delayed rectifier, dog') in ref. 6 . The collective single-channel and macroscopic properties of IK,ur, Id was stated to have 'many similarities' to those of currents carried by Kv3.1 cloned channels (this entry) and led to suggestion of a role for Kv3.1 channels in cardiac repolarization. Note: Prior to this, that Kvl.5 had also been suggested as a candidate subunit contributing to 'ultrarapid' delayed rectifiers 7-11.

Gene family 50-05-01: Vertebrate voltage-dependent K+ channel genes related to the Drosophila Shaw gene are assigned to Shaw-related subfamily 3, designated as Kv3.1-3.4 by the agreed nomenclature2 ,3. Independently identified genes/ gene products are listed in Table 1.

Trivial names 50-07-01: Vertebrate K+ channels encoded by genes related to Drosophila Shaw; Shaw-like channels. The rat Shaw clones were originally designated as the 'Raw' isolates 18 . See also Table 1 under Gene family, 50-05.

EXPRESSION For references comparing expression determinants applicable to all of the Kv channel subfamilies, see the EXPRESSION section under VLG K Kv1-Shak, entry 48.

II

Table 1. Kv gene nomenclature and other (original) names in use for Shaw-related vertebrate voltage-dependent K+ channels (ShIll, subfamily 3). The first name listed in each species/variant group (underlined) is that adopted in this entry. (From 50-05-01) Kv designation

Human isoforms

Rat isoforms

Mouse isoforms

Kv3.1 (KCNC1) gene Kv3.1a transcripts Other names in use and refs (see notes 1 and 3)

hKv3.1 hKv3.1a NGK2·KV4 (hyphenated)12 Kv3.1,813 hKv3.1b KV3.1a (== clone/isolate 'KV4,)12

rKv3.1 rKv3.1a RKShIIIB14,15 KShIIIB KV3.1,8 rKv3.1b Isolate/clone KV4 (see note 2)4 Raw2 18 (see note 4)

mKv3.1 mKv3.1a NGK2 16 mShaw22 17

hKv3.2 hKv3.2a HKShIIIA 19

rKv3.2 rKv3.2a RKShIIIA14,15 RKShIIIA.l

mKv3.2 mKv3.2a

Kv3.1b transcripts

Kv3.2 (KCNC2) gene Kv3.2a transcripts Other names in use and refs (see notes 1 and 3) Kv3.2b transcripts

hKv3.2b

Kv3.2c transcripts Kv3.2d transcripts

hKv3.2c hKv3.2d

Kv3.3 (KCNC3) gene Kv3.3a transcripts Other names in use and refs (see notes 1 and 3)

hKv3.3 hKv3.3a HKShIIID 19 hKv3.3 2O

rKv3.2b KShIIIA.3 rKv3.2c rKv3.2d Raw1 18 KShIIIA.2

rKv3.3 rKv3.3a RKShIIID 19

Others

mKv3.1b

Other mShaw12 RShaw12 (specific splice variants not found during compilation)

mKv3.2b mKv3.2c mKv3.2d

mKv3.3 mKv3.3a mKv3.3 21

Other KShIIID.l mShaw19 (specific splice variant; ref. not found during compilation)

('l)

=

f"'t'

~

CJ'1

0

Kv3.3 (KCNC3) gene Kv3.3b transcripts Other names in use and refs (see notes 1 and 3)

hKv3.3 hKv3.3b

Kv3.4 (KCNC4) gene Kv3.4a transcripts Other names in use and refs (see notes 1 and 3) Kv3.4b transcripts

hKv3.4 hKv3.4a

Kv3.4c transcripts

rKv3.3 rKv3.3b

mKv3.3 mKv3.3b 22

Other KShIIID.2

t:S

f"1"

~ CJ'1

0

hKv3.4b HKShIIIC24 hKv3.4c

rKv3.4 rKv3.4a Raw3 18,23 (see note 5) rKv3.4b RKShIIIC rKv3.4c

mKv3.4 mKv3.4a mKv3.4 21

Other

mKv3.4b mKv3.4c

Notes: 1. If the original 'clone' nomenclature is used to identify a data source in this entry, the 'adopted' systematic Kv nomenclature2,3 also appears, generally as an underlined prefix to paragraphs. 'Original' names are described as isolates or clones within the entries, e.g. 'the isolates mShab and DRK1'. 2. To ensure there is no confusion between some 'Kv' subfamily numbers and certain original names (KV4)2,3, all occurrences of the original 'KV' name are capitalized and preceded by the word 'isolate' and/or 'clone'. 3. For channel equivalents that are named differently, only supplementary or contrasting category information may appear under a field. 4. The longer 'sluggish t, splice product of the Raw2 gene has also been called Raw2a 18 . This is equivalent to gene products described under the name NGK2. 5. The sequence first published under the name Raw3 23 has been renamed Raw3-H364Q. 6. The novel Shaw-related K+ channel eDNA Kv3.3b is an alternatively spliced form of the mKv3.3 gene described in ref. 21 thus the latter is therefore also named Kv3.3a. Of these splice variants, only Kv3.3b is expressed in cerebellum (for further details, see Developmental regulation, 50-11 and mRNA distribution, 50-13). For clarification of transcript origin within the Kv3 subfamily, see Gene organization, 50-20.

II

('T)

_ ' -

e_n_try_s_O_

Cell-type expression index Cell-type expression patterns implied using molecular probes specific for Kv3 family members are described under Isolation probe, 50-12, mRNA distribution, 50-13 and Protein distribution, 50-15.

Cloning resource 50-10-01: In general, high-quality CNS cDNA libraries would be expected to contain clones encoding all Kv3 subfamily members, although the relative abundance would be related to the subregion of CNS tissue used (for comparative listing, see Table 3 under mRNA distribution, 50-13). Original RNA/DNA sources used for cloning Kv3 subfamily members have included: mKv3.1a/NGK2: mouse neuroblastoma x rat glioma hybrid cell cDNA library (probably originating from mouse DNA, see Domain conservation, 50-28); rKv3.1b/Raw2: rat brain cDNA library; rKv3.1/Isolate KV4: rat brain oligo (dT)-primed cDNA library; Kv3.2d/Rawl: rat brain cDNA library; mKv3.3: mouse genomic DNA library and mouse brain cDNA library; rKv3.4a/Raw3: rat brain cDNA library; hKv3.4/HKShmC: human brainstem cDNA library. The original cloning of the Drosophila Shaw locus appears in refs 25,26.

Developmental regulation Differential developmental ontogeny of Kv3.1a versus Kv3.1b 50-11-01: Kv3.1a == Kv3.1,B; Kv3.1b == Kv3.1a: In one systematic study l, ribonuclease protection assays t and in situ hybridization t have been used to localize the specific subsets of neurones containing Kv3.1b/Kv3.1a and Kv3.1a/Kv3.1,B mRNAs in the adult and developing rat brain (see also mRNA distribution, 50-13). Generally, Kv3.1b/Kv3.1a appears to be the predominant splice variant in adult brain, whereas Kv3.1a/Kv3.1,B appears as the predominant species in embryonic and perinatal neurones 1 . Kv3.1b/ Kv3.1a mRNA is first detected between embryonic days 11 and 17 and then increases 'gradually' until approx. post-natal day 10 (compare protein appearance, paragraph 50-11-02), when there is a large increase in Kv3.1b/ Kv3.1a transcript abundance. In comparison, Kv3.1a/Kv3.1,B expression mRNA shows only a gradual increase during the same developmental period1 .

Developmental expression of Kv3.1b in 'fast-spiking' hippocampal interneurones 50-11-02: rKv3.1 b: Double-immunostaining techniques for Kv3.1b protein and the Ca2+ -binding protein parvalbumin (see note 1) or the neuropeptide somatostatin demonstrate that Kv3.1b is present in approx. 800/0 of the parvalbumin-positive subset of GABAergic inhibitory interneurones t in the rat hippocampus (but not the somatostatin-positive subset interneurones)27. This study also found evidence for developmental regulation of Kv3.1b in this specific subpopulation of hippocampal interneurones (i.e. the developmental onset of Kv3.1b and parvalbuIllin immunoreactivity appeared

1'--_e_n_t_ry_sO

---"_

'identical'). In the parvalbumin-positive inhibitory interneurones, the predominant Kv3.lb protein is expressed selectively in the somata, proximal dendrites and axons of cells lying within or near the pyramidal cell layer 7 (i.e. consistent with locations of GABAergic t inhibitory interneurones, see notes 2 and 3). Using conventional immunohistochemical techniques, Kv3.l b protein is first detectable at post-natal day 8 (P8) in slices of developing rat hippocampus. In slices, these signals reach a maximum intensity at Pl4 and this intensity is maintained until P40. In isolated membrane vesicles, however, Kv3.lb protein signals become more intense up to P40, indicating an increase in Kv3.lb/cell protein between Pl4 and P40. Notes: 1. In addition to the hippocampus, Kv3.lb co-localizes with parvalbumin in the striatum281'29. 2. A predominant 'somatodendritic' pattern of expression has also been described for Kv4.2 subunits (see Subcellular locations under VLG K Kv1-Sha1, 51-16).3. Electrophysiological recordings of native channels in stratum pyramidale interneurones (in which Kv3.lb-positive immunoreactivity was subsequently demonstrated) resemble those of channels formed by heterologously expressed Kv3.lb. These channels have been assigned a tentative role in fast action-potential repolarization (i.e. channels containing Kv3.l b subunits may impart 'fastspiking' characteristics to expressing neurones).

Induction of rKv3.1 subtype gene expression by the El-ras oncogene 50-11-03: rKv3.la/isolate-clone KV4/NGK2: Overexpression of the EJ-ras oncogene t in the anterior pituitary-derived cell line AtT20 is associated with a developmental 'switch' to a more 'neurone-like' phenotype. Transfection of expression constructs encoding EJ-ras induce shortening of action potential duration (APD) by approx. 20-fold «lOms compared to >200 ms in control AtT20 cells)30. Changes in APD are also correlated with (i) a two- to three-fold increase in the density of voltage-dependent K+ currents which have a rate of inactivation that is decreased (by approx. twofold) in ras-transfected cells and (ii) induction of mRNA detected with probes for clone/isolate KV4 (systematic name rKv3.1) or NGK2 (an alternatively spliced product transcribed from the same gene - see Gene organization, 50-20). Since Kv3.l-specific mRNAs are not detected in control AtT20 cells, these results suggest that ras protein can modulate excitable cell phenotypes in part by selective induction of K+ channel expression.

Kv channels in T lymphocyte ontogeny (see also Phenotypic expression, 50-14) 50-11-04: Kv3.l: Patch-clamp experiments have demonstrated that T lymphocytes can express at least three distinct types of voltage-dependent K+ channel (type n, type n' and type 1, the latter probably encoded by Kv3.l). The expression pattern of these channels is largely determined by the state of T cell differentiation t and mitogenic t activation (for reviews, see refs31-35). The channels appear to play essential roles in the physiology of lymphocytes, including the regulation of membrane potential, cell volume, calcium signalling and mitogenic activation361'37. Note: For a brief comparison of type n, n' and 1 channel properties, see Developmental regulation under VLG K DR [native], 45-11.

_'--

e_n_try_5_0

-----l

Kv3.3b as a marker for terminal differentiation of Purkinje neurones 50-II-OS: mKv3.3b: A novel Shaw-related mRNA encoding Kv3.3b was isolated by a two-step hybridization/subtraction t screening procedure designed to retrieve markers for the later stages of cerebellar Purkinje cellt differentiation22 . Expression of the Kv3.3b mRNA begins in cerebellar Purkinje cells between post-natal day 8 (P8) and P10 and continues through adulthood (a period which coincides with maturation of the Purkinje cell dendritic arbor t ). There is a notably 'restricted' pattern of abundant Kv3.3b expression within developing brain (mainly confined to terminally differentiated Purkinje cells and deep cerebellar nuclei). Note: The timing of Kv3.3b expression (developmental onset, as determined from in situ hybridization) is largely maintained in mixed, dissociated primary cerebellar cell culture, suggesting it to be a useful model for studying K+ channel gene regulation in relation to brain development22 . Note: Kv3.3b activity is evident in Purkinje cells of the neurological mutant mouse strain lurcher prior to their death 38. Notes: 1. The lurcher mutation induces apoptosis t in cerebellar Purkinje cells following their maturation in post-natal cerebellum. 2. A Caenorhabditis elegans egg-laying abnormal mutation (EGL-36) encodes a Shaw-type potassium channel with altered activation properties39.

Isolation probe 50-12-01: Conventional cross-hybridization screening strategies were used to isolate prototype members of the Kv3 subfamily cDNAs. Examples of cited probes and methods include the following., mKv3.1a/NGK2: Two overlapping lOS-mer oligonucleotides (nt 862-1041 from the Kvl.l isolate MBKl) which were annealed and the termini filled-in using Klenow fragment of DNA polymerase I. Kv3.1bIRaw2: Oligonucleotides derived from the cDNA sequence of isolate NGK2. rKv3.2dIRavvl: Oligonucleotides derived from the cDNA sequence of isolate NGK2. mKv3.3: A mixture of mouse Kvl.l (MKl) and rat Kv1.5 (isolate KVl) cDNA from S3 to its 3' end. Kv3.3b: The novel Shaw-related cDNA encoding Kv3.3b was isolated by a two-step hybridization/subtractiont screening procedure designed to retrieve markers for the later stages of cerebellar Purkinje cell differentiation t 22 (see Developmental regulation, 50-11). rKv3.4a/Raw3: Oligonucleotides t derived from the cDNA sequence of isolate NGK2. Kv3.4/HKShmC: Two nucleotide probes derived from Kv3.2 (isolate RKShTIIA, nt 301-501 and 964-1296).

mRNA distribution Neuronal subset expression and co-distribution patterns for Kv3 subfamily transcripts 50-13-01: As summarized in Table 2, combined Northern blot, RNAase protection and in situ hybridization analyses have provided important information about the overlapping distribution patterns of Kv3 subfamily members. Early analyses of mRNA for the rat isoforms of Shaw-related channels (i.e. clones Raw1 to 3) indicated their marked differential distribution in the CNS 18 . In addition to the descriptions in Table 2, the

e_n_t_ry_50

1_ _

_

Table 2. Reported tissue distributions of Kv3 subfamily mRNAs. For a direct comparative listing of Kv3 subfamily transcripts in different regions of the eNS, see Table 3. (From 50-13-01) Kv3.1

50-13-02: rKv3.1: In the dorsolateral striatum, only parvalbumin mRNA-positive neurones express Kv3.1 mRNA28 (see Developmental regulation, 50-11). This co-distribution is striking in that the calcium-binding protein parvalbumin is often used as a marker for a subpopulation of fast-firing GABAergic interneurones (as distinguished from numerous GABAergic efferent neurones in the striatum). The distinct firing properties of the 'parvalbumin subset' neurones correlate well with Kv3.1's rapid activation and inactivation kinetics. Note: Parvalbumin mRNA-positive neurones also express 'very high levels' of the mRNA encoding glutamic acid decarboxylase (Mr 67000: GAD67) in the dorsolateral and striatum28 . 'Low-levels' of Kv3.1 transcripts (mainly the 8 knt band, see Transcript size, 50-17) are detectable in skeletal muscle (cited in ref. 42). 50-13-03: Kv3.1: Detected in T lymphocyte eDNA pOOlS13, where it encodes the l-type K+ channel subunit (see Phenotypic expression, 50-14). 50-13-04: Kv3.1a: Kv3.1a (not Kv3.1b) transcripts have been detected in phaeochromocytoma (PCI2) cells 12,19,43. 50-13-05: rKv3.1a == clone Kv3.1a: In adult rat brain, Kv3.1,B mRNA is generally of lower abundance than Kv3. la species (this table, above). Notably, however the overall expression pattern for the a and ,B splice variants is similar, and it is clear that both are co-expressed in several neuronal subsets 1 (for predominance' of splice variant expression during development, see Developmental regulation, 50-11). 50-13-06: rKv3.1b == clone Kv3.1a: ISH: In adult rat brain, in situ hybridization histochemistry reveals a heterogeneous expression pattern of Kv3.1a mRNA1. Highest Kv3.1b/Kv3.1a mRNA levels are expressed in the cerebellum, but also in the globus pallidus, subthalamus and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, show strong hybridization signals with Kv3. Ib/Kv3. la-specific probes 1. Cortex and hippocampus neuronal subpopulations (resembling interneurones) show strong Kv3.1b signals. This study also reported many brainstem nuclei including the inferior colliculus and the cochlear and vestibular nuclei to express Kv3.1b/Kv3.1a mRNA. In contrast, 'low' or 'undetectable' levels of Kv3.1a mRNA were reported for the caudate-putamen, olfactory tubercle, amygdala and hypothalamus 1 (see also Developmental regulation, 50-11). For likely physiological roles of Kv3.1, especially in fast repolarization in neuronal subsets, see Phenotypic expression, 50-14.

_L...----

-

_

entry 50

Table 2. Continued Kv3.1

50-13-07: rKv3.lb/isolate KV4: Northerns: Adult brain: + + + +i not detected in neonatal brain or whole embryo 44. As described in one quantitative analysis40, Kv3.lb (and Kv3.3) are the most abundant Kv3 subfamily mRNAs in rat brain. ISH: Predominantly (though not exclusively) expressed in the adult cerebellum. KV4 mRNA is high in Purkinje and granule cells, low in molecular layer and white matter. Some hybridization in the cochlear nuclear complex and in the trigeminal nucleus. KV4-expressing cells are scattered through various cortical layers, and through the pyramidal and granule cell layers of the hippocampus. There is low level KV4 expression in the medial septal nucleus, the reticular thalamic nucleus, the ventral posteriomedial thalamic nucleus, the piriform cortex and the zona incerta44 . 50-13-08: rKv3.lb/Raw2: ISH: 18 mRNA for the Raw2a 'sluggish t ' splice variant (== rat isoform of isolate NGK2, see Kv3.l in Fig. 3 under Gene organization, 50-20) is not localized to specific brain areas. 50-13-09: rKv3.1b/Raw2: Northerns: Using a 3' UTRt riboprobe t , Raw2 is expressed 'predominantly or exclusively' in brain (not heart or kidney). ISH: Using 50-mer oligonucleotide probes from 3' UTR, neurones in the cerebellum or the caudate-putamen express Raw mRNAs very distinctly (limited to one type of Raw mRNA, cf. other brain regions such as the cortex or dentate gyrus, which may express more than one Raw subtype per cell - Raw2 exhibits a 'granulated' appearance in these brain areas). The reticular thalamic and the cerebellar nuclei preferentially (if not exclusively) express Raw2 mRNA18 .

Kv3.2

50-13-10: rKv3.2d/Rawl: ISH: Kv3.2 gene transcripts are abundant in thalamic relay neurones 40,41,45. Northerns: It has been estimated that 'close to 900/0' of all Kv3.2 mRNAs in the rat eNS are found in the dorsal thalamus, including nuclei involved in auditory, visual and somatosensory processing (all suggesting the roles of Kv3.2 channels are general to thalamic function)45 (see also Protein distribution, 50-15 and Protein phosphorylation, 50-32). Northerns: Expressed predominantly or exclusively in brain (see comments on Raw2, above). Rawl also exhibits a 'granulated' appearance in cortex and dentate gyrus. mRNA for the Rawl splice variants 'do not appear to be localized' in specific brain areas 18 .

Kv3.3

50-13-11: mKv3.3a: Northerns: Brain: + +. Heart: +. Liver: + +. Thymus: + +. 'Low levels' of Kv3.3 transcripts are detectable in kidney and lung mRNA pools (see below, cited in ref. 42). ISH: mRNA encoding the Kv3.3a is not expressed in cerebellum (compare the alternative splice variant Kv3.3b, below). 50-13-12: mKv3.3b: ISH: mRNA encoding the alternative splice variant Kv3.3b becomes highly enriched in developing brain,

l_e_n_t_ry_sO

---'_

Table 2. Continued Kv3.3

particularly in the cerebellum, where it is confined to Purkinje cells and deep cerebellar nuclei22 (see Developmental regulation, 50-11). 50-13-13: rKv3.3: Kv3.3 (and Kv3.lb) form the most abundant Kv3 subfamily mRNAs in rat brain4o . Kv3.3 cDNA can be detected in phaeochromocytoma (PCI2) cells 12,19,43. 50-13-14: hKv3.3: Kv3.3 has been detected in lung (airway mucosa, pulmonary neuroepithelial bodies (NEB) and in small cell lung carcinoma cell lines) (see ref. s under Channel modulation, 50-44).

Kv3.4

50-13-15: rKv3.4a: Kv3.4 transcripts are more abundant in skeletal muscle than in brain (cited in ref.42). ISH: Raw3 (rKv3.4a) and RCK4 (rKvl.4) mRNA, which both encode channels supporting Atype K+ currents, show distinct distribution patterns in brain18 (see also Subcellular locations under VLC K Kv1-Shak, 48-16 and VLC K Kv1-Shal, 51-16). Raw3 mRNA is not detected in areas like caudate-putamen or the CAl to CA3 fields of the hippocampus, which express high levels of RCK4 (Kvl.4) mRNA. Conversely, RCK4 mRNA is not detected in the pontine nuclei and the cerebellum, which express high levels of Raw3 mRNA. Neurones in the dentate gyrus appear to express both Raw3 and RCK4 mRNA. See also Protein distribution, 50-15 and compare to Kv1.4 under VLC K Kv1-Shak, 48-15. 50-13-16: Kv3.4: Detected in phaeochromocytoma (PCI2) cells 12,19,43.

separate Table 3 compares differential expression patterns of Shaw-related channels in the rat CNS from the major quantitative hybridization study by Weiser et a1. 40 . This study invoked overlapping Kv3 mRNA distributions (and some functional co-expression data) to suggest a common occurrence of Kv3 subfamily heteromultimers in the CNS (see also Inactivation, 50-37). Three of the four Shill genes studied (Kv3.I, Kv3.2 and Kv3.3) were expressed 'mainly in the CNS', albeit Kv3.4 transcripts were more abundant in skeletal muscle40 . Subsets of neurones in the cerebral cortex, hippocampus and caudate-putamen could be distinguished by the expression of specific Kv3 subfamily mRNAs (see also Developmental regulation, 50-11 and Protein distribution, 50-15). Many neuronal populations co-express Kv3.1 and Kv3.3 mRNAs and Kv3.4 transcripts are present (at lower levels) in several neuronal subtypes that also express Kv3.1 and/or Kv3.3 mRNAs 40 . In agreement with earlier studies 1,41, Kv3 mRNAs were not detectable in glial cell populations. As further described in Table 2, a number of RNA distribution studies have employed probes capable of distinguishing alternatively spliced variants of the Kv3 genes (see Cene organization, 50-20).

II

Table 3. Comparative listing of Kv3 subfamily RNA transcript distributions in the CNS from the quantitative study of Weiser et a1.

(see text). (From 50-13-01) Olfactory bulb Periglomerular cells Tufted cells Mitral cells Neocortex Interneurones Pyramidal cells Pyriform complex Hippocampus CAl pyramidal cells Str. radiatum a Str. oriens a CA3 pyramidal cells Str. radiatum a Str. oriensa DG granule cells Borders with str. granulosum Hilus Basal nuclei Caudate-putamen Globus pallidus Subthalamic nucleus

Kv3.l (KShIllB)

Kv3.2 (KShIIIA)

Kv3.3 (KShllID)

Kv3.4 (KShIIIC)

+ + ++

+ + ++ Layers II-IV> V-VI

Layers V-VI

Layers II-IV> V-VI

Layers II-IV> V-VI

+

+

+

±

+ ++ ++ +++ + + +++

+

+ + + + + + ++

++

+

+++ ++ ++ ++

±

±

+++

+ +++

++ scattered ++ ++

±

±

++

+ ('n

Septum Diagonal band of Broca Medial septum Lateral septum

::s

M-

+ +

++ ++

± ±

± ±

~ CJ'l

0

Epithalamus Medial habenula Lateral habenula Paraventricular nuclei Pretectum Thalamus (dorsal) Anterodorsal nu. Anteroventral nu. Anteromedial nu. Laterodorsal nu. Parataenial nu. Reuniens nu. Mediodorsal nu. Intermediodorsal nu. Lateral posterior nu. Ventral posterolateral nu. Ventrolateral nu. Ventral posteriomedial nu. Ventro medial nu. Central medial nu. Posterior nu. Dorsal lateral geniculate Medial geniculate

II

(t)

=:s

r-t-

±

±

+

~ CJ1

0

++ + ± ± + ± ±

+ ++ ± ++ ± ± ± ++

±

++++ ++++ ++++ +++ ++++ +++ ++++ ++ +++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++

+

± ±

+ +

+

Thalamus (Ventral) Reticular thalamic nu. Ventral lateral geniculate nu.

++++ +

±

++ +

±

Brainstem Substantia nigra pars compacta Substantia nigra pars reticulata Zone incerta

++ +++

+ +

+ +++

± ±

II

Table 3. Continued -Brainstem Superior colliculi, optic layer Superior colliculi, IGL b Darkshevich nu. Interstitial nu. Oculomotor nu. Red nucleus Inferior colliculi Lateral lemniscus nuclei Reticulo tegmental nu. pons Pontine nuclei Pontine reticular nu. Cochlear nu. dorsal Cochlear nu. ventral Spinal trigeminal nu. Principal sensory trigeminal nu. Motor trigeminal nu. Locus coeruleus Superior olivary complex Nu. trapezpoid body Vestibular nu. lateral Vestibular nu. medial Parvocellular reticular nu. Gigantocellular reticular nu. Intermediate reticular nu. Abducens nu. Facial nu. Prepositus hypoglossal nu. Inferior olive

+++ scattered ++

+++ + scattered +

±

±

+++ ++++ +++ +++ +++ +++

++ + ++ (ventral) +

± scattered

++

++ + ++ ++ ±

++ ++ + + ++ ++

±

++ ++ scattered + + + + ++

++ scattered + ++ +++ +++ +++ +++ +++ +++ +++ + scattered +++ +++ +++ +++ ++ ++ +++ ++ ++ +++ +++ ++ +++ ±

+ scattered ± ± ± ± ±

+ ++ + ±

+ ±

++ ± ±

+ + + + ~

±

=:s

t"'t'

~ (Jl

0

±

Hypoglosal nu. Dorsal column nu. Cerebellum Molecular cell layer Purkinje cells Granular cell layer Deep cerebellar nu. Spinal cord Dorsal horn Ventral horn

+++ ++

±

r-t-

~

±

+ ++++ +++ +++ +

++

+ ++++ ++ +++ +++ +++

+ + ±

++

Notes: Table data, headings and footnotes from Weiser, M., E.C. Vega-Saenz de Miera, C. Kentros, H. Moreno, L. Franzen, D. Hillman, H. Neulosci 14: 949-972. For the interneurons in the cerebral cortex and the hippocampus, symbols Baker and B. Rudy (1994) indicate numbers of cells labelled; otherwise, the symbols indicate signal intensity as follows: - (signals undistinguishable from background); ± (very weak signals but clearly above surrounding background); + (weak); ++ (moderate); +++ (high); ++++ (very high). a Refers to labelled neurones in the indicated stratum or on their interface with the stratum pyramidalis. b IGL, intermediary grey layer.

r

II

Cb ~

CJ1

o

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_s_O_

Phenotypic expression Roles of Kv3.1 in maintenance of high-frequency action potential trains 50-14-01: Kv3.1a; Kv3.1b: Both Kv3.1a (long form) and Kv3.1b (short form) transcripts/protein subunits are abundant in neurones that generate trains of high-frequency action potentials in response to synaptic inputs 1 . Examples of such cells include (i) neurones of the auditory brainstem (e.g. medial neurones of the trapezoid body (MNTB), capable of 'phase-locking' their action potentials on to sinusoid stimulus frequencies of up to several kilohertz) and (ii) hippocampal and cortical interneurones t, which can generate trains of action potentials at several hundred hertz. The biophysical characteristics of Kv3.1 channels appear well adapted for these roles, in that they (i) undergo 'ultrafast' activation; (ii) have very rapid deactivation t kinetics in comparison to other Kv family members and (iii) open at relatively positive potentials (e.g. for Kv3.1, opening between -10 and OmV; more typically, 'delayed rectifier' channels activate at about -40mV, so most are open at the upstroke of the action potential) (see also Activation, 50-33 and Inactivation, 50-37). In cell simulation studies (e.g. ref. 46, further described under Kinetic model, 50-38), channels with the properties of Kv3.1 provide rapidly firing neurones with a high 'safety factor' for impulse propagation (e.g. preventing 'overshoot' of action potentials as a feature of its fast closure upon repolarization, and minimizing the relative refractory period t that follows individual action potentials). Comparative note: Whereas most neuronal subsets expressing Kv3.1 display narrow action potentials and high-frequency firing rates (with little or no spike adaptation, see above), the subcellular distribution patterns shown by Kv3.1b subunits are consistent with a role for Kv3.1b-containing channels in the modulation of action potentials29 (see Protein distribution, 50-15 and Subcellular locations, 50-16).

Phenotypes of Kv3.1 'gene knockout' mice 50-14-02: The mKv3.1 gene open reading frame (encoding the type 1 K+ channel of T cells, see Developmental regulation, 50-11) has been disrupted using targeted homologous recombination t techniques47. Homozygous Kv3.1 mutant mice (mKv3.1-/-) were viable and fertile, with no marked mutant phenotype in external morphology or in the gross anatomy of the brain (including the cerebellum, where the Kv3.1 gene is predominantly expressed - see mRNA distribution, 50-13 and Protein distribution, 50-15). Notably, however, Kv3.1-deficient mice perform 'significantly worse' in tests for co-ordinated motor skill, with Kv3.1-deficient muscles generating significantly smaller contractile forces during single twitches or tetanic conditions. Both fast and slow skeletal muscles of Kv3.1-/- mice display slower time-to-peak force and time-to-relax responses, properties which promote muscular tetany t at lower stimulation frequencies. Other differences between mKv3.1-/- and control mice were initially reported47, including (i) significantly lower body weights than controllittermates (by an undetermined mechanism, albeit this was not due to reduced muscle mass or hypophagia) and (ii) a reduced acoustic startle response, perhaps indicating a normal role for Kv3.1 in the auditory pathway (see Protein distribution, 50-15). Otherwise,

entry50

_

I' - - - - - - - - - -

Kv3.1 channel-deficient mice exhibit similar spontaneous locomotor and exploratory activity in the open field test, unaltered acquisition (learning) and retention (memory), and do not display increased susceptibility to seizures.

tprediction' of Kv3 channel functions from biophysical properties 50-14-03: Kv3 channels generally begin activating at 'highly depolarized' potentials (see Activation, 50-33) and are capable of forming heteromultimers where one subunit may contribute a transient-inactivating phenotype (see Protein interactions, 50-31). As emphasized by Vega-Saenz de Miera et al. (1994, see Related sources and reviews, 50-56), biophysical properties can conceivably be used to predict 'roles' that Kv3 family members would/would not be expected to play in vivo. In practice, however, many of these 'potential' roles require rigorous testing in a specific signalling context (or by reference to highly selective subunit localizations in native tissues) (see Protein distribution, 50-15). As described elsewhere in this entry, Kv3 subunitcontaining channels have been suggested to play roles in (for example) shortening of action potential durations, setting frequencies of action potential trains (by controlling interspike resistance), modulating pre-synaptic neurotransmitter release and limiting post-synaptic Ispread' of depolarization induced by receptor-channel openings. Kv3.1 expression associated with mouse autoimmune disorders 50-14-04: Kv3.1: The Shaw-related Kv3.1 encodes the type 1 K+ channel in a specific T lymphocyte subset CD4-CDS-Thyl+. Type 1 channels are found 'sparingly' in cytotoxic T cells from normal mice 13; however, Kv3.1 transcripts are detectable in T cells isolated from lymph nodes of MRL-lpr mice with systemic lupus erythematosus and in a human lymphoma cell line (Louckes) that exhibits type 1 channel activity. Supplementary note: Kv3.1 can not restore tvolume-regulatory' properties when transfected into the mouse cytotoxic T lymphocyte line CTLL-2 (compare with Kv1.3 under Phenotypic expression of VLC K Kv1-Shak, 50-14).

Protein distribution Immunolocalization of Kv3.1 protein in the auditory systems 50-15-01: Kv3.1 b: As determined by immunohistochemical localizations in rat brain slices29, Kv3.1 b subunits are selectively expressed in cerebellar granulet cells, projecting neurones of deep cerebellar nuclei, the substantia nigrat pars reticulata, the globus pallidust, and the ventral thalamust (reticular thalamic nucleus, ventral lateral geniculate and zona incertal. Kv3.1b protein is also associated with various neuronal populations involved in the processing of auditory signals, including the inferior colliculust, the nuclei of the lateral lemniscust, the superior olivet, and some parts of the cochlear nucleit. Kv3.1b immunoreactivity is also localized to several other neuronal groups in the brainstem (e.g. the oculomotor nucleus t , the pontine nucleil , the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurones in the neocortext, the

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hippocampus t and the caudate-putament (shown by double staining to correspond to neurones containing parvalbumin t )29 (see also Subcellular locations, 50-16).

Thalamic relay neurones 50-15-02: Kv3.2: Immunohistochemical studies using site-specific antibodies

(common to all Kv3.2 variants) have been used to localize Kv3.2 proteins in thalamic relay neurones 45 . These studies have demonstrated that Kv3.2 proteins localize to the terminal fields of thalamocortical projections 45 (see also mRNA distribution, 50-13 and Protein phosphorylation, 50-32).

Early immunocytochemical localizations 50-15-03: rKv3.4a/Raw3: Use of antibodies against Raw3 and Kvl.4/RCK4

proteins (which form two distinct A-type potassium channels) indicated that in the hippocampus the two channels were expressed both in different neurones and in the same ones 18 . Staining intensities with both antibodies are comparable in the outer and inner molecular layers of the dentate gyrus. Axons in the polymorph layer of the dentate gyrus and the mossy fibre system also co-expressed these channels (Raw3»RCK4)18. In this study, interneurones did not appear to express Raw3 channels, but RCK4 immunoreactivity was distributed throughout all parts of the hippocampus 18 .

Subcellular locations Distinct functional roles of Kv3.1 implied by tperi-synaptic' locations of Kv3.1 50-16-01: A polyclonal antibody specific for Kv3.1a (isolate KV4) has shown

that Kv3.1 protein is highly enriched in the nuclei of the auditory brainstem where it outlines both the cell somata and the neuropil 48 • By highresolution electron microscopic immunohistochemical methods, Kv3.1 protein is associated with lamellae of the endoplasmic reticulum and Golgi cisterna. In addition to common staining of cell somata, dendritic proceses and spines, staining inside pre-synaptic terminals, including the calyx of Held has been observed (see also Subcellular locations under VLC K DR [native], 45-16). The dual pre- and post-synaptic (Le. 'peri-synaptic') locations of Kv3.1 have suggested roles in shaping fast synaptic transmission in auditory pathways. Pre-synaptically, Kv3.1 appears to be involved in 'smoothing' amounts of transmitter release per impulse, so the terminal is not depleted; post-synaptically, Kv3.1 appears to induce a marked shortening of synaptic potentials enabling high-frequency 'phase-locking' (see Phenotypic expression, 50-14 and mRNA distribution, 50-13).

Modulation of action potentials 50-16-02: Kv3.1b: As determined by high··resolution immunocytochemical

staining27,29, Kv3.1b subunits are localized predominantly in somatic t and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations t. This distribution is consistent with a role for Kv3.1b-containing channels in modulation of action potentials. Note: Although (following oocyte expression) Kv3.1b and Kv3.2 (0 subunit)

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homomultimeric channels pass 'broadly similar' currents, in vivo, their cellular and subcellular distribution patterns are distinct29 (see also Protein distribution, 50-15).

Channel distributions surrounding cerebellar Purkinje cells 50-16-03: Kv3.3; Kv3.4: An alternately spliced t transcript of Kv3.3 (Kv3.3b) is found exclusively in terminally differentiatedt cerebellar Purkinje cells22 (see Developmental regulation, 50-11). In separate studies 49, Kv3.4 immunoreactivity was found to be uniformly distributed over the Pinceau and the pericellular basket surrounding Purkinje cell soma (i.e. not concentrated in junctional regions). Comparative note: In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment; other Kv channels (Kv1.1, Kv1.2 were predominantly co-localized with synapse-associated protein 90 (SAP90) to septate-like junctions), which connect the basket cell axonal branchlets49 . For associations of subcellular targeting t and differential cell type t -specific expression of ion channels through alternative splicingt events, see also ILG K Ca, entry 27 and VLG Ca, entry 42.

Alternatively spliced C-termini determine subcellular targeting of Kv3.2 channels 50-16-04: Kv3.2: Antibodies detecting all four alternatively spliced products of the Kv3.2 gene (Kv3.2a, Kv3.2b, Kv3.3c and Kv3.2d - see Gene organization, 50-20) show predominant distribution in axons and terminals in some brain neurones (e.g. thalamic relay neurones, see Protein distribution, 50-15) and somatic distribution in others (e.g. in the dorsal cochlear nucleus). These distribution patterns have suggested that different spliced products possess different intrinsic 'targeting' signals 50. This hypothesis has been tested within a polarized t (apical/basolateral) epithelial cell model (MDCK) which lacks Shaw-related K+ channel subunits (but displays some similarities in protein-sorting mechanisms to those seen in neurones - see note). Immunocytochemical and confocal microscopic analyses of MDCK cells transfected with various Kv3 cDNA expression constructs reveal a basolateral distribution pattern for the Kv2.3a variant, whereas Kv3.2b, Kv3.2c and Kv3.2d products localize to the apical domain 5o . Since the Kv3.2a to Kv3.2d products differ only in their C-terminal domains (see Gene organization, 5020), this suggests that the C-terminus possesses a subcellular membrane targeting domain. Note: The somatodendritic membrane of neurones is thought to be analogous to the basolateral domain of MDCK, whereas the axonal membrane represents the apical domain.

Transcript size Multiple Kv3 subfamily transcripts are represented in brain mRNApools 50-17-01: Northern blotst of rat brain poly(A)+ RNA have identified multiple transcript sizes, as listed in Table 4. The origins of these have been discussed in detail (Vega-Saenz et a1., 1994, see Related sources and reviews, 50-56); in general, however, the different transcript sizes can be accounted for by (i)

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_0_

Table 4. Summary of major and minor RNA transcript sizes detected using Kv3 subfamily gene sequence probes (From 50-17-01) Kv3.1

50-17-02: rKv3.1: 10.5,8.0 and 4.5 knt4,40; NGK2: rv7-8 kb. A probe unique to the 3' UTR of the NGK2 sequence hybridizes to transcripts of 8, 10 and 11 kb; see isolate KV4 below4. By definition, probes specific for clone KV4 sequences do not cross-hybridize with the NGK2 transcripts indicating the 8 kb and 4.5 kb RNAs do not have a 'precursor-product' relationship4. 50-17-03: Kv3.1b/Raw2: In brain, strong rv4.5kb; weak rv12-13kb.

Clone KV4: An oligonucleotide probe comprising sequences common to isolates NGK2 and KV4 hybridizes to bands of 4.5 kb (major) but also to species of 10, II, 6 and 8 kb of low abundance4,44. Kv3.2

Kv3.3 Kv3.4

50-17-04: rKv3.2: 7.0, 6.0, and 4.0 knt; the 6.0 knt band is relatively weak in thalamic RNA pools41; rKv3.2d/Raw1: In brain, prominent rv6.8 kb; less abundant rv5.6 kb. 50-17-05: rKv3.3: 8.5 and 5.5knt40,41. 50-17-06: rKv3.4: 4.5 and 3.5 knt40. rKv3.4a/Raw3: Hybridizes to two or three bands (rv 4.5, 4.2 and 3.3 kb) which occur in RNA derived from both brain and skeletal muscle due to either distinct Raw3 mRNAs in both tissues or the occurrence of Raw3 splice variants18.

alternative transcriptional start sites (there is evidence for alternative splicing of 5' UTRs, ibid., see also Gene organization, 50-20); (ii) alternative polyadenylation sites or (iii) unusually large untranslated regions. Some of the discrepancies in transcript sizes reported betweeen independent studies (for the same isoform) may be explicable in terms of different probe sizes and regional specificities, but explanations based on 'incomplete splicing' events appear unlikely. Based on other examples in the nervous system, alternative transcript sizes from each Kv3 subfamily gene could provide a mechanism for distinct subcellular localizations, specific regulation of translation or mRNA stability and turnover.

SEQUENCE ANALYSES For references comparing sequence analyses applicable to all of the Kv channel subfamilies, see the SEQUENCE ANALYSES section under VLG K Kvl-Shak, entry 48.

Chromosomal location 50-18-01: The similar organization of the Shaw-related subfamily genes (see Gene organization, 50-20) is consistent with them having arisen by duplication of a single ancestral gene (for further details, see Fig. 3, p. 419,

1'--_e_n_t_ry_5_0

---'_

and text under Chromosomal location of VLG K Kv1-Shak, 48-18). Table 5 summarizes reported chromosome locations for mouse and human Kv3 subfamily genes.

Encoding Sequence alignment of Kv3 subfamily members 50-19-01: An amino acid sequence alignment of Shaw-related isoforms rKv3.1 through rKv3.4, together with that of Drosophila Shaw is shown in Fig. 1. Variants in open reading frame lengths due to alternative splicing are indicated within the figure series Figs 4-6 under Gene organization, 50-20 and Database listings, 50-53. Other notable features of the Kv3 subfamily sequences are outlined in Sequence motifs, 50-24, Protein phosphorylation,

50-32 and Domain conservation, 50-28. Note that individual nucleotide and/or amino acid sequences may be retrieved for local analysis using the accession numbers listed under Database listings, 50-53.

Kv3 splice variants show little functional variation following oocyte expression 50-19-02: Homomeric Kv3 subfamily channels encoded by different alternatively spliced mRNA transcripts (from anyone gene) do not produce currents in Xenopus oocytes markedly different from those produced by other transcripts from the same gene. As reviewed in Vega-Saenz de Miera et al. (1994, see Related sources and reviews, 50-56), other possible functional consequences in vivo may include (i) differential responsiveness to various second messenger systems (Le. isoform-specific modulation of channel function by neurotransmitter and neuropeptide-induced cellular responses - see Receptor/transducer interactions, 50-49) and/or (ii) differential protein-protein interactions conferring isoform-specific channel localization (see ref. 50 in Subcellular locations, 50-16), mobility, and susceptibility to local modulatory influences (e.g. relying on divergent Ctermini, see Fig. 1, Protein interactions, 50-31 and Channel modulation, 5044). Other, more subtle biophysical differences (not observed thus far in the limited range of heterologous expression systems employed) may be found in due course. For a summary of evidence for heteromultimeric associations between Kv3 subfamily members, see Protein interactions, 50-31.

Gene organization Transcriptional regulation of the Kv3.1 gene 50-20-01: rKv3.1: The Kv3.1 gene exhibits a restricted tissue expression pattern (see mRNA distribution, 50-13 and Protein distribution, 50-15) and experiments in cell lines suggest that Kv3.1 transcription is regulated by Ca2+, cAMP and growth factor signal transduction cascades56 . In gene reporter t assays utilizing a 5.3 kbp fragment of the Kv3.1 5' flanking region, the fragment was active in an undifferentiated phaeochromocytoma (PCI2) cell line, but not in NIH3T3 fibroblast cells56 . Following deletion analyses,

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Table 5. Chromosome locations reported for Kv3 subfamily genes (From 50-18-01) Consensus/summary (see note 1)

Further details (see note 2)

Kv3.1 hKv3.1, KCNC1, 11p14.3-p15.2 mKv3.1, Kcncl, Chr 7 [~]

50-18-02: hKv3.1; KCNC1: In situ hybridization localizes KCNC1/hKv3.1 to region on human chromosome IlpI4.3-pI5.2 51 (see also ref.13). The region showing KCNC1 homology of synteny t in the region of mouse chromosome 7 (see below) maps close to a candidate region for the dominant LQTl marker for congenital long QT syndrome (i.e. human chromosome llp15.5 - for further background, see Chromosome location under VLC K eag/elk/erg, 46-18). See also OMIM entry 176258. 50-18-03: Isolate/clone NGK2-KV4; KCNC1: Human chromosome 11p15 (an earlier study, ref. 12 ). 50-18-04: mKv3.1: Kcnc1/mKv3.1, has been determined to lie approximately 1.8 cM from the Myod-1 gene on mouse chromosome 751 . Long-range physical mapping of the region of mouse chromosome 752 included six 'clustered' genes located within a 500 kb interval and noted that the gene content and organization within this homology segment had been highly conserved throughout evolution. The markers in this segment (just proximal of the pink-eyed dilution (p) locus (OMIM 203200) included Ldh1 (OMIM 150000), Ldh3 (OMIM 150150), Saa (OMIM 104750), Tph (OMIM 191060), Kcncl (lower case indic.ating the mouse homologue, OMIM 176258), and Myod-1 (OMIM 159970).

Kv3.2 hKv3.2, KCNC2, 19q13.3-19q13.4 or chr 12 [?] [~] mKv3.2, Kcnc2, Chr 10

50-18-05: hKv3.~: KCNC2/hKv3.2/HKShIIIA: Localized by fluorescence in situ hybridization to human chromosome 19qI3.3-19qI3.4, co-localized with KCNC3 53 . Conflict note: Also mapped to human chromosome 12 using an mShaw12 probe13,54. See also OMIM entry 176256. 50-18-06: mKv3.~: The gene homologous to KCNC2 in the mouse has been placed on mouse chromosome 10 using inheritance patterns of DNA restriction fragment length variants in recombinant inbred strains of mice 53 .

---J_

e_n_t_ry_50

1_ _

Table 5. Continued Consensus/summary (see note 1)

Further details (see note 2)

Kv3.3 hKv3.2, KCNC3, 19qI3.3-19qI3.4 [~] mKv3.3, Kcnc3, Chr 10

50-18-07: hKv3.3: KCNC3/hKv3.3/HKShIllD: Localized by fluorescence in situ hybridization to human chromosome 19qI3.3-19qI3.4 co-localized with KCNC253 . This localization was consistent with that obtained using a human genomic DNA probe containing the 3' exon of the KCNC3 gene21?54. See also OMIM entry 176264. KCNC3 has been noted as a candidate disease gene for chromosome 19q cone-rod dystrophy (CORD2)55. 50-18-08: mKv3.3: The gene homologous to KCNC3 in the mouse has been placed on mouse chromosome 1053 .

Kv3.4 hKv3.4, KCNC4, Ip21

50-18-09: hKv3.4: KCNC4/hKv3.4/HKShIllC: Human chromosome Ip21 by fluorescence in situ hybridization (FISHt )21?24?53?54. See also OMIM entry 176265.

Notes: 1. [?] symbol denotes possible conflict. [~] symbol denotes likely paralogous cluster. 2. For background, see also Tables 7 and 8 in Chromosomal location under VLC K Kv1-Shak, 48-18.

further functional elements of the Kv3.1 promoter were delineated, including: (i) the essential promoter, localized to a highly GC-rich regiont containing four SP-l t -binding sites; (ii) multiple silencing elementst i. (iii) enhancing elements t ; (iv) one cyclic AMP/calcium response element f (CRE, beginninf at position -252 relative to the start codon) and (v) one AP-l-binding site (position -508)56.

Mammalian Kv3 subfamily genes encode multiple protein variants (initial identification) 50-20-02: Kv3.1b/isolate-clone KV4: The vertebrate Shaw-related channel mRNAs undergo extensive alternative splicing t events within their proteincoding regions (for example, generating potassium channel subunits with variant C-termini, see below). The existence of alternative splicing in the Kv3 subfamily was initially identified when genomic and eDNA sequences encoding the isolate/clone KV4 diverged at the 'breakpoints' of genomic/ eDNA co-linearityt. In this example, sequences local to the 'breakroint' agreed well with the consensus sequence for a 3' splice acceptor site (YYYNCAG), suggesting the presence of intronst upstream of the splice

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Figure 1. Amino acid sequences of the Shaw-related isoforms rKv3.1, rKv3.2, rKv3.3 and rKv3.4, together with that of Drosophila Shaw. Variants in open reading frame lengths due to alternative splicing are indicated within Figs 4-6 under Gene organization, 50-20. Other ORF lengths can be traced through accession numbers and references listed under Database listings, 50-53. (Alignment kindly provided by George Gutman, University of California at Irvine.)

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Figure 2. Recognition of an exon/intron border in genomic DNA producing Kv3.1b. The figure illustrates how the exon/intron border in genomic DNA (compare to the consensus sequence at bottom) is recognized for expression of the Kv3.1b/Raw2 mRNA, whereas the Kv3.1a/NGK2 product represents a tread-through' product.

sites4 . Consistent with an authentic splicing mechanism, mature transcripts diverged at the same point in the sequence following an AG dinucleotide motif characteristic of 5' exonic donor splice junctions (see Fig. 2; for a catalogue of splice junction sequences, see ref. 57). Genomic Southern blots t confirmed the existence of alternative splicing from single-copy Kv3.1 (and later Kv3.2) genes (see Southerns, 50-25 and paragraph 50-20-03).

Overviews of intron/exon usage in the Kv3 gene subfamily 50-20-03: The linear organization, regulatory mechanisms and splicing patterns of the four independent Kv3.1 to Kv3.4 subfamily genes appear broadly similar, consistent with an evolutionary origin at some time involving duplication of an ancestral gene (for discussion, see Chromosomal location of VLG K Kv1-Shak, 48-18). For comparative purposes, Figs 3-6 summarize the basic patterns of alternative splicing that have been reported or predicted from eDNA/genomic sequencing and mRNA transcript mapping t studies. These figures emphasize length variations in C-termini.

Homologous isoforms Species origin of clone NGK2 50-21-01: Kv3.1a/NGK2; Kv3.1b/Raw2: In comparison with the Kv3.1b/Raw2 nucleic acid sequence, the Kv3.1a/NGK2 sequence derived from a mouse-rat neuroblastoma-glioma hybrid cell eDNA library contains many substitutions (50 within 1640nt)18. These substitutions do not alter the derived protein sequence but suggest that the NGK2 eDNA was derived from mouse mRNA sequences in the hybrid cell line initially used16.

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50Iaasegment K\' .l.la and Kd.lb encoded by exonsl&2

Kv3.Ib As~ l

•

Asp Ser Lys Leu Asn Gly Glu Vd

Al~

Lys

Al~

Al~

Leu

Al~

1.3n Glu 1.3p Cys Pro His Ile 1.3p Gln

Al~

Leu Thr Pro

~

[

~~~~~~~~~~~~IGA~~~~~~~~~~~~~~~ Glu Gly Leu Pro Phe Thr Arg Ser Gly Thr Arg Glu Arg Tyr Gly Pro Cys Phe Leu Leu Ser Thr Gly Glu Tyr

[

Al~

Cys Pro

J

1758

CCT GGT ~ GGA ATG IGA AN:; GAT CTT TGC MA GM 1GC CCT GTC ATT GeT AN:; TAT ATG CC'G ACA GAG GCT GTG IGA GTG ACT ~GA ••• Pro Gly Gly Gly Met Arg Lys Asp Leu Cys Lys Glu Ser Pro Vd Ile Al~ Lys Tyr Met Pro Thr Glu Ah Vd Arg V~l Thr •

585

Figure 3. Key features of Kv3.1 gene organization and variable 3' ends of Kv3.1a and Kv3.1b isolates produced by alternative splicing. (a) Partial Kv3.1 gene organization/splicing patterns predicting different C-terminal ends. (b) C-terminal ends of mKv3.1a (511 aa) and rKv3.1b (585 aa). A B, C

D

E

F, G

H

-

Putative alternative splice variants (with open reading frames as indicated) begin at presumed initiator codons (ATG). The amino end of the Kv3.1 proteins is encoded in a single exon (exon 1 [B}) which is constitutively spliced in all Kv3 subfamily genes to the downstream exon 2 (Ie}). Exon 2 encodes most of the 'core' domains, from the beginning of the Sl domain to a point beyond domain S6 (for Kv domain descriptions, see Domain functions under VLG K Kv1-Shak, 48-29). A site consistent with alternative splicing (as opposed to constitutive splicing, see above) is present at the end of exon 2 sequences which have been identified in Kv3.1, Kv3.2 and Kv3.3. Kv3.1a (and Kv3.3b, see Fig. 5) is generated by read-through of the sequence immediately following exon 2. Kv3.1b (and Kv3.3a, see Fig. 5) is generated by splicing out segment [E} and replacing it with exons 3 and 4 present downstream (as marked). Note: Mechanisms of splice choice ('splice/don't splice') have been discussed in ref.58. The 5' end of exon 3 in the Kv3.1 gene ([F}) starts with the sequence of Kv3.1b immediately following the point of divergence and extends 188 bp in the 3' direction. Exon 4 is separated from exon 3 by an intron and contains the remainder of the 3' end of Kv3.1b ([G}). Kv3.1a and Kv3.1b have identical coding sequences leading up to (but prior to) in-frame stop codons (marked with asterisks in (b)). Mature transcripts from each gene diverge at the same point in each sequence following an AG (5' exoDic splice motif7), denoted by the arrow labelled [H].

1'--_e_n_t_ry_50

I

---"_

In Kv3.1a (and Kv3.3b, see Fig. 5) the genomic nucleotide sequence (and 'read-through' cDNA sequence) following the AG starts with GT, the dinucleotide characteristic of intronic portions of 5' splice sites 57. The GT residues are denoted by the arrow labelled [I].

Further notes on Kv3.1 gene organization/exon usage cited in original literature Kv3.1a/NGK2: In comparison with the Raw2 nucleic acid sequence, the NGK2 sequence contains many substitutions but these do not alter the derived amino acid sequence (50 in 1640nt)16,18 (see Domain conservation, 50-28). Kv3.1b/Raw2: Amino acid sequence identical to isolate KV4, and is a rat isoform of NGK2 (see below). The Raw2 C-terminus diverges from the NGK2 C-terminus (a putative consensus splice site t at amino acid 502) and is 74 amino acids longer. For these reasons, the rat isoform of NGK2 has also been called Raw2a (see 'sluggish splicingt , in the glossary). Kv3.1 a/Isolate KV4: KV4 has 'unusually long' 5' and 3' untranslated regions t (1161 bp and 1061 bp respectively). The isolate KV4 poly(A)+ signalt is 21 bp upstream t of approx. 150-200 adenosine residues as based on the size of the mRNA to which rvfull-length isolate KV4 hybridizes. (Redrawn by author with permission from Vega-Saenz de Miera et al. (1994) Shaw-related K+ channels in mammals. In Handbook of Membrane Channels (ed. C. Peracchia), pp. 41-78. Academic Press, San Diego.) (From 50-20-93)

Protein molecular weight (calc.) 50-23-01: Typical calculated molecular masses (as cited in the literature) for Kv3 subfamily channels include Kv3.1a/NGK2: 57.9kDa (57925); Kv3.1b/ Raw2: 65.9 kDa (65864); Kv3.1b/isolate KV4: 65.8 kDa (65860); rKv3.2d/ Rawl: 69kDa (69015); rKv3.4a/Raw3: 68.4kDa (68443); hKv3.4b/HKShIllC: 64.5 kDa (64526).

Sequence motifs A motif substituent potentially influencing the threshold of voltage-dependent gating

50-24-01: The leucine heptad repeat motif (similar to the leucine zipper t sequence described in Sequence motifs under VLG K Kv1-Shak, 48-24) is interrupted in the Shaw-related channels (the fourth Leu is substituted by Phe). This conserved substitution may contribute to the observed shifts in voltage-activation thresholds (to more positive membrane potentials) in Kv3 subfamily channels compared to the Shaker-related K+ channels (see

Phenotypic expression, 50-14 and Activation, 50-33).

Other notable motifs in the Kv3 subfamily protein sequences 50-24-02: Several other conserved protein motifs associated with Kv3 subfamily proteins have been mentioned in the literature that either have no verified functional role or else are predicted to be relevant for certain post-translatory modifications (summarized in Table 6).

_'--

e_n_try_s_O-----'

(a)

Kv3.2 Kv3.2

(b)

G • 20aa

~gmcnt

1874 Kv3.2a

in K, ,l2a

6i 3 •

AT AN:. TGC AM c.AT GTT GTC ATT ACT GeT TN: ACX; CA1t. Gee GAG GCC N;A TCT CTT ACT TAA TGAC'TTGGG ~ Asp Asn Cys Lys Asp VAl VAl l1e Thr Gly Tyr Thr GIn Ala Glu Ala Arg Ser Leu r

1,771

H •

ATC N:iI:; AM G l1eAcgLys

591

CA~

I

~~5

I

624 • ".

TC TTG TN: /\GA ATT TAT CAT GGA TTT TTC CCT GCT GAIt. AAT GGG ACA TTG N:;A TTT IGC CAT TCC Mt:.. GAT TGT ACT GGA AAC TTC TGC TJr8J Leu Tyr Acg l1e Tyr His Gly Phe lAu Pro Ala Glu Asn Gly Thr Leu Arg Phe Ser Hi' Ser Lys Asp ':ys Thl' Gly Asn Phe Cy"

'1:1

Kv3.2b c.A~GAlt.AM~~IGC~AN:.1lN:.~~~~~~~~~N:;A~~~~~~~~AN:.~~~

GlI Tye Glu Lys Ser Arg Ser Leu Asn Asn lIe Ala ':ily lAu Ala Gly Asn Ala lAu Acg lAu Ser Pro Val Thr Se1' Pro Tyr Asn Se1' Pro eys

F

L

l

1,917

CCT CTG AGG CGC TCT CGG TCT CCC ATC CCA TCT ATC TTG TAA '" Pro Leu Acg Acg Ser Acg Ser Pro Ile Pro Ser Ile Leu •

638

Figure 4. Key features of Kv3.2 gene organization and variable 3' ends of Kv3.2a, Kv3.2b and Kv3.2d isolates produced by alternative splicing. (a) Partial Kv3.2 gene organization/splicing patterns predicting different Cterminal ends (not to scale; for 5' UTR splice variants of Kv3.2, see text). (b) C-terminal ends of Kv3.2a (613 aa), Kv.3.2b (638 aa) and Kv3.2d. A

B, C

D

E

Putative alternative splice variants (with open reading frames as indicated) begin at presumed initiator codons (ATG). The amino end of the Kv3.2 proteins is encoded in a single exon, (exon lIB}) which is constitutively spliced in all Kv3 subfamily genes to (i) the downstream exon 2 (Ie}) and (ii) a similar Iconstitutive' splice pattern between exon 2 and exon 3. Note: This extends the region shared by all Kv3.2 proteins to 165 bp beyond that typical of Kv3.1 proteins (compare Fig. 3). Exon 2 encodes most of the ~core' domains, from the beginning of domain Sl to a point beyond domain S6 (for Kv domain descriptions, see Domain functions under VLG K Kvl-Shak, 48-29). A site consistent with alternative splicing (as opposed to constitutive splicing, see above) is present at the end of Kv3.2 exon 3 sequences. Although details are presently unclear, indirect evidence suggests that Kv3.2a and Kv3.2b are generated by alternative exon usage (either exon 4 or exon 5 joining on to exon 3). The Kv3.2d transcript may arise by use of an internal acceptor site in the same exon as used to generate Kv3.2a. (95 bp downstream of the Kv3.2a sequence in (b)). Correspondingly, the presence of internal splice acceptor sites t might also generate the variant designated as Kv3.2c in the same exon that generates Kv3.2b. (Note: Kv3.2c was described as a partial cDNA, see ref. 59).

l_e_n_t_ry_s_O

_

Southerns Initial evidence for transcript/channel diversity arising from splicing single-copy gene products 50-25-01: Direct support for an alternative splicing mechanism in specifying diversity of Kv3.l 4 and Kv3.2 channels 59 initially came from analysis of rat genomic DNA in Southern blotst. Radiolabelled DNA probest derived from regions preceding points of divergence (see Gene organization, 50-20) detected single restriction fragmentt bands for each gene under high stringencyt hybridization conditions. This indicated that Kv3 subfamily genes were single-copy in the genome (as typical of other Kv genes), a finding since confirmed by independent gene mapping analyses (see Chromosomal location, 50-18).

STRUCTURE AND FUNCTIONS For references pertinent to all of the Kv channel subfamilies, see the STRUCTURE AND FUNCTIONS section under VLG K KVl-Shak, entry 48.

Domain arrangement 50-27-01: Alignments of vertebrate Shaw-related channels show high conservation of the hydrophobic core regions seen in other Kv families {the F, G, H, I

Although the AG (5' exonic splice motif, [HJ) is present in all Kv3.2 variants, none of the mature transcripts from the Kv3.2 gene begin with GT motif (as seen in Kv3.1 or Kv3.4 transcripts, see figs 3 and 6). The existence of an AT motif (in Kv3.2a, [GJ), GA (in Kv3.2b, [FJ) and TG (in Kv3.2d, [IJ) indicates that distinct splicing mechanisms may operate in the generation of Kv3.2 transcript diversity.

Further notes on Kv3.2 gene organization/exon usage cited in original literature Kv3.2/RKShIIIA: Correction 14 of a reported60 in-frame stop codon t in the RKShIIIA sequence revealed three variants that differed in the carboxyl end, probably generated by alternative splicing. In comparison with Rawl (below), RKShIIIA possibly contains an additional exon of 95 nucleotides (nt 1781-1875 in RKShIIIA cDNA) rKv3.2d/Rawl: Variant C-termini generated by alternative splicing of RNA; cDNA sequences diverge at the same nucleotide relative to RKShIIIA above (nt 1970/1971 in Rawl). The Rawl gene gives rise to several (at least four 4 ,14,15,18,59) alternative mRNAs. (Redrawn by author with permission from Vega-Saenz de Miera et al. (1994) Shaw-related K+ channels in mammals. In Handbook of Membrane Channels (ed. C. Peracchia), pp. 41-78. Academic Press, San Diego.) (From 50-20-93)

II

_'---

e_n_try_s_O_

(a)

Kv3.3 ..- Kv3.3a Kv3.3

(b)

Kv3.3a

22llaasplicecas.<;etteinK\"J.JaenClldedhye\onsJ&~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .~O

Gl, AIIn AIIp l.eu Gl, Val l.eu Glu Glu Gl, A8p Pro Art PrO AlIa Gl, AIIp

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Ala l1e AIIp Glft

.~o

Ala Met

le~

,~o

G1II AIIp L,.

'e~

Pro l1e

ft~

.~o

COl,

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ '1M Leu Val ftl~ AIIp 'I'J~ Ala .~o •• ~ ,~o AIIp Gly . . ~ 11. Art L,. Ala Tlu

H

+

1.966

C~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Pro eye Pro Gln Ala

I.~

Ala I ...

'~o

ft~

l u TIll'

TIll' Gl" Gln ,~o

I.~

Art Gly Art Ty.. Se.. Art AIIp Art Ala

G11 Ala

G11 T,I'

'''0

,~o

'''0 '1'0 ..~

Leu

,~o

eye

Pro Mia Ala CUy Val

Gly Art Ala

'''0 Ie..

Pro

'''0

~~~~GM~~~~~~~~~~~~~~~~~~~~~~~~

Gl" Ala TIl..

GM~1IN:MiG~G

,~o

ey•• ~o

Ala Ala Ala Ala l.eu Ala M18 Glu Asp

'~o

Glu Ala

.~

Ala l1e the AIIp Val

'~p

l.eu Pro

,~o

.he Ili. Act le~

M18 Gl"

,~O

'~o

Gly Ly. lia Gl" Art

Glu Ttl.. Asn Art Ala

t

6S6

660aa IeltneDl commoato Kv3.3a aDd Kv 3.3b

eDCOdedby exoul&2

===~=========================J

C~~~~~~~~~~~~~~~~~~~~~~~~~MiG~~ eye eye l.eu

11. Se.. Vd

'~o

le~

Se..

Mia Art IAu Art

,~o

Art Glu

TIl~

l.eu COlf Phe

'1'0

Leu Ie .. IAu Pro

'''0 Art

ACT GGG AAT GiGT ~ MiG GI\G TGT ~ N;A GAT CCT GGG CT1 CCT ftC CCC TCC ~ CAC TCC TCT CCT Gel. GTT TeA Tlu Glf As" Glf Gly Art Glu eye '1'0 Art Asp 'ro Glf Leu '1'0 the '1'0 le~ Art Mh Ser Se.. Pr~ Ala Val •

Leu Ala

GTAC~CC

•••

889

Kv3.3b

\9aa segment in K,' .L1h generated hv 'read through' from e\on 2

-.

-'.

2.069

lOT GI\G ~ GGA ~ MiG ACT GGA GGT GTG ~ N;A ~ GGG GGT ~ GGG TCG GOG TAG G Glf Glu Ala C011 Ala Ar, TIl~ Glf Gly Val Glf Art '1'0 Glf Glf Trp Gly '.1' Glf •

•

GGGllGGAGGC

GGOGGGTTG

Gol\iGGGGAT •••

6~

Figure 5. Key features of Kv3.3 gene organization and variable 3' ends of

Kv3.3a and Kv3.3b isolates produced by alternative splicing. (a) Partial Kv3.3 gene organization/splicing patterns predicting different C-terminal ends. (b) C-terminal ends of Kv3.3a (889 aa) and Kv3.3b (679 aa). A H, C

D

E

Putative alternative splice variants (with open reading frames as indicated) begin at presumed initiator codons (ATG). The amino end of the Kv3.3 proteins is encoded in a single exon (exon 1, [B}) which is constitutively spliced in all Kv3 subfamily genes to the downstream exon 2 ([C}). Exon 2 encodes most of the 'core' domains, from the beginning of the Sl domain to a point beyond domain S6 (for Kv domain descriptions, see Domain functions under VLC K Kvl-Shak, 48-29). A site consistent with alternative splicing (as opposed to constitutive splicing, see above) is present at the end of exon 2 sequences which have been identified in Kv3.3, Kv3.2 and Kv3.1. Kv3.3b (and Kv3.1a, see Fig. 3) is generated by contiguous readthrough of the sequence immediately following exon 2 ([E}). - Kv3.3a (by similarities in gene structure to Kv3.1b, see Fig. 3 and ref. 19) is expected to be generated by splicing out segment [E} and replacing it with exons 3 and 4 present downstream (as marked). Note: Mechanisms of splice choice ('splice/dont splice') have been discussed in ref. 58 .

l_e_n_t_ry_50

_

'SI-S6 + H5' arrangement, for background, see VLG key facts, entry 41 and VLG K Kv1-Shak, 48-27).

Domain conservation Sequence conservation and diversity in the Kv3 subfamily 50-28-01: Since Shaw-related vertebrate K+ channel proteins arise from alternatively spliced t mRNA transcripts t they display much variation in length and sequence of their N- and C-termini (see Gene organization, 50-20). Other conserved features deduced from Kv3 subfamily sequence alignments include (i) an approx. 70 residue stretch in the N-terminus separated from domain SI by a non-conserved segment of 50-70 residues (see Fig. 1 under Encoding, 50-19) and (ii) two consensus sites for N-glycosylation in the SI/ S2 extracellular loop and several motifs for protein phosphorylation (e.g. a 'universal' protein kinase C motif in the S4/S5 intracellular loop - see also Protein phosphorylation, 50-32). As noted for the RCK proteins 61 , the loop between putative transmembrane domains SI and S2 (the 81/82 loop) is the most variable sequence in the core regions of Kv3 subfamily proteins. In contrast to alignments between Drosophila Shaker and the RCK channels,

F, G

H

I

The 5' end of exon 3 in the Kv3.3 gene ([F}) starts with the sequence of Kv3.3a immediately following the point of divergence. Exon 4 is separated from exon 3 by an intron and contains the remainder of the 3' end of Kv3.3a ([G]). Kv3.3a and Kv3.3b have identical coding sequences leading up to (but prior to) in-frame stop codons (marked with asterisks in the lower sequence). Mature transcripts from each gene diverge at the same point in each sequence following an AG (5' exonic splice motif 57), denoted by the arrows labelled [H]. In Kv3.3b (and Kv3.1a, see Fig. 3) the genomic nucleotide (and 'readthrough' cDNA) sequence following the AG starts with GT, the dinucleotide characteristic of intronic portions of 5' splice sites (ref.). The GT residues are denoted by the arrows labelled [I].

Further notes on Kv3.3 gene organization/exon usage cited in original literature. mKv3.3: The presence of multiple transcripts in brain and liver suggested the existence of alternatively spliced forms of mouse Kv3.3 (see Kv3.3b, below). Kv3.3 is encoded by at least two exons (encoding rv211 N-terminalresidues and rv467 C-terminal residues with 1 residue encoded by sequences straddling the splice point). Exons are separated by rv3kb of intervening sequence as described21 . Kv3.3b: Sequence analysis of the novel developmentally regulated cDNA mKv3.3b (ref. 22, see Developmental regulation, 50-11) confirmed it as an alternatively spliced variant of Kv3.3 (described in ref. 21). (Redrawn by author with permission from Vega-Saenz de Miera et a1. (1994) Shaw-related K+ channels in mammals. In Handbook of Membrane Channels red. C. Peracchia), pp. 41-78. Academic Press, San Diego.) (From 50-20-93)

en_t_ry_5_0

_'--

----l

(a)

Kv3.4 Kv3.4

(b)

Kv3.4b

-t4aasegmenlinK\"J.-thgeneraledhy'readlhrough'fromexon2 M~~JlGG~~~~AAA~~~~~~~JlGG~~~~~~~~~~~~

Cly Clu Ua

1,603

~,

Gly Trp Glu Cly Ly. Sal' Leu ....

H •

ATG TGT

'1'0

Glft fql

~

GTG

.~

~t

Glu "'-

~ ~ ~ ~

'1'0

Asft Gly

'1'0

MG CAf AAA GM' GTG

...t ey••he Val Trp Gly ftla fro Ly. Mia Ly. Aap Val

GAG JlGG AAA C'GG Gel. C Clu ~, Ly. ~, Ala

Gln ftl' Leu ely'''' ely

~ '1'0

nA 1Q GGG Leu •

c~.

~2

Sa3

S3S

~~ ~~

= = ~::::

~:~::

~ ~

Kv3.4a

e: ~ := ~;I

~;J

~ ~ :: ~ ~ ~~ ~:c:

~

Ley.

[ CTG TCC GAf GGA GCT GGC erc N:r Leu "I' Asp Glu Glu Gly Ala Cly Leu ftl' G1A

L CGA

0

'1'0

Gee CTG Ala aiC TCA GGC ACA C'GG GAoC

Art Ala Leu

~t

~,

'ar ely Tblf Ar, Asp

~

fAA TGT GCT Ala Leu •

Lccc CTG Gee Tai Gee ccc: N:r ~ ~ ~ OGTl Leu Ala Salf ALA 'If !Ill' Glu Glu '1'0

~ ~ ~l

~,814

62S

~,

AGAl ~t

LAN;

MG AN. GCA GCT Gee 1'GC ~ CTG erc JlGT] Asn Ly. Ly. Ala Ala Ala ey. 'ha Lau Leu ....

LGCT GGG TAT Gee TGT GCT GAT ~ IlGT GTe C'GG AAA G Ala C1y Asp Tyl' Ala ey. Ala Asp Gly Sal' Val Ly. CAe

~9

607

Kv3.4c N:r 1'GC CAA GIll: GC'C ere Tao ~ AN: TAT Gee CAt GCT ~ clu Thlf ey. Gln Asp Ala ~u Sal' Sal' An Ala .11 Ala elu

"'1'

TC erc N:r erc TCT TJIG .to GCGGCNXAA Val Leu TtlI' Leu sal' •

~

1.A•••

627

Figure 6. Key features of Kv3.4 gene organization and variable 3' ends of Kv3.4a, Kv3.4b and Kv3.4c isolates produced by alternative splicing. (a) Partial Kv3.4 gene organization/splicing patterns predicting different Cterminal ends. Compare similar relationship of Kv3.4a and Kv3.4c to that between Kv3.2a and Kv3.2d (Fig. 4). (b) C-terminal ends of Kv3.4a (625 aa), Kv3.4b (583 aa) and Kv3.4c (627 aa). A Putative alternative splice variants (with open reading frames as indicated) begin at presumed initiator codons (ATG). B, C The amino end of the Kv3.4 proteins is encoded in a single exon (exon 1, [B}) which is constitutively spliced in all Kv4 subfamily genes to the downstream exon 2 (Ic)). Exon 2 encodes most of the 'core' domains, from the beginning of the Sl domain to a point beyond domain S6 (for Kv domain descriptions, see Domain functions under VLG K Kvl-Shak, 48-29). D, E hKv3.4b diverges from the Kv3.4a and Kv3.4c at an analogous point ([D}) to the Kv3.1 and Kv3.3 variants (compare Figs 3 and 5). The nucleotide sequence of Kv3.4b immediately following the divergence point (above) starts ~vith GT (as seen in Kv3.1a and Kv3.3b, ibid.) consistent with it being generated by read-through of the sequence immediately following exon 2 ([E}). F Sites at the ends of both exons 2 and 3 appear to generate Kv3.4 variants: rKv3.4a and rKv3.4c have an identical 204 bp sequence following their divergence from Kv3.4b. Sequence §imilarities of this 204 bp segment with exon 3-derived sequences in Kv3.1b, Kv3.2 and Kv3.3a suggest alternative splicing takes place at the end of exon 3 ([F}).

l_e_n_t_ry_S_O

_

the flanking N-terminal sequences are not well conserved between the Drosophila Shaw and the rat (Raw) channels (an exception to this is one stretch of 67 amino acids - residues 100-166 in rKv3.2d/Rawl)18. Furthermore, the rKv3.4a/Raw3 'inactivation ball' region (1"'J28 N-terminal amino acid residues) shares little homology with corresponding regions of other potassium channels 18 .

Protein interactions Indirect evidence for heteromultimer formation between Kv3 subfamily proteins 50-31-01: As reviewed by Vega-Saenz de Miera et al. (1994; see Related

sources and reviews, 50-56) all Kv3 subfamily channels show a similar voltage dependence of activation, and this property does not change following

H

I

I

J

K

A site consistent with alternative splicing (as opposed to constitutive splicing, see above) is present at the end of exon 2 sequences which have been identified in Kv3.1, Kv3.2 and Kv3.3. Kv3.1a (and Kv3.3b, see Fig. 5) is generated by splicing out segment [E} and replacing it with exons 3 and 4 present downstream (as marked). Note: Mechanisms of splice choice ('splice/don't splice') have been discussed in ref. 58. The 5' end of exon 3 in the Kv3.1 gene ([F}) starts with the sequence of Kv3.1b immediately following the point of divergence and extends 188 bp in the 3' direction. Exon 4 is separated from exon 3 by an intron and contains the remainder of the 3' end of Kv3.1b ([G}). Kv3.1a and Kv3.1 b have identical coding sequences leading up to (but prior to) in-frame stop codons (marked with asterisks in the lower sequence). Mature transcripts from each gene diverge at the same point in each sequence following an AG (5' exonic splice motif 57), denoted by the arrows labelled [H}. In Kv3.1a (and Kv3.3b, see Fig. 5) the genomic nucleotide (and Ireadthrough' eDNA) sequence following the AG starts with GT, the dinucleotide characteristic of intronic portions of 5' splice sites 57. The GT residues are denoted by the arrows labelled [I}.

Further notes on Kv3.4 gene organization/exon usage cited in original literature mKv3.4: Mouse Kv3.4 gene has a similar genomic organization to mouse Kv3.3 (i.e. an intron t immediately upstream of the Sl transmembrane domain coding region). hKv3.4b/HKShIIIC: hKv3.4 and rKv3.4a/Raw3 show 960/0 amino acid identity up to Ala-538, and then Idiverge completely,24. (Redrawn by author with permission from Vega-Saenz de Miera et al. (1994) Shaw-related K+ channels in mammals. In Handbook of Membrane Channels (ed. C. Peracchia), pp. 41-78. Academic Press, San Diego.) (From 50-20-93)

_ L..--

entry 50 _

Table 6. Miscellaneous Kv3 subfami.ly protein motifs (of unknown significance or else predicted to be sites of specified post-translatory modification) (see also Domain functions, 50-29 and Protein phosphorylation, 50-32) (From 50-24-02) Kv3.l/Isolate KV4:

Two N-glycosylation motifs at Asn220 and Asn229. Note: A short C-terminal sequence thought to be encoded by the variable exon 3 coding region appears relatively conserved. Compare the sequences PLAQEEI (Kv3.l, residues 489-495), PLAQEEV (Kv3.3, residues 648-654) and PPAREEG (Kv3.4, residues 526-532).

Kv3.2b:

Two N-glycosylation motifs at aa 259 and 266.

Kv3.2c:

Gly/Pro-rich insert aa 56-99; Clone RKShIIIA: Two N-glycosylation motifs at aa 259 and 266. Kv3.2, isolate RKShIIIA, a 44 aa insert in the amino end (residues 56-100) contains several stretches of consecutive prolines. These proline-rich sequences of Kv3.2/3.3 fit the consensus sequence derived for SH3t domain-binding sites, which have been established as subunit association domains in several signal transduction proteins (e.g. the hinge region of IgGs, Al basic protein and synapsin). In addition, there are putative O-glycosylation (cytoplasmic) motifs in this sequence which may be involved in regulation of phosphorylation and protein assembly/targeting (see ref. 14).

rKv3.2d/Rawl:

Two N-glycosylation motifs at Asn259 and Asn266 in the S1-S2 loop region are the only potential sites likely to be extracellular.

mKv3.3:

Three putative N-glycosylation sites conforming to Asn-X-Ser/Th:r.

rKv3.4a/Raw3:

Two highly conserved sequence motifs (in other K+ channels) are different in Raw3-H364Q23: NEYFFD to ~EEFFD in the N-terminal sequence, and MTTVGY to MTT1GY between segments S5 and S6.

hKv3.4/HKShITIC:

Two putative N-glycosylation sites in the extracellular S1-S2 linker region. In the 'proline-rich' segment of clone HKShIIIC (a homologous region to that described under Kv3.2, this table) there is a shorter sequence rich in glycines24 .

lL...--e_n_t_ry_SO

--'_

(a)

Kv3.4

(b)

Kv3.1

r---I~~O?

,1000 nA

nA

100 ms

~ms

(c)

Kv3.4/Kv3.1

11000 nA

100 ms

Figure 7. Recordings suggesting formation of Kv3.1:Kv3.4 heteromultimeric channels expressed from co-injected cRNAs in Xenopus oocytes. (a) Current following injection of Kv3.4b cRNA alone. (b) Current following injection of Kv3.1 b cRNA alone. (c) Current following co-injection of Kv3.4b and Kv3.1b in the same amount as in (a) and (b). (Current traces taken from Weiser et al. (1994) J Neurosci 14: 949-72.) (From 50-31-01) co-expression experiments (as expected). However, oocytes co-injected with Kv3 cRNAs encoding channels with markedly different inactivation rates (e.g. Kv3.1 plus Kv3.4) produce currents suggestive of heteromultimer formation40 (see Fig. 7 below). Putative Kv3.1:Kv3.4 heteromultimers thus display fastinactivating currents that are several-fold larger than those seen in oocytes injected with the same amount of Kv3.4 cRNA alone (Fig. 7). Note: As demonstrated for Kvl subfamily channels displaying fast (N-type) inactivation, single ball domains are sufficient to confer inactivating properties, although the rate of inactivation increases with the number of inactivating subunits (see VLC K Kv1-Shak, entry 48). This pattern appears to hold for Kv3.1:Kv3.4 heteromultimers 40 and channels obtained following injection with Kv3.4 and Kv3.2 or Kv3.3 cRNAs (H. Moreno, cited in Vega-Saenz de Miera et al., 1994, under Related sources and reviews, 50-56).

Other domains with potential functional roles in Kv3 subfamily proteins 50-31-02: (i) Evidence for C-terminal 'subcellular targeting' domains in alternatively spliced products of the Kv3.2 gene are described under Subcellular

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locations, 50-16. (ii) several proline-rich domains have been observed in Kv3.2 and Kv3.3 amino acid sequences (e.g. the N-terminal side of 51 in Kv3.2, the N-terminal ball domain and post-56 region of Kv3.3) (see Sequence motifs, 50-24). (iii) Subfamily-specific assembly domains, i.e. those preventing heteromultimerization between Kv subfamily proteins and other Kv subfamilies18,62 are described under Protein interactions in VLC K Kvl-Shak, 48-31. Note that some apparent exceptions to subfamily-specific association have been reported for Kv3 'subfamily members' (see next paragraph). See also Sequence motifs, 50-24, Protein phosphorylation, 50-32 and Redox modulation under Channel modulation, 50-44-01.

Indirect evidence for 'cross-subfamily' heteromultimerism 50-31-03: Kv3.1a; Kv3.4: A novel class of heteromultimeric channels (exhibiting a 20p5 conductance with three degrees of inactivation mode) has been reported following cell-attached recordings of Xenopus oocytes injected with an equal amount of mRNAs encoding NGKI (Shaker-related Kvl.2) and NGK2 (Shaw-related Kv3.1a)63. Furthermore, the novel subunit Kv8.1 has been reported able to 'selectively abolish' the functional expression of Shaw (Kv3.4) and Shab (Kv2.1 and Kv2.2) channels when co-expressed in oocytes (for further details, see ref.64 plus the discussion under Phenotypic expression of VLC K Kvx [unassigned], 52-14).

Protein phosphorylation Compilation of common phosphorylation motifs in Kv3 subfamily proteins 50-32-01: Kv3 protein phosphorylation (e.g. as part of neurotransmitter receptor signalling cascades) is likely to be an important mechanism for controlling thresholds of pre-synaptic and post-synaptic excitability (see Phenotypic expression, 50-14). Mechanisms for modulation of Kv3 protein function by description of structural transitions or subunit assemblies/disassemblies are poorly developed, but extensive primary sequence analysis has predicted multiple candidate sites for kinase modification (Table 7). While the simple consensus sequences described in Table 7 do not provide evidence for functional significance, the striking conservation of some motifs across all Kv3 members (asterisked in Table 7) suggests they were present early in the evolution of the Kv3 subfamily. Other caveats regarding the interpretation of these motifs are described in the footnotes and key to Table 7.

Attenuation of Kv3.1 current by PKC and diacylglycerols 50-32-02: Kv3.1: The amplitude of Kv3.1 currents in NIH 3T3 fibroblasts (and the probability of Kv3.1 channel openings) are reduced following activation of protein kinase C by a phorbol ester46 . This action can be blocked by pre-incubation with the protein kinase inhibitor H-7 (1-[5isoquinolinesulphonyl]-2-methylpiperazine). Dioctanoyl glycerol also attenuates Kv3.1 currents, but this inhibition cannot be completely blocked by H-7, suggesting a kinase-independent mechanism of diacylglycerol action46 .

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Potential role of PKA modulation of Kv3.2 channels in thalamocortical synapse efficacy 50-32-03: rKv3.2: Kv3.2 channels expressed in CHO cells are strongly inhibited following phosphorylation by cAMP-dependent protein kinase (PKA)45 (in contrast to Kv3.1 subunits which are unaffected by PKA treatment but otherwise exhibit 'nearly identical' electrophysiological properties in heterologous expression systems). Kv3.2 proteins can also be phosphorylated in situ by intrinsic PKA (see note) and have localizations consistent with a role in regulation of thalamocortical synaptic efficacy t (see also mRNA distribution, 50-13 and Protein distribution, 50-15). These features have suggested potential functional roles of Kv3.2 in neurotransmitter-mediated control of the thalamocortical system and, in consequence, altered global states of awareness 45 . Methodological note: The 'intrinsic' phosphorylation described in this study employed a synaptosome model (resealed synaptic terminals). This technique 66 - 68 depends on measuring the amount of protein made unavailable for phosphorylation by the exogenous catalytic subunit of PKA in the presence of 'Y-32p-ATP following solubilization of the synaptosomes.

Elimination of N-type inactivation following phosphorylation 50-32-04: hKv3.4: Protein kinase C (PKC) treatment has been shown to specifically eliminate rapid (N-type) inactivation of hKv3.4, converting the channel to a non-inactivating delayed rectifier69 . Furthermore, the hKv3.4 N-terminal domain is a substrate for 'direct' in vitro phosphorylation by PKC at two serine residues within the N-terminal inactivation gate domain69 . Notably, mutation of one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may prevent rapid inactivation by 'shielding' basic (positively charged) residues known to be critical to the function of the inactivation gate (see Inactivation under VLC K Kv1-Shak, 48-37). The implications of this mechanism for signal encoding in the eNS have been discussed69 .

ELECTROPHYSIOLOGY

Activation Characteristic thigh voltage activation' of Kv3 subfamily channels 50-33-01: All heterologously expressed Kv3 subfamily channels show a similar voltage-dependence of activation, generally requiring membrane potentials more positive than -10mV to activate significant currents (i.e. more depolarized than typical in other Kv families). For example, Kv3.1 channels (e.g. isolates Raw2, KV4, NGK2) and Kv3.2 channels (e.g. isolates rKv3.2d/ Rawl, RKShIIIA) activate slowly, first open at approx. -20 mY. In oocytes, Kv3.4 (isolate Raw3) channels activate between -20 and OmV beyond the threshold of excitation 70, approx. 20 mV more positive than the threshold of activation for RCK-type channels 18 . Similarly, activation of hKv3.4b/HKShIIIC requires large membrane depolarizations (activating from about -10 mV) and is therefore described as 'high voltage activating'24. Table 8 further compares

-

entry 50

Table 7. Phosphorylation motifs in Kv3 subfamily proteins (From 50-32-01) Protein

PKA

PKC

CKII

Kv3.1

T21 * T25* T342* 5341 * [a] == 5510 [b] == 5503 [b] ==T539 [b]==T585

T21 * T25* T73* TI08* T337* 5185* T152

T99* 5341 * T483 544 5130 [b]==T527 [b] == 5550

T21 * T25* T379* T378* T574 5563

T21 * T25* T155* T374* 5224* 5205 5564 [d] ==T609

T146* 5378* T17 T520 T546 T579 5414 5541 5553 5564 [a] ==T605

TI03* TI07* T446 * 5445* T16 [a] == T736 [a] == T852 [a] == T865 [a] == 5713 [a] == 5885 [a] == 5886

TI03* TI07* T155* T190* T441 * 5286* T16 T657

T181 * 5445* T120 [a] == 5697

T49* T53* T379* 5378* T520 515 5519 [a] == 5541 [c] == 5541 [a] == 5578 [c] == T609

T49* T53* T113* T148* T374* 5222* 59 535 5515 [a] == 5604 [c] == 5604

(see note 3)

Kv3.2

(see note 4)

S564 [a] == T613 [d] == 5613 [b] == 5615 Kv3.3

Kv3.4

(see note 5)

II

ill 589 [a] == 5731 [a] == 5769 T139* 5378* T45 T520 5515 [a] ==T568 [c] ==T568 [a] == 5552 [c] == 5552 [a] == 5594 [c] == 5594 [c] ==T609

I

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activation parameter values for Kv3 subfamily members, distinguishing characteristics between Kv3 members (see Current type, 50-34).

Current type Types of current supported by Kv3 subfamily channels in oocytes 50-34-01: For comparative purposes, Table 9 summarizes the 'type' of currents observed following heterologous cell expression of Kv3 subfamily Q subunits alone. In vivo modulation (e.g. redox modulation t , see Channel modulation, 50-44 plus VLC K Kv-beta, entry 47) and subunit associations (ibid.) may markedly influence these properties. Note: In Drosophila, the Shaw gene was often described (along with the Shah gene) as encoding the 'delayed rectifier subtypes' (e.g. ref.26) in comparison to the 'transient (A-current) subtypes' encoded by Shaker and Shale

PKA: Protein kinase A consensus sites (see note 1); PKC: Protein kinase C consensus sites (see note 1); CKII: Protein kinase IT consensus sites (see note 1). * == putative sites common to all Kv3 subfamily channels. [a], [b], [c], [d] indicates a consensus site specific to the alternatively spliced variant of the gene as denoted in the square brackets. Underlined sites are designated as 'gene-specific'. Residues predicted to be phosphorylated are denoted by S == serine or T == threonine. Residues in italics - see note 2. Notes: 1. The first four columns are derived from a comprehensive compilation in the review by Vega-Saenz de Meira (1994, see Related sources and reviews, 50-56) using the following consensus sequences: Protein kinase A, PKA == R-[R/K]-X-[TIS] »R-X2 -[SIT] == R-X-[S-/T]; some of these sites may be substrates for cGMP-dependent protein kinase (see also note 2). Protein kinase C, PKC == [R/K I _3 ]-X2_0 -[S/T]-X-[R/K] or [S/T]-X-[R/K] (sites where the S or T was separated from the C-terminal basic residue were not included). Casein kinase II, CKII == [S/T]-X2 -[D/E] (other sites have been derived for this kinase but were not included in the analysis). 2. PKA sites in italics (column 2) conform to the more stringent consensus sequence R-[R/K]-X-[T/S]; this consensus has been more frequently used in the primary literature to predict one (or a small number) of PKA sites in Kv amino acid sequences/monomer. 3. rKv3.1/NGK2: Following stable expression in human embryonic kidney (HEK 293) cells, application of the PKC activator phorbol 12,13-dibutyrate decreases Kv3.1 currents 65 • 4. Kv3.2b: aa 564 (by cAMP kinase 'stringent' consensus; see note 2). 5. rKv3.4a/Raw3: PKA: (for Raw3-H364Q) aa residues 537-541 (C-terminus) encode the putative PKA phosphorylation site KRADS 23 •

II

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Table 8. Activation parameters for Shaw-related vertebrate K+ channels following injection of single mRNAs into Xenopus oocytes (From 50-33-01)

Kv3.2d-Rawl Kv3.lb-Raw2 Kv3.4a-Raw3

V n ,1/2 (mV)

an

(mV)

5.6±5.4 18.7 ±4.l -3.4±5.l

5.9±0.7 9.7 ±4.3 8.4±0.4

tn

(ms)

9.5 ± 1.8 2.4± 1.0

Notes: V n ,1/2 is the test potential where the conductance increase has reached half its maximal value (the conductance is calculated for each test potential by dividing the current amplitude by the driving force; the potassium reversal potential was assumed to be -lOOmV). Data derived from ensemble current recording from macro-patches (or by voltage-clamp for Kv3.lb/Raw2) with voltage steps made from -80 mV holding potential. an is the slope of normalized conductance-voltage relation (the value corresponds to the mV change in test potential to cause an e-fold increase in conductance). t n is the rise time of ensemble patch currents (the time to rise from 100/0 to 900/0 of final value). Data were measured at 50mV test potential following step changes from -80 mV holding potential. Activation parameter definitions and data from ref. 18 .

Current-voltage relation Fast voltage-dependent block by intracellular Mg2+ at millimolar concentrations 50-35-01: All Kv3 subfamily currents show a decline in conductance at membrane potentials more positive than +20mV (albeit this is less evident in Kv3.l). Kv3.2a channels display a 'fast flickering' at voltages more positive than +20mV, an effect that may be due to fast voltage-dependent open-channel block by intracellular components such as Na+ or Mg2 + ions (e.g. see refs. 19,24). For example, initial characterization of the Raw currents revealed an intense rectification and a negative slope conductance at test potentials more positive than +20 to +40mV18 . This is illustrated by the current-voltage relationship of single Raw3-~28, H364Q channels (defined under Voltage sensitivity, 50-42) in Fig. 8. The inward rectification was dependent on the presence of Mg2+ in the bathing solution (ibid.). Following removal of Mg2 +, the negative slope conductance disappears in inside-out patches. Under these conditions, the conductance does not decline, but increases over a range of test potentials. It was concluded that rectification of the outward currents results from voltage-dependent block by internal Mg2+, which binds to the channel at the cytoplasmic side. Furthermore, [Mg2+h is likely to block open channels as single-channel conductance at +50mV test potentials is rv3-fold higher in the absence of Mg2 + than in its presence. Compare to mechanisms determined for fast intracellular ion block of tstrong' inward rectifier channels by micromolar {Mg 2+ J and

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Table 9. General properties of Kv3 channel currents (From 50-34-01) Kv3.1 Kv3.2

Kv3.l and Kv3.2 transcripts support 'very similar' currents in oocytes that can be generally described as slowly rising, delayed rectifier type outward currents that inactivate 'extremely slowly'. As non-inactivating delayed rectifiers (see entry 45) these channels activate with a delay, and their rise timet decreases with increasing depolarization. In general terms, the rat isoforms of Shaw-related channels (isolates Rawl to 3) share several properties with the Shaker-related (RCK) channel series when expressed in oocytes 18. A notable difference, however, is that Raw outward currents exhibit an intense rectification at test potentials higher than +20 to +40 mV (see Current-voltage relation, 50-35).

Kv3.3 Kv3.4

Conversely, Kv3.3 and Kv3.4 transcripts express A-type inactivating currents that can be broadly distinguished by (i) Kv3.4's macroscopic inactivation rates being an order of magnitude faster than Kv3.3 and (ii) Kv3.4 current rise time being shorter than other Kv3 channels. These kinetic properties enable Kv3.3 channels to conduct in the steady state over a wider range of potentials than Kv3.4. Differences in the 'coupling' between inactivation and channel opening (e.g. Kv3.4 inactivating from closed states much more readily than Kv3.3)19 may explain differences in the voltage dependence of steady-state inactivation between Kv3.3 and Kv3.4.

polycationic species as described in INR key facts, entry 29, INR K [native], entry 32 and INR K [subunits], entry 33.

Inactivation iRe-opening' phenomena during recovery 50-37-01: rKv3.4aIRaw3: Inactivated Kv3.4a channels in oocytes are able to

conduct (i.e. 're-open') during repolarization of the membrane 70. Although current responses to depolarization are rapidly inactivating, current elicited by repolarization (Le. the 're-opening current') declines slowly and produces long-lasting after-hyperpolarizations t (AHP) under current-clamp r conditions 7o. In this study, Kv3.4a channels appeared unable to enter the resting (closed) state when the membrane was hyperpolarized unless their inactivation was first removed (resulting in the long-lasting AHP during recovery at voltages much more negative than those required for activation). Experiments involving fast application of ball domaint peptides have suggested that the phenomenon may be due to voltage-dependent release of the inactivation gatet 70. The general significance of 're-opening' currents has been critically discussed 71 . If a similar mechanism operated in vivo, it would imply additional physiological roles for Kv3.4a (or other A-type channels activating positive to the threshold of excitation or influencing the membrane potential in the 'subthreshold' range). Note: For other examples of Ire-opening currents', see refs 72,73 and VLG Ca, entry 42.

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pA

Raw3Q·.128

2.S

•

cell-attached

inside-out: 2.0

C 2mM Mg o without Mg

l.S

-2S

o

2S

SO mV

Figure 8. Single channel current-voltage (1- V) relationships of Kv3.4/Raw3~28, H364 channels (see text) in different recording configurations and in the presence or absence of 2 mM Mg2+. Figure illustrates the intense rectification resulting from a voltage-dependent open-channel block by internal Mg2+ ion, binding to the channel at the cytoplasmic side. (Reproduced, with permission, from Rettig et a!. (1992) EMB() J 112: 2473-86.) (From 50-35-01)

Initial direct evidence for N-type inactivation domains in Kv3.3 and Kv3.4 channels 50-37-02: The amino acid sequences of the inactivating homomeric channels Kv3.3 and Kv3.4 differ from those that do not inactivate by possession of an Nterminal1ball' segment of 78 residues (in Kv3.3) and 28 residues in Kv3.4. At the C-terminal end of these segments is a methionine that can be aligned with the starting methionine of Kv3.1 and Kv3.2 (see Encoding, 50-19). Deletion of the N-terminal 78 amino acid residues from Kv3.3 or the N· terminal 28 residues from Kv3.4 results in the expression of channels that do not inactivate18 . The fast inactivation of Raw3-H364Q (see Voltage sensitivity, 50-42) was thus described as being'converted to slow inactivation' by deletion of 28 N-terminal amino acids (mutant Raw3-~28, H364Q)18 in a manner similar to the Kr3.2djRawl and Kv3.1bjRaw2 wild-type isolates.

Properties of the hKv3.4 N-terminal irlactivation domain/peptide 50-37-03: Kv3.4: On the basis of measured effects on rapid (N-type) inactivation following application of various peptides on homomeric Kv1.1 currents in

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CHO cells, it has been proposed that (i) the hydrophobic N-terminal region of the hKv3.4 inactivation peptide (see below) blocks the channel pore and (ii) the adjacent positively charged region interacts with negative charges on the channel protein 74 . These features are analogous to those determined for fastinactivating channels in the Shaker subfamily (e.g. see Kv1.4 in entry 48). Furthermore, the 28-mer 'inactivation peptide' (hKv3.4-IP, 320 JlM in the patch electrode) based on part of the N-terminal sequence of the human Kv3.4 K+ channel can endow a rapidly inactivating A-current phenotype on noninactivating mKvl.l channels expressed in CHO cells 75 . Notes and crossreferences: 1. For similar experiments performed with Shaker B inactivation peptides (ShB-IP), see Inactivation under VLC K Kv-Shak, 48-37. 2. For differences in sensitivity to chemical modification and redox modulation of the hKv3.4-IP to the ShB-IP, see Channel modulation, 50-44. 3. For effects of protein kinase C specifically eliminating N-type inactivation of hKv3.4 (converting this channel to a non-inactivating delayed rectifier), see ref. 69 and Protein phosphorylation, 50-32.

tLow-level expression' of Kv3.4 affecting heteromultimeric channel properties 50-37-04: Small amounts of Kv3.4 cRNA (expressing small, fast-inactivating currents when injected into oocytes alone) produces fast-inactivating currents that are 'severalfold larger' when co-injected with an excess of Kv3.1 or Kv3.3 cRNAs4o . This amplification appears to result from increases in singlechannel conductance (in the heteromultimeric channels) and the observation that less than four (possibly even a single) Kv3.4 subunits are sufficient to impart fast-inactivating properties to the heteromultimeric complex40 .

Kinetic model Kv3.1 functions suggested by cellular-electrical modelling 50-38-01: Computational representations of the kinetics and voltage dependence of Kv3.1 have been incorporated into a cellular model with a sustained inward current46 . In contrast to other delayed rectifier currents (e.g. Kv1.1 and Kv1.6, see entry 48) the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. These results support the proposal that Kv3.1 provides rapidly firing neurones with a high 'safety factor' for impulse propagation (for background, see Phenotypic expression, 50-14).

Selectivity In-press update: see also conclusions from the first K+ -channel crystal structure (ref. 98).

Utility of domain chimaeras in determination of K+ channel pore properties 50-40-01: Kv3.1: Studies of Kv3.1 wild-type channels or chimaeras containing segments of Kv3.1 were important in defining the HSt or P-domain t function in K+ channels. In Kv3.1/NGK2, a 21 aa segment was shown to confer pore

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properties such as selectivity, single-channel conductance and sensitivity to internal/external TEA 76 to a Kv2.l-based 'recipient' (DRKl) (see Selectivity under VLC K Kv2-Shab, 49_40)77,78. In later studies, exchange of P-domains between Kv3.l and Kvl.3 (both T cell channels but with widely divergent pore properties) have also been important for identifying single residues in the deep pore acting as a K+ -Rb+ conductance switch 77 and that the Pregion and 86 domain contribute to the ion conduction pathway79 (see also ref. 80, which also concludes that the S6 segment forms part of the pore following transfer of the Shaker B S6 segment into a Kv3.la/NGK2 'recipient').

'Slight deviation' of Kv3.4 selectivity from a pure' K+ channel 50-40-02: Kv3.4: In one study24 of Kv3.4b/HKShmC, a plot of reversal potentials as a function of external K+ concentration (between 10 and 100 mM) gave a slope of 52 mV for a tenfold change in external K+ concentration (Le. smaller than that predicted (56 mV) by the Nernst equation t for a purely K+ -selective channel) and indicating some permeability to other ions81 . In general, Shaw-related channels show high selectivity for K+ over Na+ ions.

Voltage sensitivity Voltage sensing in Kv3.4 involves residues outside the S4 segment 50-42-01: rKv3.4aIRaw3: A substitution mutant of the Kv3.4 isolate Raw3

contains a His364Glu substitution in segment S4 (Raw3-H364Q)18, previously described23 simply as Raw3). Mutant Raw3-H364Q channels undergo shifts in threshold of voltage activation to more positive test potentials. This observation indicated that the conductance-voltage relationships of voltage-dependent ion channels are influenced not only by the number of basic residues in segment S4, but also the neighbouring amino acids 82 . Note: The Raw3-H364Q mutant is unaltered with respect to singlechannel conductance, apparent slope of conductance curves or the rapid inactivation kinetics of the Raw3 'wild type'.

Voltage sensitivity of 'time to peak' and current decline 50-42-02: For transient Shaw-related subtypes (e.g. hKv3.4b/HKShillC) the parameter of 'time-to-peak' (the time for current to reach a maximum following a depolarization) decreases with increasing depolarization. The rate at which the current declines or inactivates during a pulse is also voltage dependent, increasing with increasing depolarization24.This is a property of some (but not all) fast-inactivating K+ currents 83- 86 .

Kv channel surface potential related to activation midpoint potential 50-42-03: Kv3.4: The effects of strontium ions (Sr2+j 7-S0mM) in shifting of the Kv3.4 G(K)(V) curve along the voltage axis (8 mV at 50 mM Sr2+) has been interpreted in terms of lscreening' of fixed surface charges. The variable shift observed between channel subtypes expressed in oocytes (Kvl.l, Kvl.5, Kvl.6, Kv2.l and Kv3.4) is partially attributable to (i) net extracellular charge and (ii) variable amino acid composition in the loop between the S5 segment and the pore-forming segment of each channel87. The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.

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PHARMACOLOGY

Blockers Kv3 subfamily-selective pharmacology is poorly developed 50-43-01: The pharmacology of Kv3 subfamily channels using presently available compounds is very similar between subfamily members. Kv3 channels are 'relatively sensitive' to both 4-aminopyridine (4-AP) and tetraethylammonium ions (e.g. IC so rv 0.1-0.9 mM for the Raw isolates - compare the data in Table 10 with the 'high millimolar' sensitivities of other Kv channels in Blockers under VLC K Krl-Shak, 48-43). Conversely, Kv3 subfamily channels are relatively insensitive to toxins that block other Kv channels (Table 10, ibid.). Kv3.1 was included as part of a comparative study88 of the pharmacology of five Kv channel subtypes (see also Blockers under VLC K Kvl-Shak, 48-43). In separate comparative studies89, whole-cell analyses showed that 4-AP sensitivity of Kv3.1 was approximately '150 times greater' than that of Kv2.1; however patch-clamp analysis suggested the mechanism of 4-AP block in both channels to be qualitatively similar. Kv3.1's high 4-AP sensitivity relative to Kv2.1 was associated with both a slower 'off rate' (therefore increasing stability of the blocked state) in addition to a faster 'on rate' (and therefore increased access to the binding site)89.

Determinants of high-affinity tetraethylammonium- and 4-aminopyridine-binding sites 50-43-02: Various segmental exchanges and point mutations of non-conserved residues between high-affinity TEA+ binding sites in Kv3.1 and other Kv channels with distinct pore properties have been used to determine

Table 10. Examples of blocker sensitivities in Kv3 subfamily channels (From 50-43-01)

Kv3.1b, isolate Raw2 Kv3.2d, isolate Raw1 Kv3.4a, isolate Raw3

TEA+ (mM)

4-AP (mM)

Quinine (mM)

DTx (mM)

MCDP

0.1

0.25

1.0

0.1

0.9

0.13

0.3 (see note 3)

0.5

0.5

>100 (no effect) >100 (no effect) (-)

>200 (no effect) >200 (no effect) (-)

(nM)

Notes: 1. The table compares examples of IDso values (500/0 inhibition of peak current at 40mV test potential using two microelectrode voltage clamp configuration). 2. For compound abbreviations, see Blockers under VLC K Kvl-Shak, 48-43. 3. The human Kv3.4b channel (isolate HKShIllC) was reported24 with even higher sensitivity to TEA (half-block rv88 ± 121lM TEA).

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critical amino acid residues affecting block (e.g. between Kv3.1 and Kv2.1 79,8o or between Kv3.1 and Kvl.3 78 ). These types of studies were the first to define protein regions forming the inner mouth of voltage-gated K+ channel pores (suggesting that transmembrane domains 55 and 56 contribute to this region - for further details, see VLC K Kvl-Shak, entry 48). High 4-AP sensitivity could not be transferred from Kv3.1 to Kv2.1 via the 55-56 linker (pore or P region)9o. However, a chimaera of the cytoplasmic half of domain 86 increased block 20-fold, without affecting gating. Furthermore, a 'double chimaera' of the cytoplasmic halves of 55 and 56 'fully transferred' 4-AP sensitivity. It has thus been concluded that 4-APbinding determinants lie in the 86 segment forming the cytoplasmic vestibule of the pore, possibly overlapping a quaternary ammonium (QA) site (see next paragraph).

QA ions bind strongly to hydrophobic electron-rich functions in Kv3.1 50-43-03: Application of a series of external hydrophilic quaternary ammonium (QA) ions to oocyte outside-out patches containing Kv3.1 wild-type, Kv3.1 mutants and the 'non-inactivating mutant' ShakerB T449Y have determined that QA ion selectivity is determined by the physical properties of the QA ion, not the channel type 91 . The affinity for TEA+ derivatives is reduced in Kv3.1 Y407T (supporting the hypothesis that cation/1f-electron interaction is involved. Furthermore, binding affinities of QA ions were higher in Kv3.1 Y407F than in the wild type, suggesting that the hydroxyl groups of the tyrosines reduce QA binding91 ).

Channel modulation Removal of the Kv3.4 fast-inactivation process by redox modulation 50-44-01: Kv3.4: Exposure of the cytoplasmic side of inside-out membrane patches containing fast-inactivating Kv3.4 or Kv1.4 channels to mild oxidizing conditions (e.g. air exposure) results in removal of the inactivating process 92 . Inactivation can be restored by exposure to reducing agents (e.g. dithiothreitol or glutathione) or by reinsertion of the patch into cells ('patch-cramming'). These modulatory effects have been shown to be critically dependent upon oxido-reduction of a cysteine residue in the respective N-terminal 'ball' domains of these channels (for further background, see Channel modulation under VLC K Kvl-Shak, 48-44 and under VLC K Kv-beta, 47-44). See also the following paragraphs.

A candidate oxygen sensor enzyme/channel complex in airway chemoreceptors 50-44-02: Kv3.3: A role for co-expressed Kv3.3a and NADPH oxidase functioning as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines has been proposed5 . KV3.3a and membrane components of NADPH oxidase (gp91 phox and p22phox) are co-expressed in the pulmonary neuroepithelial bodies (NEB) cells in the airway mucosa of

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en t

_

foetal rabbit and neonatal human lungs. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted byproduct of NADPH oxidase) under normoxic t conditions results in an increase of the outward K+ current, suggesting that H 20 2 is a transmitter modulating the '02-sensitive K+ channel'. Notes: 1. The oxidase in NEB cells can be significantly stimulated by exposure to phorbol esters (0.1 JlM) and inhibited by diphenyliodonium (5 JlM). 2. Oocytes expressing Kv3.3, Kv3.4 (or Kvl.4) channels to 'high micromolar' H202 result in a reversible removal of inactivation 93; this effect is concomitant with a significant increase in current magnitudes (as above, but notably strong in Kv3.4-injected oocytes) and dependent upon a conserved cysteine-containing sequence in the N-termini (as in paragraph 50-44-01 and cross-references therein).

Selective effects of chemical modification on the hKv3.4 Nterminal 'inactivation peptide' 50-44-03: hKv3.4: The inactivation 'conferred' to mKvl.l channels expressed in CHO cells by hKv3.4 inactivation peptide (hKv3.4-Ip, see Domain functions, 50-29) can be removed by brief exposure to the chemical modification agents N-bromoacetamide (NBA, 100 JlM) or chloramine-T (CL-T, 500 JlM) 75. In contrast, these treatments have no effect on the fast inactivation of CHO-expressed mKvl.l induced by Shaker B inactivation peptide (ShB-IP, ibid.). Chemical modification of mKvl.l in the presence of hKv3.4-IP results in a hyperpolarizing shift of -8 mV (CL-T) and -11 mV (NBA) in the voltage dependence of activation 75 . These effects were 'critically dependent' on the presence of a cysteine residue at position 6 (but not postion 24) of the hKv3.4-IP, consistent with a selective action on the hKv3.4 inactivation peptide itself. Note: NBA and CL-T treatments cause only a slight inhibition of 'unmodified' mKvl.l current (with no significant effects on the voltage dependence or channel deactivation properties)75.

Alcohol inhibition of Shaw-related channels at a discrete saturable site 50-44-04: Low concentrations of ethanol (17-170 mM) selectively inhibit noninactivating cloned K+ channel encoded by Drosophila Shaw2 94 . Drosophila Shaw2 channel whole-cell currents are also inhibited by other members of this homologous series of n-alkanols (i.e. ethanol to I-hexanol)95. In these studies, equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action. This discrete site (possibly a hydrophobic pocket) appeared to be part of the protein since nalkanol sensitivity could be transferred to novel hybrid K+ channels (Shaw2and homologous ethanol-insensitive subunit chimaeras). Significantly, a hydrophobic point mutation within a cytoplasmic loop of an ethanolinsensitive hKv3.4 was sufficient to allow significant inhibition by n-alkanols (with a dose-inhibition relation matching wild-type Shaw2)95. These studies also reported apparently 'direct' effects on channel gating by a homologous alcohol (I-butanol) during selective inhibition of long-duration single channel openings. Supplementary note: From four types of voltage-gated channels tested in this study, only Shaw2 channels were sensitive to 'block' by halothane (1 mM)94.

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Receptor/transducer interactions Differentiation of Kv3.1 and Kvl.3 by selective coupling to a 5-HT receptor 50-49-01: Comparative note: Kv 3.1 and Kvl.3 are co-expressed in T

lymphocytes (see Phenotypic expression, 50-14). Kv3.1 currents are not susceptible to 5-hydroxytryptamine agonist suppression when co-expressed with 5-HT1C receptor in oocytes 96 (compare with Kv1.3 in Table 28 within Receptor/transducer interactions under VLG K Kv3-Shak, 48-49). 50-49-02: A small number of densely labelled neurones for GAD67 mRNA also express the mRNA encoding the dopamine D 2 receptor with none expressing detectable levels of dopamine D 1 receptor mRNA28 (for significance, see

GAD67 under mRNA distribution, 50-13).

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 50-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:)

which should not be typed (see Introduction eiJ layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in the Introduction eiJ layout of entries, entry 02, at the front of the book). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouringt analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers), author/reference or nomenclature. Following direct accession, however, neighbouring t analysis is strongly recommended to identify newly reported and related sequences. Kv nomenclature

Species, original isolate name (bold)

Database locus, ORF

Accession

Sequence/ discussion

hKv3.1

Human, cDNA

gb: HUMKCNCI

gb:M96747

Grissmer, T Bioi Chern (1992) 267: 20971-9. Yokoyama, FEBS Lett (1989) 259: 3742.

hKv3.1

mKv3.1a

Mouse, cDNA NGK2

gb: MMRNNGK2 gb: Y07521 pr: CIKD_MOUSE sp:P15388 pir: S07095 ORF: 511 aa

entry 50

-

Kv nomenclature

Species, original isolate name (bold)

Database locus, ORF

Accession

Sequence/ discussion

rKv3.1a

Rat, cDNA RShIIIB high voltageactivating, TEA-sensitive, type A Rat, cDNA KV4

gb: RATKCHANB

gb:M84211

Vega-Saenz de Miera, Proc R Soc Lond [Biol] (1992) 248: 9-18.

gb:M68880 pir: A39395 sp:P25122

Luneau, Proc Natl Acad Sci USA (1991) 88: 3932-6.

gb: X62840

Rettig, EMBO '(1992)11: 2473-86.5

gb: not found

Vega-Saenzde-Miera, Proc R Soc Lond [Biol] (1992) 248: 9-18. McCormack, Proc Natl Acad Sci USA (1990) 87: 5227-31. McCormack, Proc Natl Acad Sci USA (1991) 88: 4060. Vega-Saenz de Miera, Proc R Soc Land [Biol] (1992) 248: 9-18. Rudy, Proc Natl Acad Sci USA (1992) 89: 4603-7. Luneau, FEBS Lett (1991) 288: 163-7.

rKv3.1b

Rat, cDNA Raw2

gb: RRKV4 ORF: 585 aa (NB positions 512585 are missing in splice variants) gb: RRPCP3145 ORF: 585 aa

hKv3.2a

HKShIIIA

ORF: 613 aa

rKv3.2a

Rat, cDNA gb: RRSHIIIA sp: CIG_RAT RKShIIIA alternative splice ORF: 595 aa form of the Kv3.2 gene

gb:M34052 sp:P22463

rKv3.2b

Rat, cDNA KShIIIA.3

gb: RATSHTIIC ORF: 638 aa

gb:M84203

rKv3.2b

Rat, cDNA K+ channel3.2b alternative splice form of the Kv3.2 gene Rat, cDNA Shaw-like, Kv3.2b divergent 3' end

gb: RATPCKV32B sp: CIKE_RAT ORF: 638 aa

gb:M59211 sp:P22462

ORF: 638 aa

only gim As above. number found (see Introduction eJ layout, entry 02) 210-563 gb:M59313 Luneau, FEBS sp:P22461 Lett (1991) 288: 163-7.

rKv3.1b

rKv3.2b

rKv3.2c

Rat, cDNA gb: RNPCKV32C K+ channel 3.2c sp: CIKF_RAT alternative splice ORF: 635 aa form of the Kv3.2 gene

-

entry SO

Kv nomenclature

Species, original isolate name (bold)

rKv3.2c

not found Rat, cDNA only gim As above. Shaw-like, Kv3.2c (encoding a 208 aa number found (see divergent 3' end segment) Introduction etJ layout, entry 02) 210565 gb: RRPCP3120 gb: X62839 Rat, cDNA Rettig, EMBO Rawl ORF: 624 aa T(1992) 11: 2473-86.

rKv3.2d

Database locus, ORF

Accession

rKv3.2d

Rat, cDNA KShIIIA.2

hKv3.3

Human, genomic gb: HSCH19PSG gb: 211585 hKv3.3 partial Human sequence chromosome 19 Shaw-type K+ channel gene Mouse, genomic gb: MMPOTCHAI gb: X60796 mKv3.3 exon 1 pir S22046 (splicing removes (spliced a ",-,3 kb intron in version) cds (see Gene organization, 5020)

mKv3.3

mKv3.3

mKv3.3b

rKv3.3a

mKv3.4 exon segment from gene

gb: RATSHIllB ORF: 624 aa

gb:M84202

Mouse, genomic gb: MMPOTCHA2 gb: X60797 pir S22046 mKv3.3 exon 2 (spliced (as above) version) gb:S69381 Mouse Expressed in cerebellum terminally cDNA (B6C3 Fe- differentiated cerebellar Purkinje a/a-pcd). cells and deep cerebellar nuclei ORF: 679 aa gb: RATKCHANA gb:M84210 Rat, cDNA RKShIIID. Cell High voltage activating, TEAtype cited as pheochromosensitive, type-A cytoma Mouse, genomic DNA (Mus musculus strain AKR/J)

gb: MUSSRPCG gb:M81253 1370 bp sequence Intron: nt 1 to 396; exon:nt397tol341

Sequence/ discussion

Rudy, Proc Natl Acad Sci USA (1992) 89: 4603-7. Lee/Garbutt/ Phillips/ Garbutt/ Roses, unpublished. Chandy, Biophys T (1990) 57: 110a. Ghanshani, Genomics (1992) 12: 1906. As above.

GoldmanWohl, T Neurosci (1994) 14: 51122. Vega-Saenz de Miera, Proc R Soc Lond [Biol] (1992) 248: 9-18. Ghanshani, Genomics (1992) 12: 1906. Chandy, Biophys T (1990) 57: 110a. (partial cds)

I

entry 50

-

Kv nomenclature

Species, original isolate name (bold)

Database locus, ORF

Accession

rKv3.4a

Rat, cDNA Raw3

gb: RRPCP2858 ORF: 625 aa

gb: X62841

rKv3.4a

Rat, cDNA Raw3-H364Q

hKv3.4b

Human, cDNA hKShIIIC

not found ORF: 625 aa (mutant, see note 5 in Table 1) gb: HUMSHIIIC gb:M64676 ORF: 583 aa

Drosophila Shaw

Shaw locus (Shaw2 protein), Chromosome 2 left arm at locus 24B-C

FlyBase: FBgnOO03386 ORF: 498 aa

Lobster Shaw

Panulirus interruptus Shaw K+ channel (shaw 1)

gb:L48691 Lobster shaw: Cloning, sequence analysis and comparison to fly Shaw ORF: 489 aa

Sequence/ discussion Rettig, EMBO

T(1992) 11:

gb:M32661

2473-86. Schroter, FEBS Lett (1991) 278: 211-16. Rudy, T N eurosci Res (1991) 29: 40112. Butler, Nucleic Acids Res (1990) 18: 2173-4. Wei, Science (1990) 248: 599-603. Baro, Gene (1996) 170: 267-70.

Gene mapping locus designation 50-54-01: Chromosomal locus names assigned by the Human Gene Mapping Workshopt (HGMW) for the Shaw-related subfamily are as follows: Kv3.1 KCNCl j Kv3.2 - KCNC2 j Kv3.3 - KCNC3 j Kv3.4 - KCNC4 (see also Chromosomal location, 50-18).

Related sources and reviews 50-56-01: A comprehensive review on the Kv3 subfamily appears in VegaSaenz de Miera et al. (1994). For other review references covering Kv subfamilies, see Related sources and reviews under VLC K Kv1-Shak, 5056. Biophysical and regulatory aspects of lymphocyte potassium channels (including the role of cloned Kv3.1 and the native type 1 channel) have been reviewed 97.

Book references: Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand- and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton. Vega-Saenz de Miera, E., Wesier, M., Kentros, C., Lau, D., Moreno, H., Serodio, P. and Rudy, B. (1994) Shaw-related K+ channels in mammals. In Handbook of Membrane Channels (ed. C. Perrachia), pp.41-78. Academic Press, San Diego.

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.

e_n_try_5_0_1

For other book references pertinent to Kv channels, see this field under VLC K Kvl-Shak,48-56.

Feedback Error-corrections, enhancements and extensions 50-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource J - Search criteria). For this entry, send e-mail messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 50-14-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback etJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

REFERENCES 1 2 3 4 5

6 7

8 9 10

11 12 13 14 15

16 17 18 19

20 21 22 23

24 25 26 27

Perney, J Neurophysiol (1992) 68: 756-66. Chandy, Nature (1991) 352: 26. Gutman, Semin Neurosci (1993) 5: 101-6. Luneau, Proc Natl Acad Sci USA (1991) 88: 3932-6. Wang, Proc Natl Acad Sci USA (1996) 93: 13182-7. Yue, J Physiol (1996) 496: 647-62. Feng, Circulation (1996) 94: 3088. Wang, J Pharmacol Exp Ther (1995) 272: 184-96. Rampe, J Pharmacol Exp Ther (1995) 274: 444-9. Li, Circ Res (1996) 78: 903-15. Crumb, Mol Pharmacol (1995) 47: 181--90. Ried, Cenomics (1993) 15: 405-11. Grissmer, J Biol Chem (1992) 267: 20971-9. McCormack, Proc Natl Acad Sci USA (1990) 87: 5227-31. McCormack, Proc Natl Acad Sci USA (1991) 88: 4060. Yokoyama, FEBS Lett (1989) 259: 37-42. Pak/Salkoff, Unpublished data. Rettig, EMBO J (1992) 11: 2473-86. Vega-Saenz-de-Miera, Proc R Soc Lond [Biol] (1992) 248: 9-18. Lee/Garbutt/Phillips/Roses, Unpublished data. Ghanshani, Genomics (1992) 12: 190-6. Goldman-Wohl, J Neurosci (1994) 14: 511-22. Schrater, FEBS Lett (1991) 278: 211-16. Rudy, J Neurosci Res (1991) 29: 401-12. Butler, Nucleic Acids Res (1990) 18: 2173-4. Wei, Science (1990) 248: 599-603. Du, J Neurosci (1996) 16: 506-18.

l_e_n_try_5_0

28 29 30 31 32

33 34

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74 75

----'_

Lenz, Synapse (1994) 18: 55-66. Weiser, TNeurosci (1995) 15: 4298-314. Hemmick, TNeurosci (1992) 12: 2007-14. Gardner, Annu Rev Immunol (1990) 8: 231-52. Premack, T Clin Immunol (1991) 11: 225-38. Lewis, Annu Rev Physiol (1990) 52: 415-30. Cahalan, Curr Top Membr Trans (1991) 39: 358-86. Cahalan, Semin Immunol (1990) 2: 107-17. Hess, T Immunol (1993) 150: 2620-33. Lewis, Annu Rev Immunol (1995) 13: 623-53. Norman, Development (1995) 121: 1183-93. Wei, Biophys T (1997) 72: TUPM4. Weiser, TNeurosci (1994) 14: 949-72. Rudy, Proc Natl Acad Sci USA (1992) 89: 4603-7. Vega-Saenz-de-Miera, In Handbook of Membrane Channels (1994). Academic Press, San Diego, pp.41-78. Vega-Saenz-de-Miera, Biophys T(1991) 59: 197a. Drewe, T Neurosci (1992) 12: 538-48. Moreno, TNeurosci (1995) 15: 5486-501. Kanemasa, T Neurophysiol (1995) 74: 207-17. Ho, Proc Natl Acad Sci USA (1997) 94: 1533-8. Perney, Soc Neurosci Abstr (1993) 19: 494.14. Laube, Mol Brain Res (1996) 42: 51-61. Ponce, Soc Neurosci Abstr (1995) Abstr 121.3. Wymore, Genomics (1994) 20: 191-202. Stubbs, Genomics (1994) 24: 324-32. Haas, Mammalian Genome (1993) 4: 711-15. McPherson, Cytogenet Cell Genet (1991) 58: 1979 (Abstr). Bellingham, Invest Ophthalmol Vis Sci (1996) 37: 4566. Gan, TBioi Chem (1996) 271: 5859-65. Mount, Nucleic Acids Res (1982) 10: 459-72. McKeown, Annu Rev Cell BioI (1992) 8: 133-55. Luneau, FEBS Lett (1991) 288: 163-7. McCormack, Nature (1989) 340: 103-4. Stiihmer, EMBO T (1989) 8: 3235-44. Shen, Neuron (1995) 14: 625-33. Shahidullah, FEBS Lett (1995) 371: 307-10. Hugnot, EMBO T (1996) 15: 3322-31. Critz, TNeurochem (1993) 60: 1175-8. Forn, Proc Natl Acad Sci USA (1978) 75: 5195-9. Rossie, TBiol Chem (1987) 262: 12735-44. Costa, T Biol Chem (1984) 259: 8210-8. Covarrubias, Neuron (1994) 13: 1403-12. Ruppersberg, Nature (1991) 353: 657-60. Jones, Nature (1991) 353: 603-4. Demo, Neuron (1991) 7: 743-53. Slesinger, Neuron (1991) 7: 755-62. Stephens, TPhysiol (1996) 496: 145-54. Stephens, TPhysiol (1995) 484: 1-13.

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87 88 89 90

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Hartmann, Science (1991) 251: 942-4. Kirsch, Biophys T(1992) 62: 136-44. Aiyar, Biophys T (1994) 67: 2261-4. Shieh, Biophys T (1994) 67: 2316-25. Lopez, Nature (1994) 367: 179-82. Hille, Ionic Channels of Excitable Membranes, 2nd edn (1992). Sinauer Associates, Sunderland, Mass. Lopez, Neuron (1991) 7: 327-36. Solc, T Gen Physiol (1990) 96: 135-65. Connor, TPhysiol (1971) 213: 21-30. Rudy, Neuron (1988) 1: 649-58. Rudy, Neuroscience (1988) 25: 729-49. Elinder, T Gen Physiol (1996) 108: 325-32. Grissmer, Mol Pharmacol (1994) 45: 1227-34. Kirsch, T Gen Physiol (1993) 102: 797-816. Kirsch, Neuron (1993) 11: 503-12. Jarolimek, Mol Pharmacol (1996) 49: 165-71. Ruppersberg, Nature (1991) 352: 711-14. Vega-Saenz-de-Miera, Biochem Biophys Res Commun (1992) 186: 1681-7. Covarrubias, Proc Natl Acad Sci USA (1993) 90: 6957-60. Covarrubias, T Biol Chem (1995) 270: 19408-16. Aiyar, Am TPhysiol (1993) 265: CI571--8. Lewis, Annu Rev Immunol (1995) 13: 623-53. Doyle, Science (1998) 280: 69-77.

Vertebrate K+ channel subunits related to Drosophila Shal (Kvo: subunits encoded by gene subfamily Kv4) Edward C. Conley

Entry 51

See note on coverage at the head of VLG K Kv1-Shak, entry 48. General properties of Kv channel subunits expressed in heterologous cells are summarized in the fields of entry 48.

NOMENCLATURES

Abstract/general description 51-01-01: Vertebrate K+ channel subunits whose primary amino acid sequences are most closely related to those of Drosophila Shal are grouped in the voltage-gated K+ channel subfamily 4 (Kv4, this entry). Shal-related channel-a subunits generally support outward, K+ -selective currents in oocytes. Despite the 'evolutionary distance' between them, Drosophila (fShal) and vertebrate Kv4-subfamily channels (e.g. mShal, ratShal) show remarkably similar properties when expressed in oocytes (e.g. see below and Fig. 2 under Activation, 51-33). 51-01-02: All three presently known Kv4 gene subfamily members (Kv4.1 to Kv4.3) produce Ifast-inactivating' outward K+ currents in oocytes which are similar (but not identical) to native transient I A-type' currents described in heart and neurones (e.g. ITO and I SA - see VLG K A-T [native], entry 44). mKv4.1/mShal cDNAs express 'low-threshold' A-type channels, displaying inactivation that is markedly faster at more negative potentials, typical of native A-channels which recover from inactivation in a time- and voltagedependent manner upon repolarization. Notably, hybrid arrest with antisense oligonucleotides complementary to a sequence common to all known mammalian Shal-related mRNAs (Kv4.1, Kv4.2 and Kv4.3) can suppress the expression of a native ISA component supported from total brain mRNA pools (see Protein interactions, 51-31). 51-01-03: Several comparative 'spatio-temporal' immunolocalization studies of different Kva subunit proteins forming fast-inactivating channels in heterologous expression systems (e.g. Kv4.2 and Kv1.4) have shown distinct protein distributions in subpopulations of neurones. In addition, these studies have revealed differential subcellular localizations; e.g. Kv4.2 is concentrated in neuronal dendrites and somata (suggesting predominantly post-synaptic roles) compared to Kvl.4, which is associated with in axonal/nerve terminal pre-synaptic sites) (see Protein distribution, 51-13, Subcellular locations, 51-16 and cross-references therein). Notably, some Kv4 channel mRNA distribution patterns (see mRNA distribution, 51-13) exhibit different patterns from the corresponding protein distribution (see Protein distribution, 51-15). 'Reciprocal' gradients of expression of Kv4.2 versus Kv4.3 transcripts have been described in some brain areas (e.g. the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum). Notably, Kv4.2 and Kv4.3 have also been reported to display 'complementary' expression in adult rat heart, with Kv4.3 being more abundant in cardiac atria and Kv4.2 in ventricle (see mRNA distribution, 51-13).

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51-01-04: The complete conservation of the 'K+ channel signature' sequences in their 85{P) region predicts that Kv4 subfamily channels are highly selective for K+ ions, an expectation that has been confirmed by experiment. However, the slope of the voltage dependence of conductance curves is generally much less steep for Shal-related channels than for Shaker-related homologues. In rKv4.2 (isolate RK5) the 54 'voltage sensor' region has five basic residues with a phenylalanine in position 303 rather than a leucine seen in the corresponding position in all other families of voltage-gated ion channels (see also Voltage sensitivity, 51-42). Inactivation properties of mKv4.1 appear to be influenced by protein domains at both the N- and C-terminus (for details, see Inactivation, 51-37). 51-01-05: In oocyte systems, co-injection of low molecular weight (LMW) fractions (2-4 kb) of poly(A)+ mRNA can result in a significant (fourfold) increase in the surface expression of some Kv4 channels (see Channel density, 51-09); furthermore, a number of modifications of gating kinetics and pharmacological sensitivities have been reported following expression with LMW mRNA fractions (see Channel modulation, 51-44). These observations are consistent with those induced by association of Kv4o: subunits with one or more Kv,8 subunits (see VLG K Kv-beta, entry 47). 51-01-06: Direct comparison of arachidonic acid (AA) modulation on 12 Kv channel subtypes has shown that only Kv4 subfamily members (Kv4.1 and Kv4.2) are inhibited by the free fatty acid (for details, see Channel modulation, 51-44). This is of some significance since AA (an established 'retrograde messenger', see Receptor/transducer interactions under ELG CAT GLU NMDA, 08-49 and ILG K AA [native], entry 26) potently suppresses A-current in native sympathetic neurones and is implicated in the mechanism of muscarinic inhibition of A-current in cultured coeliac ganglion neurones. 51-01-07: Like most 'cloned' K+ channels, the selective pharmacology of Kv4 subfamily channels is poorly developed. 4-Aminopyridine (4-AP) has been extensively reported as blocker of Kv4 subfamily channels, although the degree of block is variable and 'high millimolar' concentrations of 4-AP are generally required for half-block. Unusually, binding of 4-AP to Kv4.2 expressed in Xenopus oocytes has been shown to occur exclusively in the closed state. 4-AP blocking of Kv4.2 channels from the intracellular side appears to be at or near the domains involved in channel inactivation, such that 4-AP binding and inactivation are 'mutually exclusive'. Both Kv4.1 and Kv4.2 are insensitive to block by tetraethylammonium (TEA) ions and a number of other 'classical' K+ channel blockers (see Blockers, 51-43). 51-01-08: Regulation of native 'A-type' channels by neurotransmitters and second messengers has been extensively studied, and these mechanisms may be important in learning and memory (see VLG K A-T [native], entry 44). However, knowledge of receptor-transducer coupling to 'cloned' Kv4 subfamily channel effectors was largely uncharacterized at the time of compilation. However, the postulated roles of these channels in modulation of synaptic plasticity (see above and Phenotypic expression, 51-14), discrete subcellular localization patterns (Subcellular locations, 51-16) and possession

l_e_n_t_ry_S_l

---l_

of multiple phosphomodulation sites (Protein phosphorylation, 51-32) make 'receptor-coupled' control of excitability by Kv4 effectors of functional significance.

Category (sortcode) 51-02-01: VLG K Kv4-Shal, i.e. a subunits of vertebrate voltage-gated potassium channels encoded by Kv gene subfamily 4 whose primary (amino acid) sequences show highest identity to those of Drosophila Shal (see Gene family, 51-05). Homomultimeric t channels formed from Kv4a subunits are normally named in accordance with the cDNA name (see Channel designation, 51-03). Assigned names of subfamily Kv4 gene (chromosomal) loci are listed under Cene mapping locus designation, 51-54.

Information sorting/retrieval aided by designated gene family nomenclatures 51-02-02: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: Kv4 where n is a designated !!umber in the systematic nomenclature (e.g. Kv4.2). Within this entry, paragraph 'running orders' (sort orders) are largely determined alphanumerically by systematic nomenclatures, i.e. denoting species and gene product prefix combined with any trivial or clone name(s) where these have been used in the source reference (e.g. rKv4.2/RKS:). Where properties are likely to apply to all or several subfamily proteins (i.e. irrespective of species or isolate) the 'species' term may be omitted. Entry updates covering novel isoforms will index information using these conventions established from systematic nomenclatures based on gene family relationships.

Channel designation 51-03-01: Vertebrate voltage-dependent K+ channel genes/cDNAs/channels related to Drosophila Shal are presently designated as Kv4.1 to Kv4.3 in keeping with the published nomenclature 1,2 (see Table 1 under Cene family, 49-05; for general background, see this field under VLC K Kv1-Shak, 48-03). Confusion may arise between Kv subfamily numbers and the clone (isolate) name for the Shaw subfamily clone named KV4 (ref. 3 , systematic name Kv3.1, as described under Channel designation of VLC K Kv-Shaw, 50-03). Additional instances of potentially confusing nomenclatures (for isolate/ clones KVl, KV2 and KV3) are described in note 2 under Table 1 of VLC K Kvl-Shak, 48-05.

Current designation Kv4 subfamily gene products as candidate contributors to native cardiac ITO and neuronal ISA 51-04-01: The cardiac ventricular 'transient outward' current component ITO has been reported to have properties that 'resemble' those shown by heterologously expressed Kv4.2 (isolate RKS, e.g. refs. 4,5) and (later, independently)

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e_n_try_5_1_ _

Kv4.3 6 (for further details, see Phenotypic expression, 51-14). Xenopus oocytes injected with rat brain mRNA express a transient current similar to the A-current in somatic recordings from neurones that activates transiently near the threshold for Na+ action potential generation (IsA ). For further observations identifying Kv4 subfamily proteins as candidate channels underlying native A-type currents, see Channel density, 51-09; Developmental regulation, 51-11; Phenotypic expression, 51-14; Protein distribution, 51-15; Subcellular locations, 51-16; Activation, 51-33; Inactivation, 51-37; Current type, 51-34; Voltage sensitivity, 51-42 and Channel modulation, 51-44. See also Table 1 under Cene family, 51-05 and for general background, see Current designation under VLC K Kv1-Shak, 48-04.

Gene family 51-05-01: Vertebrate voltage-dependent K+ channel genes related to the Drosophila Shal gene are assigned to Shal-related subfamily 4, presently designated as Kv4.1 to Kv4.3 by the agreed nomenclature1 . To indicate their relatedness to the Sh superfamily t of genes, some authors have used the designation ShIV to designate mammalian homologues of Shal. Separately identified genes in this subfamily are listed in Table 1. See also Category (sortcode), field 51-02 for conventions regarding tsort order' for information in this entry.

Table 1. Kv nomenclature and original names in use for Shal-related vertebrate voltage-dependent K+ channel genes/cDNAs (subfamily 4) (From 51-05-01)

Kv designation

Human isoforms

Rat isoforms

Mouse isoforms

Other species

Kv4.1 Names in use and refs

hKv4.1

rKv4.1

mKv4.1 mshal1 7

Other

Kv4.2 Names in use and refs

hKv4.2

rKv4.2 Kv4.2 RK5 8 Rat shal1 9

mKv4.2

Other

Kv4.3 Names in use and refs

hKv4.3

mKv4.3 rKv4.3 Kv4.3 RKShIVB 1O- 13

Other Canine Kv4.3 6

Notes: 1. See also Lobster Shal1 14 . For accession numbers, see Database listings, 51-53. 2. Details on the channel clone KV4 (are listed under VLC K Kv3-Shaw, entry 50 (see Cene family under VLC K Kv3-Shaw, 50-05).

entry 51

_

I' - - - - - - - - - - Trivial names

51-07-01: The terms rShal or rat Shal (or other species equivalents) are alternative trivial names that have been used in the literature for describing Shal subfamily proteins. Italicized versions of these names e.g. fShal, mShal, rShal etc. usually make reference to the gene. In this entry, trivial names such as these have been preserved as used in original articles.

EXPRESSION For references comparing expression determinants applicable to all of the Kv channel subfamilies, see the EXPRESSION section under VLG K Kvl-Shak, entry 48.

Cell-type expression index Cell-type expression patterns identified using molecular probes specific for Kv4 family members are described under Cloning resource, 51-10, Isolation probe, 51-12, mRNA distribution, 51-13 and Protein distribution, 51-15.

Channel density Apparent control of Kv4.1 surface membrane densities by subunit associations 51-09-01: mKv4.1/mshall; rKv4.2; rKv4.3: Low molecular weight (LMW) fractions (2-4 kb) of poly(A)+ mRNA (both from rodent brain) results in a significant (fourfold) increase in the surface expression of Kv4.1/mshall K+ channels in oocytes 15 (see Channel modulation, 51-44; for significance, see Cloning resource under VLG K Kv-beta, 47-10). For predicted protein interactions mediating subcellular segregation patterns of different Kv subunits, see Subcellular locations, 51-16 and Protein interactions, 51-31. Furthermore, Kv4.3 currents in oocytes have also been reported as resembling native rat brain A-currents following co-injection with a 'LMW' mRNA fraction from rat brain (which did not express detectable currents on its own)12 (see Inactivation, 51-37).

Consequences of reduced Kv4.2 channel densities following seizures 51-09-02: mKv4.2: Reduction in Kv4.2 channel density in dendrites has been proposed following seizure activity based on (i) observed reductions in Kv4.2 mRNA density in dentate gyrus under these conditions16 (see Developmental regulation, 51-11) and (ii) the somatodendritic distribution of Kv4.2 subunits17 (see Subcellular locations, 51-16). Note: From Kv4.2's predicted role in hippocampal long-term synaptic plasticity (see Phenotypic expression, 51-14), reductions of Kv4.2 channel activity in the dentate gyrus might be predicted to result in a prolonged enhancement of post-srnaptic excitability of granule cells, in turn contributing to hyperexcitable states in vivo (for further background, see LTPt and kindling l under ELG CAT GLU NMDA, entry 08).

II

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Cloning resource 51-10-01: Kv4.1/mshal: Mouse brain cDNA library. rKv4.1/ratshal: Rat hippocampal cDNA library. Note: rKv4.1 cDNA abundance in this library was relatively high as judged by restriction analysist (Le. 64 hybridizationpositivet cDNA clones showed that the majority of the cDNAs recovered were identical)9. rKv4.2/RK5: Rat adult heart cDNA library.

Developmental regulation Kv4.2 expression in developing hippocampal neurones 51-11-01: Kv4.2: In the hippocampal formation, RNA transcripts encoding rat Shal and other Kv subfamily channels (e.g. RCK2, RCK3, RCK4, RCK5, Raw3)18 are heterogeneously expressed and regulated during post-natal development (for further details, see Developmental regulation under VLC K Kv1-Shak, 48-11). 'Repression' of Kv4.2 mRNA in the dentate granule cell layer of the hippocampus has been reported following induction of seizure activity16 (see also Developmental regulation under VLC K Kv1 Shak, 4811). Comparison of the spatio-temporal expression patterns of Kv4.2 with four other K+ channel polypeptides (Kvl.4, Kvl.5, Kv2.1 and Kv2.2) has also been made for rat hippocampal neurones developing in situ and in vitro 19 . In this study, Kv4.2 (and Kvl.4) proteins (both forming channels conducting Atype currents) were present distally on distinct subpopulations of neurones (see also Subcellular locations, 51-16 and Protein distribution, 51-15). Comparative note: Kva subunit proteins associated with 'delayed rectifiertype' channels when expressed alone (e.g. Kvl.5, Kv2.1 and Kv2.2) were present on all neuronal somata and proximal dendrites 19 (see also the EXPRESSION section fields under entries 48 to 50).

Adaptive changes in Kv4.2 gene expression following induced myocardial infarction 51-11-02: Kv4.2: Following experimental induction of myocardial infarction (MI), left ventricular (LV) remodelling t is associated with hypertrophyt of non-infarcted myocardium and electrophysiological alterations. Post-MI hypertrophied LV myocytes display prolonged action potential duration (APD) and generate 'triggered' activity from early after-depolarizationst . Prolonged APD has been attributed to decreased density of the two outward K+ currents, Ito.fast and Ito.slow (as opposed to changes in the density and/or kinetics of the L-type Ca2 + current). In post-MI groups, Kv4.2 channel subunit mRNAs (putatively encoding components contributing to Ito.fast) are 'significantly decreased' (by approx. 530/0), although 'no significant change' can be observed in Kvl.2 and Kvl.5 mRNA levels20 . In addition, Western t blotting has demonstrated reduced immunoreactive signals in post-MI left ventricle for Kv4.2 and Kv2.1 proteins (approx. 43 and 67% respectively) but no significant change in Kvl.5 immunoreactive protein level.

Isolation probe 51-12-01: Kv4.1/mshall: Drosophila Shal2 cDNA probe. rKv4.2/RK5: PCRtgenerated fragments corresponding to Kvl.l, isolate RBKI (nt 681-1007) and

1__e_n_t_ry_5_1

----'_

Kvl.2, isolate RBK2 (nt 685-1014). rKv4.2, isolate rat Shall: Originally detected in hippocampal mRNA by RT-PCRt using K+ channel-specific degeneratet primers corresponding to amino acid sequences highly conserved in all known K+ channel sequences (aa 440-446 and 473-479 of Shaker K+ channels). This screen identified several sequences consistent with K+ channels not previously observed, including one homologous to Drosophila Shal which was used as isolation probe for rat Shall.

mRNA distribution 51-13-01: Table 2 summarizes reported tissue distributions of Kv4 subfamily mRNAs {see also general notes in Table 2 under mRNA distribution of VLC K Kvl-Shak, 48-13}. Note: Established mechanisms of Kv4 subfamily subunit segregation in vivo {see for example, Subcellular locations, 51-16, VLC K Kvl-Shak, 48-16 and Protein interactions, 51-31} may suggest why some Kv channel mRNA distribution patterns {see mRNA distribution, 51-13} exhibit distinct patterns from protein distribution {see Protein distribution, 51-15}.

Comparative mRNA expression analyses using RNAase protection assays in the CNS and heart 51-13-02: From a quantitative expression analysis of 18 different KVQ and KvrJ subunit mRNAs in rat sympathetic ganglia using an RNAase protectiont assay21 it was concluded that members of the Kv4 subfamily are likely to underlie the low-threshold A-current in sympathetic neurones. RNAase protection has also been used to compare expression of 15 different potassium channel genes in rat cardiac atrial versus ventricular muscle22 . Of these genes, only five, Kv4.2 (plus Kvl.2, Kvl.4, Kvl.5 and Kv2.1) are expressed at 'significant levels' in cardiac muscle {see Phenotypic expression, 51-14}. In direct comparisons of cardiac atrial and ventricular RNA samples, transcripts from Kv4.2 (and Kvl.2) genes show the largest differences in relative abundance22 . Interestingly, a large gradient of Kv4.2 expression can be observed across the cardiac ventricular wall, with Kv4.2 expression in epicardial muscle being 'more than eight times higher' than in papillary muscle. In comparison, other Kv channel genes are expressed at 'relatively uniform levels' across the ventricular wall. According to this study22, there is an approximately twofold decrease in total Kv4 subfamily mRNA expression in atrial muscle relative to ventricular muscle.

Lobster Shall gene expression in identified neurones 51-13-03: Different (identified) neurone types within the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster {Panulirus interruptus} have distinguishable native A-currents 14. Following description of a full-length open reading frame for lobster Shall {related to fShal, mShal and rShal, see Table 1 under Cene family, 51-05} RT-PCRt has been used to localize lobster Shall mRNA in the lateral pyloric (LP) and pyloric dilator (PY) cells of the pyloric network. Detailed comparisons between native lAS in pyloric neurones and Shall or fShal heterologously expressed in oocytes has shown differences in kinetic and voltage-dependent parameters14.

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Table 2. Reported tissue distributions of Kv4 subfamily mRNAs. For key, see notes in Table 3 under mRNA distribution of VLC K Kv1-Shak, 48-13. (From 51-13-01) Kv4.1

Kv4.I/ferret: Many ferret cardiac myocytes contain transcripts for Kv4.1 23 . For comparisons with other Kv channels expressed in heart, see this field under other Kv-series entries (fields 47-13, 48-13, 49-13, 50-13).

Kv4.2

rKv4.2/RK5: Northerns: Relative abundance: Brain » cardiac ventricle = cardiac atrium with 'no signal' in skeletal muscle24 . rKv4.2/ratshall: ISH: 9,25. Widely distributed in brain; expressed at a similar level in the cortex and hypothalamus, but at 'much higher levels' in the hippocampus, dentate gyrus and the habencular nucleus of the thalamus. Concentrated expression in the cell bodies of the granule cells of the dentate gyrus and the pyramidal cells of CA3 and CAl; expressed in neurones and possibly in CNS glial cells with relative abundance according to the series CAl ~ dentate gyrus CAl. Not expressed in the deep cerebellar nuclei (cf. Kvl.l and Kvl.2) within hippocampal subregions, Kv4.2 mRNA is approximately equally abundant in the pyramidal cells and excitatory dentate granule cells (cf. Kvl.l and Kvl.2). Highly expressed only in the granule cells but not in the Purkinje cells or cells in the molecular layer (cf. Kvl.l and Kvl.2). ISH: In the heart, in situ hybridization reveals uniform expression of mRNA, with no selective accumulation in the atria or ventricles 9,16 (compare this result with ref.12, below and ref.22 in paragraph 51-13-02).

Kv4.3

rKv4.3/RKShIVB: 'Reciprocal' or 'complementary' expression patterns of Kv4.2 versus Kv4.3. ISH: Direct comparisons of expression of the three known Kv4 genes show Kv4.2 and Kv4.3 (but not Kv4.1) to be abundant in the adult rat brain, with each displaying a 'specific, but sometimes overlapping' pattern of expression12. A 'reciprocal gradient' of expression of Kv4.2 versus Kv4.3 transcripts is seen in some brain areas (e.g. the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum). Notably, Kv4.2 and Kv4.3 also display 'complementary expression' in adult rat heart, with Kv4.3 being more abundant in cardiac atria and Kv4.2 in ventricle 12 . rKv4.3: ISH: Retrosplenial cortex, medial habenula, anterior thalamus, hippocampus, cerebellum, lateral geniculate and superior colliculus 13 .

Phenotypic expression Kv4 subfamily channels as candidates supporting neuronal A-type

currents 51-14-01: mKv4.I/mShal; rKv4.2/RK5: Shal currents from both fly (fShal) and mouse (mShal) resemble typical neuronal A-type currents of various

l_e_n_t_ry_5_1

_

species26 - 29, e.g. in voltage sensitivity of activation. As noted in several entries (e.g. paragraph 51-14-02), conclusions of 'equivalence' between native and 'cloned' A-type currents should be interpreted with caution, noting the 'interconversion' of 'A-type' and 'delayed rectifier-type' properties that could occur in vivo subject to contributions of Kv,L3 subunits (see entry 47) and/or local redox modulation (ibid., see cross-refs in paragraph 51-14-02).

Potential post-synaptic roles for Kv4.2 subunit-containing A-type channels in the eNS 51-14-02: rKv4.2/rKv1.4: Differential subcellular and regional distributions of the A-type f channels encoded by rKv4.2 and rKv1.4 implies distinct roles for these channel proteins in ViV0 17 (see also Protein distribution, 51-13, Subcellular locations, 51-16 and cross-references therein). In particular, the somatodendritic localization of Kv4.2 (ibid.) predicts native channels containing Kv4.2 may regulate neuronal transmission at post-synaptic loci in defined brain regions (i.e. facilitating reception and integration of synaptic signals). Postsynaptic modulation of excitatory input by A-type channels has been demonstrated in both invertebrate (e.g. snail30), mammalian sympathetic ganglion neurones31 and cultured hippocampal and spinal neurones32 (see also VLC K A-T [native], entry 44). Furthermore, neurotransmitter regulation by receptor-linked intracellular second messenger systems (coupled to phosphomodulation of Kv4.2-containing A-type channels) could conceivably contribute to synaptic plasticity phenotypes (see Protein phosphorylation, 51-32 and Receptor/transducer interactions, 51-49). In this regard, A-type currents have been shown to be regulated by cAMP in Aplysia neurones33,34 and by various neurotransmitter receptor/transducer systems expressed in hippocampus, e.g. acetylcholine at muscarinic receptors3S,36 and 5-HT (serotonin) at 5-HTIA receptors37. For predicted Ihyperexcita bility, phenotypes subsequent to reductions in Kv4.2 channel density in dendrites following seizure activity, see Channel density, 51-09. Comparative notes: 1. From their predominant distribution in axons and possibly terminals, A-type channels containing Kv1.4 subunits are likely to have predominantly pre-synaptic roles (see cross-refs above and for further details, Phenotypic expression under VLC K Kv1-Shak, 48-14). 2. The comparative in vivo distribution studies of Kv4.2 and Kvl.4 further underline the difficulties in 'predicting' the in vivo functions of 'phenotypically similar' currents from measurements in heterologous cell expression t systems alone. 3. A-current 'phenotype' may also be directly influenced by local 'use-dependent' redox modulation (see Channel modulation under VLC K Kvbeta, 47-44, VLC K Kv1-Shak, 48-44 and VLC K Kv3-Shaw, 48-44) and/or external K+ concentration, which may change significantly during prolonged neuronal activity (e.g. in axons and terminals, see Channel modulation under VLC K Kv1-Shak, 48-44).

Accounting for observed heterogeneities in tsubthreshold' A-current between neurones 51-14-03: Neurones such as pyramidal cells in the hippocampus and granule cells in the cerebellum represent heterogeneous cell populations in terms of their 'subthreshold' A-current (ISA ) and hence in their firing patterns. Comparative studies of Kv4 subfamily mRNA distribution patterns in rat

_L...-

e_n_try_S_l_

brain (e.g. ref.12, see also mRNA distribution, 51-13) suggest Kv4 subfamily proteins form heteromultimeric channels with distinct subunit composition in different neurones, and may in part explain the observed functional heterogeneity12.

Accounting for cardiac anatomical subregion and species variations of ITO 51-14-04: Kv4.2/Kv4.3: From tissue-distribution studies, vertebrate Shalrelated K+ channel genes are likely to encode subunits contributing to some A-type K+ channels expressed in the mammalian heart (see Current type, field 34 under the VLC K Kv series, entries 48 to 51 and compare to known expression patterns under mRNA distribution, field 13). It has been noted that the heterologously expressed rKv4.2/RKS channel current resembles the native cardiac transient outward current (ITO) as described by Josephson et a1. 4 . Similarly, 'functional and pharmacological correspondence' between Kv4.2 and cardiac transient outward currents have been noted38 . In a later, independent study6 however, a canine Kv4.3 channel subunit was specifically reported to have 'biophysical and pharmacological properties similar to the native canine transient outward current (ITO)'. Significantly, it was concluded that while the Kv4.3 channel 'underlies the bulk of ITO in canine (and probably human) ventricular myocytes, both the Kv4.3 and Kv4.2 channels are likely to contribute to the Ca2 +-independent ITO in rat heart39 . Furthermore, differential expression of the Kv4.2 and Kv4.3 genes can account for observed differences in the kinetic properties of the ITO in different regions of rat cardiac ventricle. This interpretation is strengthened by observation of significant differences in patterns of K+ channel expression in canine versus rat heart6 . These authors suggested that these species differences are due to adaptive changes required for cardiac function in mammals of markedly different sizes. For example, the significantly longer ventricular action potential duration in canine heart (compared with rat heart) is associated with lower levels of Kvl.2, Kv2.1 and Kv4.2 gene expression in canine heart 6 . Note: Suppression of neuronal and cardiac transient outward currents has been achieved using a dominant negative t Kv4.2 construct driven by viral genetic elements 4o .

Comparative genetic analyses of Drosophila Shal to Shaker, Shab and Shaw

51-14-05: The Drosophila genome encodes multiple transcripts t derived from single genes (for background, see VLC key facts, entry 41 and VLC K Kv1Shak, entry 49) and therefore all gene variants can be eliminated with mutations in a single gene (this is to be compared to the comparative difficulty of eliminating gene family members in vertebrates, ibid., see also Phenotypic expression under VLC K Kv2-Shab, 49-14). Taking advantage of this feature, many analyses have been made of native currents in embryonic neurones from wild-type Drosophila compared to those in Drosophila Shal, Shaker, Shab and Shaw mutant flies. On the basis of these studies, Shal has been suggested to be 'as important' in neuronal cell bodies as Shaker is in muscles (where the presence of Shaker is 'obvious')41. Whole-cell Shal currents display a wide variety of inactivation rates which is due to single Shal channels assuming different gating modes (as opposed to alternative

l_e_n_t_ry_S_l

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heteromultimer formation)41. From single-channel data, only three 'genetically separable' currents are found in embryonic neurones, which appear to be Shal (encoding a 4 pS transient channel, this entry), Shab (11 pS, non-inactivating, see entry 49) and Shaw (42 pS, non-inactivating, see entry 50). Shaker currents are 'not detectable' in embryonic neurones 41 .

Protein distribution See also Subcellular locations, 51-16. 51-15-01: Kv4.2: Several independent 'spatiotemporal' immunolocalization studies of KVQ subunit proteins forming fast-inactivating (A-type) channels in heterologous expression systems (e.g. Kv4.2 and Kvl.4) have shown distinct distributions in subpopulations of neurones (in addition to differential subcellular localizations (for details of the latter, see Subcellular locations, 51-16, Protein distribution under VLC K Kv1-Shak, 48-15 and Protein interactions, 51-31). Subunit-specific antibodies for Kv4.2 (see Protein molecular weight (purified), 51-22) show strong protein immunoreactivites17 (see Table 3) that are similar to the mRNA distribution patterns of Kv4.2 16 (see mRNA distribution, 51-13).

Subcellular locations ISubcellular segregation' of rKv4.2 and rKvl.4 A-type channels in the CNS 51-16-01: rKv4.2: As pointed out by Sheng et a1. 17 neurones represent the most extreme examples of cellular asrmmetry in higher animals, with different compartments l (e.g. dendrites, soma t , axon t and terminals t ) being specialized for distinct signalling functions. 'Differential segregation' or 'targeting' of Kv and other channel proteins between these subcellular domains is likely to be of functional significance in neuronal processes such as those modulating (i) pre-synaptic action potential waveforms (i.e. in axons and terminals) and (ii) post-synaptic lintegrative' responses (i.e. in dendrites and cell bodies). In a comparative study investigating the differential subcellular targeting of Kv channel subunits forming fast-inactivating K+ channels in oocytes 17, Kv4.2 subunit immunoreactivity was shown to be concentrated in dendrites and somata (the 'somatodendritic compartment'; this distribution has also been observed for mKv4.2 in mouse retinal neurones 43 ). In contrast, Kvl.4 protein immunoreactivity (see VLC K KvShak, entry 48) was observed to be predominantly associated with axons (and possibly terminals; in comparison, Kvl.2, Kvl.3, and Kv2.1 show variable subcellular distribution43 ). More general distributions across the brain (i.e. without reference to their subcellular locations) are listed under Protein distribution, 51-15. For possible mechanisms underlying differential subcellular distributions, see Protein interactions, 51-31.

Comparative note: Subcellular segregation of other channel types (cross-references) 51-16-02: Many examples of localized subcellular distribution of ion channel proteins have been described in neuronal and non-neuronal cell types based

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_S_l-----J

Table 3. Comparative distributions of Kv4.2 proteins. See also Subcellular location under this entry, 51-16 and VLC K Kv1-Shak, 48-16. (From 51-15-01) Kv4.2

51-15-02: rKv4.2: Whole brain: Antibodies Kv4.2N and Kv4.2C raised against peptide epitopes at opposite ends of rKv4.2 (see Protein molecular weight (purified), 51-22) detect qualitatively indistinguishable patterns of staining on rat brain sections, notably in cerebellum, hippocampus, thalamus and the medial habenular nucleus 17. Throughout these regions, immunostaining is consistent with Kv4.2 protein being concentrated in the somatodendritic compartment of expressing neurones (see Subcellular locations, 51-16 and next paragraph).

51-15-03: rKv4.2: Cerebellum: Kv4.2 immunoreactivity is of greatest concentration in the granule cell layer of the cerebellar cortex and is absent from Purkinje cells, in good agreement with Kv4.2 mRNA localization studies determined by in situ hybridization (see ISH: symbol under Table 2, mRNA distribution, 51-13). Notably, the Kv4.2 hybridization signal is relatively diffuse overlying the granule cells, while it is concentrated in numerous tangled globular structures (cerebellar glomeruli) that occupy the spaces between the clusters of granule cells. Interpretations based on these and other results 17 showing Kv4.2 immunoreactivity outlining the somata of granule cells (and in dendrites that cross over these cell bodies to enter the multisynaptic cerebellar glomerulus) also support a somatodendritic compartment localization for Kv4.2 proteins (see above and Subcellular locations, 51-16). Comparative note: Kv4.2 immunoreactivity is absent in the molecular layer of cerebellum, in which (i) the axons (parallel fibres) of the granule cells are packed and (ii) brainstem nuclei that give rise to ascending mossy fibres are found (contrast with Kv1.4 immunoreactivities, listed in Table 5 under Protein distribution of VLC K Kv1-Shak, 48-15).

51-15-04: rKv4.2: As part of a comparative study42, examining differential expression of five Kv subunits (Kvl.2, Kv1.4, Kvl.S, Kv2.l and Kv4.2) immunohistochemistryt on isolated adult rat cardiac ventricular myocytes has revealed 'strong labelling' with anti-Kv4.2 (and anti-Kvl.2) antibodies. Using these tools, Kv4.2 was determined to be 'more abundant' in cardiac ventricular than in atrial membranes42 . For further details, see Protein distribution under entries 48 to 50.

on electrophysiological, radioligand-binding t or immunocytochemical t data (see Subcellular locations under other entries). Differential subcellular localization may apply to (i) functionally distinct classes of ion channel protein (e.g. Na+ and K+ channel segregation in myelinated nerve fibres, reviewed in ref. 44) or (ii) different isoforms r within single gene families (e.g. see refs 45,46 and VLC Ca, 42-16; see also refs 47,48 and VLC Na, 55-16). These and similar results within the Kv gene family (this .field) are consistent with lateral

l_e_n_t_ry_S_1

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diffusion t studies which suggest that 'targeted' ion channels are generally immobile t in excitable membranes49- 51 (for likely mechanisms, see Protein interactions, 51-31). In the squid Loligo pealeneuronal, evidence supporting lin transit' synthesis, transport, and degradation of native voltage-gated Na+ and K+channels within axoplasmic organelles has been described52 .

Transcript size Reported transcript sizes by Northern analysis 51-17-01: rKv4.2/RKS: Transcripts of ",,6 and 7kb in brain; 6kb and 4kb transcripts in heart. rKv4.2/Rat Shall: A ",,6 kb mRNA present in different regions of the rat brain and in the heart, together with weaker (smeared) bands of 1-3 kb probing undegraded mRNA. The weaker bands may result from a higher sensitivity of the rat Shall transcript to degradation or alternative polyadenylation 9 .

SEQUENCE ANALYSES For references comparing sequence analyses applicable to all of the Kv channel subfamilies, see the SEQUENCE ANALYSES section under VLC K Kvl-Shak, entry 48.

Chromosomal location 51-18-01: Table 4 summarizes chromosome locations of Shal-related K+ channel genes in the human and mouse genomes. For further background to

these studies and their evolutionary implications, see Chromosomal location under VLC K Kvl-Shak, 48-18.

Encoding Sequence alignment of Kv4 subfamily members 51-19-01: An amino acid sequence alignment of the Shal-related isoforms mKv4.1 and rKv4.2, together with that of Drosophila Shal is shown in Fig. 1. Some notable features of the Kv4 subfamily sequences are outlined in Sequence motifs, 51-24, Protein phosphorylation, 51-32 and Domain conservation, 51-28. See also general features of Kv channel primary sequences denoted in entry 48. Note that individual nucleotide and/or amino acid sequences may be retrieved for local analysis using the accession numbers listed under Database listings, 51-53.

Gene organization 51-20-01: rKv4.2/RKS/rat Shall: The different transcript sizes detected with probes specific for Kv4.2, isolates RKS and/or rat Shall (field 17) may indicate that the Kv4.2 gene may be transcribed or processed differently in brain and heart24 . Note: No consensus sequence for polyadenylationtwas found in the rat Shall cDNA9 .

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Table 4. Chromosome locations reported for Kv4 subfamily genes. (From 51-18-01) Consensus/summary (see note 1)

Further details

Kv4.1 hKv4.1, KCND1, Chr Xp mKv4.1, Kcnd1, Chr X

hKv4.1: By homology to mouse (see Table 8 under Chromosomal location of VLC K Kv1-Shak, 48-18), the gene encoding hKv4.1 resides on the petite (p) arm of human chromosome X53 . mKv4.1: The mKv4.1 gene resides on mouse chromosome X53 .

'Kv4.1-related sequences' in the mouse genome53 Kcnd1rs, Chr 3 [~]

Kcndlrs: A locus on mouse chromosome 3 has been designated as the 'mKv4.1-related sequence' Kcnd1rs in the M. spretus genome (Kcnd1rs 53, as part of a paralogous cluster of K+ channel genes). See also Chromosomal location in VLC K Kv4-Shak, 48-18.

Kv4.2 hKv4.1, KCND2, Chr 7q mKv4.1, Kcnd2, Chr 6

hKv4.2: Human chromosome 7q, inferred by homology to mouse (see Table 8 under Chromosomal location of VLC K Kv1-Shak, 48-18). mKv4.2: The mKv4.2 gene resides on mouse chromosome 6.

Notes: 1. [~] Symbol denotes likely paralogous cluster. 2. This column lists species equivalents (see also Table 8 under VLG K KvlShak, 48-18), notes on phenotypes (if known) and other cross-references.

Homologous isoforms Notes on Shal subfamily and interspecies sequence conservation 51-21-01: Kv4.1/mshal: Sequence conservation for Shal proteins has been noted as 'unusually high' at the N-terminus (for significance to inactivation properties, see Inactivation under VLC K Kv1-Shak, 48-37). Rat Shal/mShal shares ~ 72-82 % amino acid sequence identity with Drosophila Sha1 7 (as compared to ~62-71 % for 'interspecies' Shaker subfamily members 54, ~750/0 for Shab subfamily members55,56 and ~51-52% for Shaw subfamily members57,58. rKv4.2/RK5: Compared to Drosophila Shal, 222 aa of rKv4.2 are 85 % identical in the SI-S6 region and the S4 region is identical.

Protein molecular weight (purified) Anti-peptide antibodies detecting in vitro translated (but not native) Kv4.2 products 51-22-01: rKv4.2: 'Specific' polyclonal antibodies raised to a synthetic peptide sequence (residues 23-42) from the N-terminal of rat Kv4.2 (antibody Kv4.2N)

~

= r+

~

CJl ~

n«v4.1(mshal) rKv4.2(Shal1 ) Shal

MAAGVAT\ JL~FARAAAVG\l pLAQQPlPplpEVKASR - - - -- -A- - --- - - - -I--M-V-SG-M-AP-R

FET\I (NTloRYPDTlLG~SEKE

FYoA~SGEY

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~GDCCLEEYR~RKKENAERLl

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EoEEAEQAGJGPALPAGSslRQRUIRAFE~PHTSTAALVhYVTGFFI~~SVIANYVdjpCRGTPR\lP~KEQSCGDRF~TA'FCMOTACVl~~TGEYLlRlFJpSRC!FLRSvAsLIDVVAIl~~YIGLFVPKAoOV~GAFVhRYFR

n«v4.1 (mShal) rKv4.2(Shal1 ) Shal

D-AOToNT- - S- --TMTA ---V-- - --- -- ---M- - - -- - -- -- - - -- - - - - - -- - -Y- -GSS-GHI - -LP- -E-YAV- - - -L- - - - -M-- -Y- - - ---A-- ---Y- -V- - - - - I -- - - - - - - - - - --VMTO-E-- - - - - -- - - - -D-KLS-N -0 QN-QQLTNM--I(M-- - - - - -- - --S- -- - - - - - -- - - -- - -M- - -- - -Y- -GHR-GRA GTLP--E-YKIV- - -L - - -- -M- - -A- - - --- - ---0- -K-V- - - - -1--- - - -M- - - - --GI TO- --- - - --- - - - - --

mKv4.1CmShal) rKv4.2(Shal1 ) Shal

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IIlKv4.1 (mShal) rKv4.2CS:.al1 ) Shal

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n«v4. 1(mshal) rKv4.2(Shal1 ) Shal

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Figure 1. Amino acid sequences of the Shal-related isoforms mKv4.1 and rKv4.2, together with that of Drosophila Shal. Open reading frame lengths for the Kv2 subfamily clones shown are mKv4.1/mShal: 651 aa and rKv4.2/rat Shall: 630aa. Note: The Kv4.3 eDNA (ref. 12, ORF length 611 aa 13) is not shown, but see Database listings, 51-53. (Alignment kindly provided by George Gutman, University of California at Irvine.) (From 51-19-01)

II

_1.-

e_n_try_5_1__

detects a full-length polypeptide of rv72000Da when Kv4.2 mRNA is translated in vitro and subjected to SDS-PAGEt analysis 17. This band is also identified by polyclonal antibodies raised to a synthetic peptide sequence from the Kv4.2 C-terminus (residues 48-502, antibody Kv4.2C). Specific immunoprecipitation can be blocked by an excess of the appropriate peptide immunogens but not by unrelated competitor peptides. Despite this apparent specificity, the Kv4.2N and Kv4.2C antibodies could not detect a specific polypeptide species corresponding to the Kv4.2 protein on immunoblots of brain membranes (presumably because they did not recognize their native epitopest under conditions of SDS denaturation and 'immobilization' on nitrocellulose membranes )17.

Protein molecular weight (calc.) 51-23-01: Kv4 subfamily molecular weights estimated by amino acid composition analysis include Kv4.1/mshal: 72kDa; rKv4.2/RK5: 55kDa and rKv4.2/RatShall: 71 kDa.

Sequence motifs Notable lack of (functional) N -glycosylation motifs between studies 51-24-01: mKv4.1/mShal: mShal1lacks the N-linked glycosylation consensus site present in the SI-S2linker region of several other cloned K+ channels (e.g. compare refs55~56~59). rKv4.2/RK5: Two potential N-glycosylation sites exist at residues 46 and 408, but according to likely domain orientations, both would be intracellular, and therefore carbohydrate-free24 . rKv4.2/rShall: All consensus N-linked glycosylation motifs are non-functional as they are located in the intracellular N- and C-terminal regions of the protein (Asn46, Asn408, Asn536).

STRUCTURE AND FUNCTIONS For references pertinent to all of the Kv channel subfamilies, see the STRUCTURE AND FUNCTIONS section under VLG K Kv1-Shak, entry 48.

Domain arrangement 51-27-01: Kv4 subfamily protein domains are typical of the Kv family, i.e. all conform to the 'SI-S6 + H5' ('SI-S6 + P') arrangement (see VLG key facts, entry 41 and this field under VLG K Kv1-Shak, 48-27).

Domain functions (predicted) See Inactivation, 51-37, Voltage sensitivity, 51-42 and cross-references in this field under VLG K Kv1-Shak, 48-29.

1L..-_e_n_t_ry_5_1

------"_

Protein interactions Kv{3-like component interactions modifying properties of Kv4 subfamily currents 51-31-01: Hybrid arrest t with antisense t oligonucleotides complementary to a sequence common to all known mammalian Shal-related mRNAs (Kv4.1, Kv4.3) suppresses the expression of the I SA supported from total brain mRNA pools56. These experiments show that properties of ISA expressed from brain mRNA depend on Shal-related proteins (encoded by high molecular weight 6-8 kb fractions) modified by additional molecular components consistent with properties induced by a Kv,8 subunit (encoded by low molecular weight 2-3 kb fractions) by analogy to protein interactions established for the Kvl subfamily a subunits (see VLC K Kv-beta, entry 47). mKv4.1/mShall: Modulation of expression and gating properties by low molecular weight regulatory factors (which might be predicted to 'interact' with a-subunit channel proteins) have been independently reported for mKv4.1 channels 15. Similar experiments showing Shal-modulatory activity induced by low molecular weight poly(A)+ mRNA fractions are described under Channel density, 51-09, Inctivation, 51-37 and Channel modulation, 51-44. See also paragraph 51-31-02.

Protein interactions affecting channel biosynthesis and subcellular targeting 51-31-02: rKv4.2: Ceneral comparative notes: It is clear from a number of studies that (i) subunit assembly is a relatively early event in channel biosynthesis (see Protein interactions under VLC K Kv1-Shak, 48-31) and that (ii) subcellular sites of channel mRNA and protein localization can be distinct (specific examples are noted in fields 13, 15 and 16 of several entries; see comparative note under Subcellular locations, 51-16). The striking patterns of differential immunoreactivities observed for the distinct 'A-type' subunits Kv4.2 and Kvl.4 (described under Protein distribution, 51-15 and Subcellular locations, 51-16) indicate the existence of 'targeting signals' facilitating subcellular segregation. Conceivably, these 'sorting' signals might be expected to be present in the non-conserved N- and Cterminal regions of Kv proteins (see Domain conservation under VLC K Kv1-Shak, 48-28). Segments of channel subunit protein may thus be involved in interactions with specific chaperon t proteins (see glossary) or Kv,8 subunits which influence surface expression properties (see for example Kvj32 under VLC K Kvb, entry 47). Furthermore, lateral diffusion t studies in a number of excitable membranes suggest that 'targeted' ion channels are generally immobile t 49-51. Specific elements mediate channel interactions with proteins like ankyrinf (effectively 'anchoring' Na+ channels to the cytoskeletont 61 - see VLC Na, entry 55). Of further significance for understanding mechanisms of protein sorting and targeting to specific subcellular compartments are notable 'co-distributions' of functionally distinct proteins. In this regard, Sheng et a1. 17 (1992) noted the overall patterns of Kv4.2 immunoreactivity in the hippocampus resembled (i) the selective dendritic distribution of the cytoskeletal marker protein MAP2 62 and (ii) 5-HT1A receptor at post-synaptic sites63 (compare Subcellular locations,

_ entry 51 ~-------

I

51-16). Note: For mechanisms of subfamily-specific interactions between Kvo subunits, see Protein interactions under VLC K Kv1-Shak, 48-31.

Protein phosphorylation Potential phosphorylation motifs and regulatory mechanisms 51-32-01: rKv4.2/Rat Shall: A single potential cAMP- and cGMP-dependent protein kinase phosphorylation site is present in the N-terminal region (Thr38). There are (i) nine potential protein kinase C phosphorylation sites at S70, T101, T166, T291, T316 (in the sequence between S4 and H4), S447, S531, S537 and S548; (ii) 12 potential casein kinase II phosphorylation sites at T54, S70, Sl13, S263, T280, S459, S460, S472, T489, S502, S552 and T606 and (iii) one tyrosine kinase phosphorylation site at Y592 9 • rKv4.2/RK5: rKv4.2 activity is depressed by stimulation of PKC and its subunits can be phosphorylated by PKC in vitro 64 . An initial comparative study of Kv4.2 and Kv4.3 channel modulation by PKC has appeared65 .

ELECTROPHYSIOLOGY

Activation Comparison of vertebrate and Drosophila Shal currents 51-33-01: As illustrated in Fig. 2, both Drosophila Shal (fShaI2) and its vertebrate homologue mShal support transient outward currents in oocytes with markedly similar features (e.g. in activation thresholds, peak current-voltage relations, steady-state inactivation properties). The fShal current lacks an additional slow component of current decay present in mShal current 7 (for further comparison, see Table 5 under Current type, 51-34).

Current type General descriptions of Shal-related currents in heterologous expression systems 51-34-01: Table 5 summarizes descriptions of currents supported by Kv4 subfamily channel 0 subunits (alone) following expression in oocytes. In vivo modulation (e.g. redox modulation t and subunit associations - see VLC K Kv-beta, entry 47 and Channel modulation, 51-44) may influence these properties in vivo.

Current-voltage relation 51-35-01: mKv4.2/rat shall: Rat Shall does not exhibit open channel rectification, Le. outward and inward currents evoked during depolarizing and hyperpolarizing phases of the voltage step are approximately equal in amplitude 9 .

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Figure 2. Comparison of vertebrate (mShal, Kv4.1) and Drosophila Shal (fSha12) currents following their expression in Xenopus oocytes. Current elicited in oocytes expressing (a) mShal and (b) fShal from a holding potential of -100mV or -90mV respectively, with voltage steps applied from -80 to +20 m V in 10mV increments at 5 s intervals. Peak current amplitudes versus test potential from normalized (linear-leak subtracted) peak current-voltage relations show marked similarity for mShal (c, .) and the fSha12 (e, 0). Peak conductances (derived from peak amplitudes using measured reversal potentials) are plotted against voltage for mShal (d, .) and fSha12 (d, 0). Fitting of the mShal curve with a Boltzmann equation assuming four transitions in the activation process shows its conductance is half-maximal at -7 m V The steady-state inactivation properties for mShall (e, .) and the fSha12 (e, 0) and are indicated by a plot of peak current (I/Imax ) versus holding (pre-pulse) potential, with the data fitted with a Boltzmann equation. For mShal, the peak current is reduced to half-maximal at a holding potential of -69 m V For (e), oocytes were held at -100mV (mShal1) or -90mV (fSha12) before pre-pulsing in 5 m V increments as shown. Following each pre-pulse, a test potential was given to +20mV (Reproduced with permission from Pak et al. (1991) Proc Natl Acad Sci USA 88: 4386-90). See also descriptions of properties in Table 5 under Current type, 51-34. (From 51-33-01)

_1...-

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Table 5. Descriptions of current Itypes' and miscellaneous properties reported following oocyte expression of Kv4 subfamily Q subunits. (From 51-34-01) Kv4.1

mKv4.l/mShall: Transient, 'low-threshold' A-type K+ current with a hyperpolarized steady-state inactivation midpoint (see Voltage-sensitivity, 51-42). mShal currents activate at voltage steps ~ositive to -50mV or -60mV (i.e. in the 'subthreshold' range) (see also Fig. 2 under Activation, 51-33).

Kv4.2

mKv4.2/ratShall/RK5: Functional expression in oocytes generates 4-AP-sensitive K+ channels displaying rapid inactivation kinetics. RK5 channels activate 'near -40 mV' with a rise timet (10-90% peak current) of 2.8ms and an activation midpoint t of -1 mV5 . Kv4.2, isolate Rat Shall: Rat Shall channels activate at membrane potentials 'above -40mV' with an activation midpoint f of -4.0mV. mKv4.2/RK5: Typically, RKS currents are '900/0 inactivated' within ?Oms following a depolarization to 20mV. The time course for RK5 channel inactivation can be described by the sum of two exponentials, with inactivation time constants of 15 ± 2 ms and 61 ± 6 ms (fractional amplitudes of 0.68 and 0.32 respectively at 20 mV). Inactivation time constants are only weakly voltage dependent5 . mKv4.2/rat shall: Displays 'lnoderately rapid' inactivation, e.g. raising membrane potential from a holding potential of -lOOmV to +60mV or more depolarized potentials, the current inactivates rapidly and completely (major time constant rv155ms at 110e). At -lOOmV, recovery from inactivation requires several seconds (recovery time constant rvl s). Inactivation midpoint for rat Shall is -48 mV with all channels becoming inactivated by holding the membrane potential at or above 0 mV. Rat Shall inactivation rates are much greater at higher temperatures. See also effect of small N-terminal deletions under Inactivation, 51-37.

Kv4.3

rKv4.3: Injection of Kv4.3 cRNA transcripts into Xenopus oocytes generates a rapidly inactivating (A-type) K+ current, with 'small but physiologically significant' differences from the currents expressed by Kv4.2 and Kv4.l mRNAs 12. These results were similar to those shown for canine Kv4.3 6 (see Phenotypic expression, 51-14).

Others (for In Drosophila, the Shal and Shaker genes have been frequentlJ; comparison) described as encoding transient (A-current) 'subtypes' (e.g. ref. 9) in contrast to the delayed rectifier 'subtypes' encoded by Shab and Shaw. Difficulties involved in determining the'contribution' of defined Kv subunits to native cell current phenotypes (such as inactivation properties) are cross-referenced in Phenotypic expression under VLC K A-T [native], 44-14 and Channel designation under VLC K Kvl-Shak, 48-03. In addition to Kv4 subfamily members (this entry) Kvl.4 and Kvl.? (entry 48) are characterized by rapid inactivation kinetics when expressed alone (i.e. as homomultimers).

_

1~_e_n_t_ry_5_l

Inactivation tConcerted action' of N- and C-terminal domains affecting mKv4.1 inactivation 51-37-01: Rapid inactivation t of mKv4.l channels follows a complex time course approximated by the sum of three exponential terms 66 • Notably, the inactivation properties of mKv4.l appear to be influenced by domains at both the N- and C-terminus. Deletion of an amphipathic t region at the N-terminus (residues 2-71) (i) abolishes the rapid phase of inactivation (r = l6ms) and (ii) alters voltage-dependent gating. Both these effects can be mimicked by deletions affecting the hydrophilic t C-terminus. Some basic amino acids at mKv4.l's N-terminus do not influence inactivation, but the positively charged domain at the C-terminus (aa 420-550) appears necessary to support rapid inactivation. It has been hypothesized that 'concerted action' of the N- and C-terminal domains may maintain the Nterminal inactivation gate near the inner mouth of the channel66 . In a separate study, removal of conserved N-terminal residues (alone) from mShal (aa 2-32) modified macroscopic inactivation but (unexpectedly) preserved the transient nature of the current. Other properties, such as activation threshold t , steady-state inactivation t, recovery from inactivation, and ion selectivity were not measurably altered in channels with the aa 2-32 deletion 7 . Notes: 1. The fastest component of inactivation can be 'slowed' by deletion of three basic residues in the N-terminal region (aa 35-37) of rat Shal1 9 (compare the effect of the larger deletion of mShal, above). 2. Replacement of basic residues 35-36 with glutamine (neutral) does not alter the inactivation rate significantly, suggesting that more than one positively charged residues are involved in the process of fast inactivation 9 .

Steady-state inactivation properties of Kv4 subfamily channels

51-37-02: Steady-state inactivation t properties of A-channels are important in determining the voltage range in which they are active (see Fig. 2 under Activation, 51-33). For example, mKv4.l/mShal subunits form a 'lowthreshold' A-type channel in oocytes (compare rKvl.4/RHKl under VLC K Kv1-Shak, entry 48 which has a more-depolarized steady-state inactivation midpoint and a 'shallow' steady-state inactivation-voltage relationship 67). The recovery of Shal-related currents from inactivation is markedly faster at more negative potentials 7 (Le. typical of A-current channels which recover from inactivation in a time- and voltage-dependent manner upon repolarization). mKv4.l/mShaI1 displays a marked similarity of macroscopic inactivation and steady-state inactivation to the Drosophila homologue, fShal but with a slightly hyperpolarized steady-state inactivation midpoint 7 (see Fig.2e).

Modification to tnative properties' by LMW mRNA fractions 51-37-03: Kv4.3 currents in oocytes can be 'modified' to resemble native rat brain A-currents by co-injection with a LMW mRNA fraction from rat brain (which does not express detectable currents on its own)12. Co-injection of Kv4.3 plus LMW fractions in Xenopus oocytes induce (i) a 7- to 10-fold

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increase in the rate of recovery from inactivation; (ii) a 5- to 10-fold increase in current magnitude and (iii) a 3- to 4-fold increase in sensitivity to 4aminopyridine block (4-AP) as compared to Kv4.3 alone12. See also insights on K+ channel selectivity mechanisms from crystal structure determinations (ref. 7s ).

Selectivity Kv4 subfamily channels are highly selective for K+ 51-40-01: The complete conservation of the 'K+ channel signature' sequences in their H5 region (see VLC key facts, entry 41 and Encoding, 51-19) predicts that Kv4 subfamily channels would be highly selective for K+ ions. Appropriate shifts in reversal potential with varying external K+ concentrations confirm this expectation (e.g. as cited for mShal ""S9mV per e-fold [K+] change 7 or ratShal1 ",,52mV per 10-fold [K+] change)9.

Voltage sensitivity Distinctive features of Shal subfamily voltage sensor regions 51-42-01: mKv4.I/mShall; rKv4.2/RK5: The voltage dependence of activation of fly Shal and mouse Shal channels are very similar (see Fig. 2e under Activation, 51-33). The slope of the voltage dependence of conductance curves is generally much less steep for Shal-related channels than for Shaker-related homologues. In rKv4.2/RKS, the S4 'voltage sensor' region has five basic residues 24 with a phenylalanine in position 303 rather than a leucine seen in the corresponding position in all other families of voltagegated ion channels5 . Comparative notes: 1. Substitution of valine for this leucine in Shaker decreases the slope and shifts the midpoint of the conductance-voltage curve to a more positive voltage 68 • 2. An 'interrupted' leucine heptad repeat has been described in the Shaw-related Kv channel subfamily (see Sequence motifs under VLC K Kv3-Shaw, 50-24).

PHARMACOLOGY

Blockers tHigh-millimolar' block of Kv4 subfamily channels by 4-aminopyridine 51-43-01: 4-Aminopyridine (4-AP) has been extensively reported as a blocker of Kv4 subfamily channels, although the degree of block is variable and 'high-millimolar' concentrations of 4-AP are generally required for half-block (e.g. for mKv4.I/mShall: ICso ",,9mM; mKv4.2/RK5: ICso of 5.0mM; mKv4.2/ rat shall: 460/0 ± 8% of channels blocked at 5 mM 4-AP, more effective at blocking the component with the fastest rate of inactivation9). Comparative note: Native cardiac ITO (see Phenotypic expression, 51-14 and VLC K A-T [native], 44-43) is 'completely blocked' by 2-4mM 4-AP; 4-AP sensitivity of KA channels expressed from brain poly(A)+ mRNA pools in oocytes can be

l_e_n_try_5_1

_

increased three- to four-fold by co-expression of low molecular weight mRNA fractions 12 (see also VLG K Kv-beta, entry 47, Inactivation, 51-37 and Channel modulation, 51-44). Both Kv4.1 and Kv4.2 are insensitive to block by (i) tetraethylammonium (TEA+) ions (e.g. '00/0 of channels blocked at IOmM 7 ); (ii) several dendrotoxins up to 300nM; (iii) lidocaine (up to 1 mM) and procainamide (up to 5 mM)5.

tMutually exclusive' 4-AP binding and inactivation of Kv4.2 51-43-02: In most Kv channel subtypes, 4-AP binding and unbinding has been concluded to occur mainly in the activated state. By contrast, 4-AP binding to Kv4.2 expressed in Xenopus oocytes occurs exclusively in the closed state69. In oocytes, 4-AP slows the rate of Kv4.2 decay during depolarization (consistent with channel inactivation occurring only after 4-AP dissociation) while inactivating Kv4.2 channels prevent 4-AP binding. These and other observations showing 4-AP blocks Kv4.2 channels from the intracellular side have been used to suggest the cytoplasmic 4-AP-binding site is at or near the domains involved in channel inactivation69 .

Channel modulation Kv4 family currents are selectively inhibited by arachidonic acid in oocytes 51-44-01: Arachidonic acid (AA) potently suppresses A-current in native sympathetic neurones 70 (see also ILG K AA [native], entry 26) and is implicated in the mechanism of muscarinic inhibition of A-current in cultured coeliac ganglion neurones 71 (see also ILG Ca AA-LTC4, entry 15). Direct comparison of AA modulation on 12 Kv channel subtypes has shown that Kv4 subfamily members (Kv4.1 and Kv4.2) can be selectively inhibited by arachidonic acid 72 . These properties (illustrated in Fig. 3a) are similar to those observed in the native preparations (refs as above). The AA effect appears to be 'direct' or with a 'closely associated component' (see below) but does not change the voltage dependence of inactivation or the rate of activation or inactivation. In inside-out macropatches, Kv4.2 current is reversibly reduced by >500/0 by 2 mM arachidonic acid, with the inhibition developing in <40 s (Fig. 3b). Inhibition of Kv4 channels cannot be prevented by cyclooxygenase, lipoxygenase, or cytochrome P-450 inhibitors but can be mimicked by 5,8,II,14-eicosatetraynoic acid 72 (an arachidonic acid analogue that is not metabolized by these pathways - for background see ILG Ca AA-LTC4, entry 15). Notes: 1. Other cis-unsaturated fatty acids with more than two double bonds can produce a similar effect to arachidonate (Fig. 3d,e). 2. The Kv4.2 N-terminal is not required for the AA inhibition, but effects on channel chimaeras show that the 84-85 loop may influence the effect. 3. In comparison, currents supported by members of the Kvl, Kv2 and Kv3 families show little or no inhibition by fatty acids in oocytes; Shaker currents show a modest increase in peak amplitude following application of AA 72 . 4. Compare the established 'retrograde messenger' role of AA described in Receptor/transducer interactions under ELG CAT GLU NMDA, 08-49.

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Figure 3. (a) Inhibition of Kv4 subfamily channels by arachidonic acid. Arrowed traces show the relative effect of 25 J-LM arachidonic acid on the channel as shown expressed in Xenopus oocytes compared to currents recorded in standard solution (for solutions and voltage protocols, see ref. 72). (b) Time course of Kv4.2 inhibition by arachidonic acid. BSA (bovine serum albumin) was employed as a lwashout agent' exploiting its arachidonate-binding property; BSA alone induces a marginal augmentation of Kv4.2 (compare data points 4 and 5). (c) Representative traces of Kv4.2 AA-inhibited current. Taken from time points 1 to 5 in (b). (d) Potency of Kv4.2 inhibition induced by different fatty acids at 25 J-LM. Names of fatty acids are followed by number of carbon atoms, then the number of double bonds, then by the nature and position of the first double bond. (e) Potency of Kv4.2 inhibition induced by fatty acid analogues with triple

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double bonds at 5 J-lM. Fatty acids are named as above but the letter n is followed by a number indicating the position of the first triple bond. (d) and (e) indicate a lack of correlation between observed potency of fatty acid inhibition of Kv4.2 and predicted (non-specific) effects on membrane fluidity. Note that the extent of inhibition produced by 5 J-lM of the fatty acid analogues BDYA, BTl and BTYA (ibid.) is much greater than arachidonate (although their potency may be related to the fact that they cannot be metabolized). For background to tfluidic' effects, see Protein interactions and Channel modulation, under lLG K AA, 26-31 and 26-44 respectively. (Reproduced with permission from Villaroel and Schwarz (1996) J Neurosci 16: 2522-31.) (From 51-44-01)

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_5_1______

Modulation of expression and gating properties by low molecular weight regulatory factors 51-44-02: mKv4.1/mShal1: Co-injection of Xenopus oocytes with (i) cRNA encoding the 'subthreshold' A-type K+ channel mShal1 and (ii) a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain) results in a significant (fourfold) increase in the surface expression of mShall K+ channels15 (for significance, see Cloning resource under VLC K Kv-beta, 47-10). Although mShal channels in co-injected oocytes are unaltered in open-channel conductance, several further modifications in mShal1 gating kinetics are observed, compared to sham-injected oocytes. (i) Both fast and slow time constants of inactivation are accelerated at all membrane potentials in co-injected oocytes following fitting of macroscopic inactivation (of whole-oocyte currents) with the sum of two exponential components: If = 47.2 ms versus 56.5 ms (control) at 0 mV and 's = 157ms versus 225ms (control) at Omy15. (ii) Corresponding ratios of current amplitude terms were shifted toward 'domination' by the fast component: Af/A s = 2.71 versus 1.17 (control) at omY. (iii) Macroscopic activation (in terms of the time-to-peak current) was found to be more rapid at all membrane potentials: 9.9ms versus 13.5ms (control) at omY. (iv) Coexpression also led to more rapid recovery from inactivation by approx. '2.4fold' in co-injected oocytes at -100mV. Notably, the co-expressed K+ currents in oocytes resembled currents expressed in mouse fibroblasts (NIH3T3) following transfection with mShal1 cDNA alone 15 . Notes: 1. This study implies that low molecular weight regulatory/modulatory factors (encoded by LMW mRNA species) may be 'missing' or expressed at low levels in the Xenopus oocyte heterologous expression system 15 (see VLC K Kv-beta, entry 47). 2. 4-Aminopyridine sensitivity of A-current channels expressed from size-fractionated brain mRNA in oocytes increases when the channels are co-expressed with a 2-4 kb RNA fraction that expresses no K+ channel activity alone (ibid. 73).

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 51-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction eiJ layout of Entries, entry 02).

Database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in CenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in Introduction eiJ layout of entries, section 02, at the front of the book). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouringt analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries

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_e_n_t_ry_S_I

deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers) author/reference or nomenclature. Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly-reported and related sequences. Kv Species, nomenclature DNA source

Original isolate

Database locus

Accession

mKv4.1

Mouse, cDNA mShal

MUSMSHAL gb:M64226

rKv4.2

Rat, cDNA

RK5

RATCK5A

rKv4.2

Rat, cDNA

Shall

rKv4.3

Rat, cDNA

RKShIVB j Kv4.3

rKv4.3

Rat, cDNA

Kv4.3 ORF 611 aa

Canine Kv4.3

Canine cardiac cDNA

Kv4.3

Drosophila Shal

Shallocus

ORF: 491 aa

(example only, Shal2 protein mRNA)

Sequence/ discussion

Pak, Froc Natl Acad Sci USA (1991) 88: 4386-90. gb:M59980 Roberds, Froc (see note 1) Natl Acad Sci USA (1991) 88: 1798-802. gb:S64320 Baldwin, Neuron (1991) 7: 471-83. gb: U42975 Serodio, T Neurophysiol (1996) 75: 2174-9. gb: U75448 Tsaur, FEBS Lett (1997) 400: 215-20. gb: not found Dixon, Circ Res (1996) 79: 659-68. gb:M32660 Butler, FlyBase: Nucleic Acids FBgnOOO5564 Res (1990) 18: 2173-4. Wei, Science (1990) 248: 599-603.

Note: 1. Initially contained a 2 bp deletion resulting in early termination compared to the Shall sequence under S64320.

Gene mapping locus designation 51-54-01: Human chromosomal locus names assigned by the Human Gene

Mapping Workshopt (HGMW) are as follows: Kv4.1 - KCNDI; Kv4.2 KCND2; Kv4.3 - KCND3 (see Chromosomal location, 51-18). By convention of the International Committee on Mouse Genetic Nomenclature 74 mouse Kv loci are designated in lower case; e.g. Kcnd1, Kcnd2, Kcnd3. Note: Italicized designations are generally used on printed maps but are usually not preserved in online resources such as OMIM.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_ 1

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Related sources and reviews Book references: Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand- and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton. For other book references pertinent to Kv channels, see this field under VLC K Kvl-Shak, 48-56.

Feedback Error-corrections, enhancements and extensions 51-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource J - Search criteria eiJ CSN development). For this entry, send email messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 51-14-02). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback eiJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN web site (www.le.ac.ukjcsnj).

REFERENCES 1

2 3 4

5 6

7

8 9 10 11

12 13 14 15 16 17 18 19 20 21

Chandy, Nature (1991) 352: 26. Gutman, Semin Neurosci (1993) 5: 101-6. Luneau, Proc Natl Acad Sci USA (1991) 88: 3932-36. Josephson, Circ Res (1984) 54: 157-62. Blair, FEBS Lett (1991) 295: 211-13. Dixon, Circ Res (1996) 79: 659-68. Pak, Proc Natl Acad Sci USA (1991) 88: 4386-90. Roberds, Proc Natl Acad Sci USA (1991) 88: 1798-802. Baldwin, Neuron (1991) 7: 471-83. Vega-Saenz-de-Miera, Biophys J (1991) 59: 197a. Rudy, Mol Cell Neurosci (1991) 2: 89-102. Serodio, J Neurophysiol (1996) 75: 2174·-9. Tsaur, FBBS Lett (1997) 400: 215-20. Baro, J Neurosci (1996) 16: 1689-701. Chabala, J Cen Physiol (1993) 102: 713--28. Tsaur, Neuron (1992) 8: 1055-67. Sheng, Neuron (1992) 9: 271-84. Kues, Bur J Neurosci (1992) 4: 1296-308. Maleticsavatic, J Neurosci (1995) 15: 3840-51. Gidhjain, Circ Res (1996) 79: 669-75. Dixon, Bur J Neurosci (1996) 8: 183-91.

l_e_n_try_5_1

22 23

24

25 26

27 28 29 30

31 32

33 34

35 36

37

38 39

40 41 42 43

44 45

46

47 48

49

50 51 52 53 54

55 56

57 58 59

60

61 62 63

64 65

66

67 68 69 70

_

Dixon, Circ Res (1994) 75: 252-60. Brahmajothi, Circ Res (1996) 78: 1083-9. Paulmichl, Nature (1992) 356: 238-41. Karschin, FEBS Lett (1994) 348: 139-44. Connor, 1 Physiol (1971) 213: 21-30. Neher, 1 Gen Physiol (1971) 58: 36-53. Solc, Science (1987) 236: 1094-8. Baker, Neuron (1990) 4: 129-40. Daut, Nature (1973) 246: 193-6. Cassell, 1 Physiol (1986) 374: 273-88. Segal, 1 Neurosci (1984) 4: 604-9. Kaczmarek, 1 Neurophysiol (1984) 52: 340-9. Strong, 1 Neurosci (1984) 4: 2772-83. Atkins, Nature (1990) 344: 240-2. Nakajima, Proc Natl Acad Sci USA (1986) 83: 3022-6. Aghajanian, Nature (1985) 315: 501-3. Yeola, Biophys 1 (1996) 70: TU261. Fiset, Biophys 1 (1997) 72: MP143. Johns, Biophys 1 (1997) 72: THP06. Tsunoda, 1 Neurosci (1995) 15: 1741-54. Barry, Circ Res (1995) 77: 361-9. Klumpp, Vis Neurosci (1995) 12: 1177-90. Black, Trends Neurosci (1990) 13: 48-54. Ahlijanian, Neuron (1990) 4: 819-32. Westenbroek, Nature (1990) 347: 281-4. Westenbroek, Neuron (1992) 9: 1099-115. Westenbroek, Neuron (1989) 3: 695-704. Angelides, 1 Cell BioI (1988) 106: 1911-25. Angelides, Nature (1986) 321: 63-6. Weiss, 1 Gen Physiol (1986) 87: 955-83. Wonderlin, Proc Natl Acad Sci USA (1991) 88: 4391-5. Lock, Genomics (1994) 20: 354-62. Tempel, Nature (1988) 332: 837-9. Frech, Nature (1989) 340: 642-5. Pak, 1 Neurosci (1991) 11: 869-80. McCormack, Proc Natl Acad Sci USA (1990) 87: 5227-31. Yokoyama, FEBS Lett (1989) 259: 37-42. Wei, Science (1990) 248: 599-603. Ser6dio, 1 Neurophysiol (1994) 72: 1516-29. Srinivasan, Nature (1988) 333: 177-80. Garner, Nature (1988) 336: 674-7. Pompeiano, 1 Neurosci (1992) 12: 440-53. Blair, Biophys 1 (1992) 61: A382. Nakamura, Biophys 1 (1997) 72: TUP28. Jerng, Biophys 1 (1997) 72: 163-74. Tseng-Crank, FEBS Lett (1990) 268: 63-8. McCormack, Proc Natl Acad Sci USA (1991) 88: 2931-5. Tseng, 1 Pharmacol Exp Ther (1996) 279: 865-76. Villarroel, FEBS Lett (1993) 335: 184-8.

_ 1...---

71

72 73

74 75

Blair, Soc Neurosci Abstr (1993) 295: 9. Villarroel, TNeurosci (1996) 16: 2522-31. Rudy, Neuron (1988) 1: 649-58. Ried, Genomics (1993) 15: 405-11. Doyle, Science (1998) 280: 69-77.

entry 51

I .

Listing of cDNA clones encoding Kv channels with unassigned gene family relationships Edward C. Conley

Entry 52

Note on coverage: This entry briefly summarizes sources of information about four cDNA clones (originally designated clone aKv5.1 1 , clone IK~, clone K132 and clone hamster Kv8.1 3 ) which appear to be part of the extended Kv channel gene family, but which have not been definitively assigned to the Kv1 to Kv4 gene subfamilies. Without exhaustive sequence comparisons and dendrogram t analysis, the 'premature' allocation of alphanumeric gene family designations to clones could be misleading, so has been avoided. Information is presently limited to simple annotations and database accession numbers tagged with the clone name (as an underlined prefix:). Nearly all of the presently available information about the clones can be obtained from the original citations (see table under Database listings, 52-53). Furthermore, no 'systematic' comparison should be inferred (unless taken from the original work directly comparing IKB versus K13) as the clones are representative of different gene subfamilies. Further novel sequences also requiring independent K+ channel gene family comparison/assignation include KvLQT1 4 , the cGMP-gated K+ channel clone Kcnl/gen 3 and the clones encoding the potassiumselective SKca family 7. Sequence database links to these and other unique (or newly discovered) chemical gene family members will appear on the CSN website (as updates, see entry 12) upon completion of the book series.

NOMENCLATURES

Abstract/general description 52-01-01: In 1992, clones IK8 and K13 were both isolated from a rat brain eDNA library screened with a rat Kv2.1 (DRK1) probe2 . On the basis of their

predicted primary amino acid sequences, IK8 and K13 appeared to define two further Kv gene subfamilies (Kv5 and Kv6, beyond the Kvl to Kv4 designations extant in 1992). Both sequences possessed an '81-86 + H5' ('S1-S6 + P') domain arrangement typical of that found for other voltagegated K+ channels in the Kv family (see VLG key facts, entry 41). Limited phylogenetic analysis (in comparison to the extended Kv family) showed IK8 and K13 to be themselves unrelated, with IK8 being placed on the branch leading to Shah, and K13 being placed on the branch leading to Shal (see Gene family, 52-05); however the final 'placement' of IK8 and K13 awaits further analysis. Since the original report2 , little additional information has appeared regarding the distribution, co-distribution or hypothetical functions of the IK8 and K13 eDNA or protein products in vivo, although novel interactions between Kv2.1 and Kv6.1 have been reported (see in-press update under Protein interactions, 52-31). 52-01-02: In 1994, a eDNA clone named aKv5.1 (cf. IK8 nomenclature in use) was isolated from the nervous system of Aplysia, and encoded a K+ channel activated at low voltage and capable of contributing to the resting potential and firing patterns of neurones. In some limited aKv5.1 showed similarities to native M-current, a voltage-gated 'non-inactivating' K+ current contributing to neuronal resting potential (compare VLG K M-i [native], entry 53).

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_2_

Expression of the aKvS.l 'non-inactivating' channel in the identified Aplysia neurone R15 increased the resting potential by >20mV and abolished the spontaneous bursting activity of the cell. This ability (to suppress the endogenous rhythm of R15) differed from transient, inactivating K+ channels such as aKvl.la, an Aplysia homologue of Shaker (see VLG K Kv1-Shak, entry 48). Generally, aKvl.la was able to shorten spike duration and increase the afterpotential, but it could not not suppress bursting behaviour. Attempts to retrieve mammalian homologues of aKv5.1 are in progress, but were not published at the time of entry compilation.. 52-01-03: As part of the 1992 description of IK8/KI3, extensive controlled expression trials in Xenopus oocytes (Le. injection of single-subunit cRNA derived from these clones) did not give rise to any measurable voltagedependent currents (but see Protein interactions, 52-31). Indeed, since 1992, several other 'non-expressible' clones in other gene families have been reported (see, for example, GIRK3 under INR K subunits, entry 33 and hamster Kv8.1, this entry). There may be many possible reasons for apparent null phenotypes (see Phenotypic expression, 52-14), including a mutated or truncated coding region, functional expression depending on specific protein interactions (e.g. with endogenous or co-transfected protein components) or requiring additional factors for their gating (e.g. intracellular ligands). These explanations do not necessarily apply to IK8 and K13, and it is still not clear why these cDNAs are 'non-expressible'. An alternative interpretation, cited for the hamster Kv8.1 subunit3 (this entry) is that it has specific inhibitory properties, and is able to selectively abolish the functional expression of Shab (Kv2) and Shaw (Kv4) channels when co-expressed in oocytes (see Phenotypic expression, 52-14). Co-immunoprecipitation studies have suggested that inhibition may occur by formation of heteropolymeric channels, and results obtained with Kv8.1 chimaeras indicate that association of Kv8.1 with other types of subunits occurs via its N-terminal domain.

Category (sortcode) 52-02-01: VLG K Kvx putative voltage-gated K+ channel cDNAs which share limited homology with subfamilies Kvl-Kv4 (typically <400/0 identity) and/or may not have been functionally expressed. Absence of functional data precludes a 'definitive' description of channel type and characteristics, but their similarities with other Kv family members (see Gene family, 52-05 and Encoding, 52-19) makes it reasonable to assign a 'Kv' designation. Family prefixes that are bracketed (e.g. K(v)5.1, K(v)6.1) have sometimes been used to indicate that functional data (e.g. confirmation of voltage gating) are not available. f'..J

Clones described together in this entrJl are unrelated 52-02-02: Clone aKv5.1; clone IK8; clone K13; clone hamster Kv8.1: The proteins predicted by these cDNAs are themselves unrelated and are listed together for convenience. As with other entries, where 'clone names' in use can be mistaken for alphanumeric gene family designations, original terms are prefixed with the term 'clone'.

l"---e_n_t_ry_S2

_

Information sorting/retrieval aided by designated gene family nomenclatures 52-02-03: The gene product (information retrieval) prefix (unique embedded identifier or DEI) for 'tagging' of new articles of relevance to the contents of this entry in the CSN pages will be of a form related to the established gene family positions (see thius field in other entries for details): Should additional phylogenetic analysis of these sequences and/or functional data establish a clear gene family relationship for these isolates (possibly in combination with reporting of novel sequences) then the UEI designations will change to reflect the assigned gene family position.

Gene family The finalized gene family tplacements' of IKB/K13 await further analysis 52-05-01: Clone aKvS.l; clone IK8; clone K13; clone hamster Kv8.1: The cDNA

isolates IK8 and K13 have been described as prototypict members of the KvS and Kv6 subfamilies, respectively (see Chandy and Gutman, 1994, under Related sources and reviews, 52-56). Later publication and designation of an Aplysia cDNA clone as aKvS.l 1 was in conflict with this nomenclature, but the 'KvS.l' term has entered use (e.g. ref. 3 ) for comparing mammalian and Aplysia channels. Lack of exhaustive analysis has left some uncertainty regarding the gene family relationships/nomenclatures for each of these cDNAs.

Presence of common K+ channel tsignature'sequences

52-05-02: Clone IK8; clone K13: Hydropathy analyses t of amino acid

sequences predicted from these cDNA sequences indicate a domain arrangement typical of that found for other voltage-gated K+ channels in the Kv family. In particular, they have 'signature sequences' characteristic of K+-selective pore regions. According to one comparative review (Chandy and Gutman, 1994, see Related sources and reviews, 52-56), adding IK8 and K13 to a phylogenetic analysis for the extended Kv gene family places IK8 on the branch leading to Shab, and K13 on the 'branch leading to' Shal.

Initial comparisons of IKB/K13 sequences with other Kv family members 52-05-03: Clone IK8; clone K13: In the original descriptions of IK8 and K13 2 ,

their predicted amino acid sequences were aligned to K+ channels in defined subfamilies (see Encoding, 52-19). Relatedness between homologous positions was further reported as an identity matrix (Table I, with inset dendrogram mainly for comparative purposes). This and other analyses (earlier to those described above and with fewer sequences) suggested that K13 and IK8 could be prototypes for new K+ channel subfamilies. In the latter case, IK8 was 'placed' as a distantly related member of the Kv2 (Shab-related subfamily).

Later comparisons of IKB/K13/aKv5.1 and KvB.l sequences with other Kv family members 52-05-04: Clone aKvS.l; clone IK8; clone K13; clone hamster Kv8.1: Hugnot

et a1., who isolated and named the hamster Kv8.1 cDNA3 , included a new

fI

Table 1. Percentage identities in homologous positions of isolates IK8 and K13 to K+ channels representing different subfamilies. Following alignment of predicted coding regions the matrix indicates amino acid identities in homologous positions between the IK8 and K13 sequences compared to (i) examples of Shaker-related channels (Drosophila ShBl, Aplysia AKOl, RCKl/RCK2/ XSha2, see VLG K Kvl-Shak, entry 48); (ii) Shab-related channels (Drosophila Shab and DRKl, see VLC K Kv2-Shab, entry 49); (iii) Shaw-related channels (Drosophila Shaw and RKShill, Raw3, isolate KV4, see VLC K Kv1-Shaw, entry 50) and (iv) the Shal-related channel RK5 (Kv4.1, see VLC K Kv4-Shal, entry 51). Percentage amino acid identities determined for K+ channels from the same subfamily are underlined in the table. The inset figure shows the gene family positions of IK8 and K13 predicted by this analysis. (Data and figure modified with permission from Drewe, J.A. et al. (1992) J. Neurosci. 12: 538-48.) (From 52-05-03) Kv2.1 (DRK1)

Dros (Shah)

Kv3.1 Kv3.2 (Kv4) (RKShllI)

100

74 100

44 44 100

.L" +0 «--u.... ~

f;:- ~

~"(

45 45 86 100

~ .& ~ ~ ~ .L' ~ .LCO '"::> 0....'S:-.'"::> #~'lf ~""rs-qj ~ ~ ~ ~ CO <;) -..I

Kv3.4 (Raw3)

Dros (Shaw)

Kv1.1 Kv1.2 (RCK1) (RCK2)

Kv1.2 (XSha2)

Aplysia Dros (AK01) (ShB1)

Kv4.1 (RK5)

Dros (Shal)

Rat (IK8)

Rat (K13)

44 45 86 87 100

45 43 56 56 54 100

45 44 46 47 47 45 100

46 43 46 47 46 45 86 83 100

47 45 47 49 48 47 81 78 79 100

41 39 41 41 42 42 42 44 43 43 44 100

41 39 42 42 43 43 42 44 42 42 43 83 100

48 47 41 42 42 40 38 39 37 38 38 37 37 100

45 43 38 38 39 36 38 39 38 37 36 37 36 40 100

.L'~ ....

~_

~J~qj

~

'"::>

~

47 45 48 49 48 47 82 100

45 44 46 47 47 45 80 78 78 82 100

Kv2.1 (DRK1) Dros (Shah) Kv3.1 (Kv4) Kv3.2 (RKShllI) Kv3.4 (Raw3) Dros (Shaw) Kv1.1 (RCK1) Kv1.2 (RCK2) Kh1.2 (XSha2) Aplysia (AK01) Dros (ShB1) Kv4.2 (RK5) Dros (Sha1) Rat (IK8) Rat (K13)

I~~ c.n

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_

amino acid alignment comparing this sequence with other representatives of the Kv family, including Aplysia aKv5.l and IK8 (which they designated as Kv6.l) and K13 (designated as Kv7.l). Using the sequence segment spanning aa 48 (NVGG in the Kvl.8 query line) to the end of the S6 TM segment (NDRF in the Kvl.8 query line) Hugnot et al. determined Kv8.l to have percentage identities with other Kv members as shown in brackets: Kvl.3 (37), Kv2.l (43.6), Kv3.4 (37.6), Kv4.l (39), Kv5.l (59.8), Kv6.l (64.4)and Kv7.l (66.9).

EXPRESSION

Cloning resource 52-10-01: Clones IK8 and K13: Rat brain cDNA libraries enriched for long inserts t (3.3-5.3 kb); clone aKv5.l: Aplysia CNS cDNA library; clone Kv8.l: hamster insulinoma cell line (HIT-T15) cDNA library.

Isolation probe 52-12-01: Clones IK8 and K13: Nick-translatedt DRKI probe (see VLC K Kv2Shab, entry 49). clone aKv5 .1: Fourfold degenerate oligonucleotide probes based on H5 K+ selectivity regions of previously known Aplysia channel sequences (cited in ref. 1 as aKvl.la, aKv2.l, aKv3.l, aKv5.2). Clone hamster Kv8.l: Nested, degenerate PCR primers based on K+ channel H5 (sense) and Walker-A ATPbinding site (antisense) motifs; subsequent re-screening of a HIT cell cDNA library (see Cloning resources, 52-10) with amplified, subcloned fragments.

mRNA distribution Differential mRNA expression patterns 52-13-01: Clone IK8 versus K13: K13 mRNA is widely expressed throughout the brain, though it is more abundant in the superficial cortical layers (cf. IK8, which is preferentially expressed in the deeper cortical layers). K13 is expressed at relatively high levels in hippocampus, dentate gyrus and zona incerta whereas IK8 shows low expression these regions. K13 mRNA is found at high levels both in the Purkinje and granular layers of the cerebellum and in the brainstem, where IK8 message is 'low or undetectable'. IK8 is expressed in the medial amygdaloid nuclei and the lateral amygdaloid area. Both IK8 and K13 mRNAs are present in the piriform cortex, olfactory tubercule and medial habencular nucleus (see also Transcript size, 52-17).

Comparative mRNA distribution in the HI-T15 cell (cDNA source) line versus other tissues 52-13-02: Hamster Kv8.l: By Northern blott analysis Kv8.l can be detected in brain and HIT-TI5 cells (see Transcript size, 52-17); this analysis showed little or no expression in mRNA pools derived from hamster heart, skeletal muscle, pancreas, kidney, liver, or the insulin-secreting lines RINmF5 and NIT-1 3 . Note: These authors suggested the 'selective' expression of Kvl.8 in pancreas-derived HIT-T15 was a manifestation of the hyperproliferative

II

_ ' - -

en_t_ry_S_2_

(tumorigenic, dedifferentiated) status of this cell line. ISH: In situ hybridization analysis of sagittal sections of adult hamster brain reveals Kvl.8 mRNA to be heterogeneously expressed, with highest levels in the neocortical and allocortical regions, hippocampus, habenula of the epithalamus and the cerebellum.

Phenotypic expression Possible reasons for tnon-expressible phenotypes' in heterologous expression systems 52-14-01: Clone IK8; clone K13: Initial attempts to express cRNA transcripts synthesized from IK8 and K13 in Xenopus oocytes in 1992 were not successful2 . The underlying reason(s) for this remains unknown, but potential termination codons in S'-untranslated regions were eliminated (see Gene organization, 52-20). Furthermore, the cRNAs were capable of supporting translation in vitro to yield protein products of expected size, and thus an explanation based on cloning artefacts was deemed unlikelr. Other formal possibilities offered for 'non-expression' were (i) that the oocyte expression system was unable to process primary translation products of these cDNAs and/or to transport/assemble constituent subunits to yield functional channels; (ii) that IK8 and K13 channels are voltageoperated channels that have open-closed state transitions t which are ligand dependent, Le. require G proteins or cyclic nucleotides; (iii) that IK8 and K13 subunits alone do not form homomultimeric channels typical of other K+ channel subunits but require a hetero-oligomeric arrangement with other, unknown subunits; or (iv) that IK8 and K13 are subunits of Ca2+activated channels, although the latter is unlikelr. See also Domain

conservation, 52-28.

An apparent tsubfamily-selective' inhibitory phenotype for clone Kv8.1 52-14-02: Clone hamster Kv8.l: As for IK8 and K13 (above) several possible reasons for the null phenotype of Kv8.l expressed alone can be suggested (but are difficult to test), including a mutated or truncated coding region, functional expression depending on specific protein interactions (e.g. with endogenous or co-transfected protein components) or requiring additional factors for their gating (e.g. intracellular ligands). These explanations do not necessarily apply for Kv8.l, and as for IK8/K13, it is still not clear why these cDNAs are 'non-expressible'. An alternative interpretation, cited for the Kv8.l subunit3 is that it has no K+ channel activity by itself, but instead mediates specific inhibitory properties. It has been shown that Kv8.l is able to selectively abolish the functional expression of Shab (Kv2.l and Kv2.2) and Shaw (Kv3.4) channels when co-expressed in oocytes (see comparative note, below). Co-immunoprecipitation studies have suggested that inhibition may occur by formation of heteropolymeric channels. Results obtained with lexpressible' Kv8.1 chimaeras (made with the slow-inactivating channel Kvl.3, see entry 48) has confirmed that association (Le. assembly and presumably the putative 'inhibitory' function) of Kv8.l with other types of subunits occurs via its N-terminal domain3 . Comparative note: These

Il....-.-e_n_t_ry_52

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putative KvS.l:Kv2.1, KvS.l:Kv2.2 and KvS.l:Kv3.4 'inhibitory co-assemblies' appear to break the 'rule' stating 'heteromultimeric subunit co-assembly only occurs within the same Kv subfamily' (for references, see Protein interactions under VLC K Kvl-Shak, 48-31). In this regard, it is notable that an independent report has indicated that Kvl.2 and Kv3.1 can actually form functional heteromultimeric assemblies (ibid., for details, see ref. s).

52-14-03: Clone aKv5.1: A detailed description of the functional characteristics of the (non-vertebrate) aKv5.1 channel is beyond the scope of this entry, but see the summary under Abstract/general description, 52-01.

Transcript size 52-17-01: Clone IK8 versus K13: IK8 probes hybridize to transcriptst of ",,5.3 kb (highest in neonatal brain) and 5.0 kb (highest in adult brain). Neonatal and adult heart and neonatal kidney and skeletal muscle show weak expression. Clone K13: K13 probes detect an abundant 4.7kb transcript in adult lung and liver with fainter bands detected in neonatal and IS-day embryonic hearts and adult brain and kidney. Weak bands for higher transcript sizes are also observed on Northern blotst of mRNA from adult liver, lung and skeletal muscle2 . Hamster Kv8.1: Four independent eDNA clones with a 2.9 kb insert size were recovered from the HIT cell library screens3 indicating that they encompassed a full-length eDNA. Northern blot T analysis revealed (only) a weak 2.9 kb mRNA in HIT-TIS cells (see Cloning resource, 52-10), but two abundant transcript sizes represented in brain mRNA pools (2.9 kb plus 3.3 kb)3.

SEQUENCE ANALYSES

Chromosomal location 52-18-01: Human Kv8.1: Human chromosome 8q22.3-8q24.1 (using the hamster clone Kv8.1 to perform in situ hybridization on human chromosome metaphase spreads)3.

Encoding 52-19-01: Clone aKv5: 577aa; clone IK8: 505 aa; clone K13; 514 aa; hamster clone Kv8.1: 504 aa. For sequences, use database accession numbers under Database listings, 52-53.

Gene organization Analysis of start and stop codons in the tnon-expressible' clones IKB and K13 52-20-01: Clone IK8: Chromosomal versus eDNA sequences have not been characterized, but the longest open reading framet on the sequenced eDNA (1515 nt) has a 5'-untranslated regiont (UTR) of 505 nt containing two ATG codons followed by termination tripletst. Following the TGA termination

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triplet, 458 nt of 3' UTR have been sequenced. Clone K13: The longest open reading frame (1542 nt) has a 5' UTR (245 nt) that carries several in-frame upstream termination triplets including one ATG codont immediately followed by a termination triplet TGA. The 3' UTR is rvl00nt in length. cRNAs from these cDNAs and their derivative constructs which had their 5' UTRs removed (and hence the alternative ATGs/termination triplets) were not expressible in Xenopus oocytes (for further background, see Abstract-general introduction, 52-01).

Indirect evidence for alternative exon usage in the Kv8.1 gene 52-20-02: Hamster clone Kv8.1: Differential transcript sizes using a single probe in Northern analyses may suggest the existence of alternative splicing t of primary transcripts from the Kv8.1 gene, with a (2.9knt variant, HIT-TIS cell) and 2.9 plus 3.3 knt variants (brain) being represented (see Transcript size, 52-17).

STRUCTURE AND FUNCTIONS

Domain conservation Conservation and divergence of common Kv channel functional elements 52-28-01: Clone IK8 and K13: Both IK8 and K13 possess a pore (H5) region closely related to known functional K+ channels (see VLC K Kv1-Shak, entry 48). The S4 'voltage sensor' domain does show some changes compared to sequences conserved in all other K+ channels (e.g. in K13 a central conserved Arg is changed to a Tyr; also a conserved'Lys is changed to an Arg). The S4 region in IK8 contains five positive charges, but there are three alanines at positions where other known K+ channels have large hydrophobict side-chains. In the 86 domain, both IK8 and K13 contain a serine at a position where all other K+ channels carry a conserved cysteine residue. All other K+ channel subunits also carry a conserved cysteine in the S2 domain. It is therefore possible to speculate that IK8 and K13 are incapable of forming a potentially important disulphide bridget (S-S) between S2 and S62 . IK8 and K13 also show more divergence in sequences relatively conserved in the S6 segment of other Kv channels.

Predicted protein topography 52-30-01: Clone aKv5.1; clone IK8; clone K13; clone Kv8.1: All isolates follow the general 'SI-S6 plus H5 domain' arrangement typical of the voltage/second messenger-gated superfamilyt of channel sequences (see VLC key facts, entry 41).

Protein interactions 52-31-01: In-press update: Protein-protein interactions between Kv2.1 and the 'electrically silent' Kv6.1 a subunits have been characterized using the

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yeast two-hybrid t system6 . Although N-terminal portions of Kv6.1 could not form homomultimers, they were able to interact specifically with N-termini of Kv2.1. Xenopus oocytes co-injected with Kv6.1 and Kv2.1 cRNAs exhibit a novel current with decreased rates of deactivation, decreased sensitivity to TEA+ block, and a hyperpolarizing shift of the half-maximal activation potential when compared to Kv2.1 6 .

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 52-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:)

which should not be typed (see Introduction ft} layout of entries, entry 02). Database locus names and accession numbers immediately follow the colon. See other general notes pertaining to sequence database accessions in this field of other Kv entries (e.g. Database listings under VLC K Kv4-Shal, 51-53). Nomenclature

Species, DNA source

Original isolate

Accession

Sequence/ discussion

Unassigned

Rat, brain cDNA

IK8

gb:M81783

Drewe, T

(see Gene family, 52-05)

Neurosci (1992)

12: 538-48.

Names in use: Kv5.1 and Kv6.1 Unassigned Rat, brain cDNA

K13

gb:M81784

(see Gene family, 52-05) Names in use: Kv6.1 and

Neurosci (1992)

12: 538-48.

Kv7.1 Unassigned

Aplysia 'nervous (see Gene family, 52-05) tissue' cDNA

Name in use: aKv5.1 Unassigned

Hamster,

aKv5.I

gb:L35766

Kv8.1

gb: not found

(see Gene family, 52-05) insulinoma cell

Name in use: Kv8.1

Drewe, T

line cDNA (HIT T15)

Zhao, Neuron (1994) 13: 120513. Hugnot, EMBO T (1996)15: 332231.

Gene mapping locus designation 52-54-01: Human locus names assigned by the Human Gene Mapping Workshopt (HGMW) are as follows: IK8 as Kv5.1 - KCNFl j K13 as Kv6.1 KCNGI.

Related sources and reviews Book reference: Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand- and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton.

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Feedback Error-corrections, enhancements and extensions 52-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource I, Search Criteria). For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 52-28-01). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback eiJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.ukjcsnf).

REFERENCES 1

2 3 4

5 6

Zhao, Neuron (1994) 13: 1205-13. Drewe, I Neurosci (1992) 12: 538-48. Hugnot, EMBO 1(1996) 15: 3322-31. Wang, Nature Genet (1996) 12: 17-23. Shahidullah, Proc Natl Acad Sci USA (1995) 261: 309-17. Post, FEBS Lett (1996) 399: 177-82.

IMuscarinic-inhibited' K+ channels underlying 1M (M-current in native cell types) Edward C. Conley

Entry 53

NOMENCLATURES

Abstract/general description 53-01-01: The M-current (1M , see Current designation, 53-04) is a voltage-gated and time-dependent 'non-inactivating' outward K+ current. M-current was originally described in bullfrog sympathetic neurones as being 'suppressed' following muscarinic receptor stimulation by cholinergic agonists, e.g. muscarine activating muscarinic M 1 or M 3 receptors. Effector-channel activities with properties similar to 'classical M-current' can be suppressed by a variety of other phospholipase C-coupled transmitter receptors, including those activated by substance P, bradykinin and nucleotide triphosphates (amongst others, largely dependent on cell type) (for full listings, see Receptor/ transducer interactions, 53-49). The 'non-inactivating' channel family members represented by eag and aKv5.1 are candidates for M-channel effectors (see Cloning resource, 53-10). Distinctions between 'classical' 1M , l K and lA components are outlined/cross-referenced under Activation, 53-33. 53-01-02: Open M-channels provide an outward, K+ -selective (hyperpolarizing) current under 'resting' conditions (i.e. in the absence of receptor agonists that induce its suppression) and thus play an important role in stabilization of cell excitabilityt (see Phenotypic expression, 53-14). 1M has been referred to as a Ibraking' current, effectively bringing action potential firing frequency under 'receptor control'. Thus suppression of M-current following activation of several G protein-linked receptors (see paragraph 53-01-01 and Receptor/ transducer interactions, 53-49) results in membrane depolarization and an increase in membrane input resistance (i.e. making it more likely that a cell will fire action potentials t). When neurones are depolarized (i.e. towards the 'threshold for firing' action potentials) Islow activation' of M-channels tends to hyperpolarize the cell membrane back towards rest. In the absence of agonist-induced inhibition, M-channels do not inactivate t under maintained depolarization, and thus are often described as being 'tonically active', remaining 'steadily activated' or 'persistent' at potentials positive to -70 mY. Upon membrane hyperpolarization, M-channels are deactivatedt ('turned off'), exhibiting slow relaxationst in response to voltage jumps. 53-01-03: In sympathetic neurones, M-current plays a major role in spike adaptationt phenotypes: synaptic suppression of M-current (following application of muscarinic-cholinergic agonists) underlies 'slow' and 'late slow' excitatory fost-synaptic potentials (slow EPPs). Induction of slow EPPs reduces adaptation and permits prolonged, repetitive firing of action potentials, for example in sympathetic ganglia. The localization of M-channels suggests an important role coupling synaptically released transmitters (acetylcholine for example) to increased excitability. M-current is transiently augmented following removal of receptor agonist in frog sympathetic neurones, a phenomenon termed 'over-recovery'· 'Over-recovery' is [Ca2+h dependent and a similar phenomenon has been shown following removal of agonists that

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induce suppression of voltage-gated calcium currents (for details, see Protein phosphorylation, 53-32, Single-channel data, 53-41 and Channel modulation, 53-44). Note also that M-current augmentation by coupling to ,a-adrenergic receptors has been reported (see Receptor/transducer interactions, 53-49). 53-01-04: Although it has been described as 'widely distributed' in mammalian

sympathetic and central neurones, M-channels do not appear to be ubiquitous. M-current is particularly well-characterized in sympathetic and parasympathetic ganglia of frog and rat, coeliac neurones, rat hippocampus (CAl pyramidal neurones), olfactory cortical neurones, NGI08-15 differentiated mouse neuroblastoma x rat glioma hybrid cells, gastric smooth muscle cells, pituitary lactotrophs, and rod photoreceptor inner segments (for predicted physiological roles of 1M in these tissues, see Phenotypic expression, 53-14). Based on their response to a maintained depolarizing current stimulus, neurones in rat pre-vertebral and paravertebral sympathetic ganglia can be classified as 'phasic' or 'tonic' neurones. Notably, M-current has been characterized as present in all phasic neurones, but is 'very weak' or absent in 'tonic' neuron~ -53-01-05: Some studies have proposed that calcium-dependent phosphorylation/

dephosphorylation cycles determine macroscopic M-channel amplitudes in bullfrog sympathetic neurones by controlling transitions between gating modes 1 and 2 (for illustration, see Fig. 1, under Protein phosphorylation, 53-32 and Single-channel data, 53-41). Agonists such as muscarine appear to decrease M-channel activity by selective reduction of the long open time, high open probability gating mode 2 (ibid.). At moderate [Ca2+h (50-150nM) calciumdependent phosphorylation appears to promote gating mode 2 (ibid.), producing characteristic macroscopic M-current relaxations t . At higher [Ca2 + h (>200 nM) calcium-dependent dephosphorylation of M··channels is proposed to increase the short open time, low open probability gating mode 1 activity. The calcium/ calmodulin-dependent phosphatase calcineurin (protein phosphatase 2B, see Related sources and reviews, 53-56) has been implicated as a candidate dephosphorylating activity in M-channel responses. In bullfrog sympathetic neurones, a candidate kinase promoting nl0de 2 gating behaviour is myosin light chain kinase (ibid.). Independent proposals for 'tonic regulation' of M-channels by variations in resting intracellular [Ca2+] (i.e. without any role for a dephosphorylating activity) have appeared (see below). 53-01-06: The 'precise' mechanisms modulating M-channels in vivo are still unclear (see Channel modulation, 53-44)., Notably, additional observations

suggest that activation of calcineurin (see above) does not mediate receptorcoupled suppression of M-current (see Protein phosphorylation, 53-32). Furthermore, although several receptors that couple to phospholipase C and the production of InsPa and diacylglycerol may inhibit K M (see ILG Ca InsP;j, entry 19), the agonist-induced suppression of M-current in frog sympathetic neurones has been reported to be independent of the phospholipase C second messenger cascade. Much work has been done on specific modulatory mechanisms of M-currents (mostly in sympathetic neurones and NGI08-15 cells). In many of these studies (particularly those involving arachidonate and its metabolites and calcium ions) results have shown lack of consensus

l_e_n_t_ry_S_3

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between studies or have otherwise had conflicting conclusions (or are incomparable due to different recording conditions or lack of controls). Several of these apparently disparate observations could be due to cell-type specificity and consequentially, effector-channel heterogeneity. Breakdowns of M-current modulator and agonist (receptor ligand) actions (with any special conditions or caveats for their interpretation) are tabulated in the fields Channel modulation, 53-44 and Receptor/transducer interactions, 53-49. 53-01-07: Much collated evidence indicates that M-current suppression involves diffusible (probably cytoplasmic) messenger(s) and there is general agreement on this, although not about its/their identity(ies), which remain unknown (uncertain) at the time of compilation (see Protein phosphorylation, 53-32, Rundown, 53-39, Channel modulation, 53-44 and Receptor/transducer interactions, 53-49). However, some authors have proposed that intracellular Ca2+ ions may fulfil all of the roles of the 'M-current messenger', based on reductions in M-channel open probability following application of Ca2+ ions to excised inside-out patches of rat sympathetic neurones (see Table 3 under Channel modulation, 53-44). Since this effect occurred in the absence of ATP, it further suggested independence from protein phosphorylation/dephosphorylation cycles (cf. this field, above) and was used as the basis for proposing a role for Ca2 + ions as a 'direct' inhibitor of M-channels following receptor activation (for further discussion and a perspective on these results, see Table 3). 53-01-08: While the 'molecular identities' of M-channel effectors presently remain as candidates (see Cloning resource, 53-10) some progress has been made defining the likely G protein transducers in the muscarinic (Md receptor and bradykinin (B2 ) receptor responses in rat ganglia (Goq /11, which also activates phospholipase C). Although several exogenous agents can suppress M-currents in a receptor-independent manner (e.g. phorbol esters, see discussion under Protein phosphorylation, 53-32), there are no well-characterized, highly selective pore blockers available for M-channels. M-channels are sensitive to block by external barium ions (e.g. K i == 300 ~M in sympathetic ganglion cells); external Ba2+ shows a preferential block of outward current (for details, see Blockers, 53-43).

Category (sortcode) 53-02-01: VLG K M-i [native]. i.e. Voltage-gated K+ channels (principally) inhibited by muscarinic agonists in native cells (for full receptor range, see Receptor/transducer interactions, 53-49). The tnon-inactivating' channel cDNAs aKv5. 1 and r-eag are candidates for M-channel effectors, as described under Cloning resource, 53-10. However, since no structural 'identity' has been confirmed to date, most properties described in this entry are limited to descriptions of native cell preparations and many are subject to reinterpretation. Although KM channels pass outward 'delayed rectifier-type' currents, their distinctive regulatory properties (ibid.) have not been reproduced in heterologous expression systems with any of the presently known 'delayed rectifier-type' channels formed from Kvl to Kv4 gene products (entries 48 to 51 inclusive).

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Information sorting/retrieval aided by designated gene product nomenclatures 53-02-02: Should the gene(s) encoding the KM channel be cloned, a gene product (information retrieval) prefix (Unique Embedded Identifier or VEl) will 'tag' new articles of relevance to the contents of this entry in the CSN pages according to convention (see this field in other entries for details).

Channel designation 53-03-01: Channel proteins with regulatory and kinetic properties similar to the 'classically defined' 'M-channel' (see Abstract/general introduction, 5301 and Phenotypic expression, 53-14) have been designated as K(M)1 K M or K(M). Since the 'molecular basis' or subunit composition of channels underlying M-current is presently unclear, the generic term 'M-channel' is used in this entry. The designation Kx has been used for channels in the inner segment of rod photoreceptors which share the kinetic properties of the M-current observed in other cell types (see Phenotypic expression, 53-14).

Current designation 53-04-01: Usually designated as IK(M), I K .M or 1M . The designation I Kx , for the '1M -like' voltage-dependent K+ current in photoreceptors (see crossreferences in Phenotypic expression, 53-14). The designation IKM,ng has been used for M-current in differentiated NGI08-15 mouse neuroblastoma x rat glioma hybrid cells 1 (see Blockers, 53-43 and Channel modulation, 53-44). As outlined in the entry, 1M is generally designated as meeting criteria such as being a non-inactivating time- and voltage-dependent outward current which is suppressed by muscarinic agonists (e.g. oxotremorine> muscarine > bethanechol); 1M activates at membrane potentials more positive than -60 mV and is slowly turned off when the membrane is hyperpolarized back to -60mV (data for 1M in acutely dispersed coeliac-superior mesenteric ganglia (C-SMG) from adult rat2 ). An extensive comparative study of ion permeation, conduction and blocking properties of native M-current (1M ) versus delayed rectifier (I DR ) in isolated bullfrog sympathetic neurones has appeared3 .

Gene family Similarities and distinctions between K M and cloned K+ channels 53-05-01: Comparative note: The gene family relationships for gene(s)

encoding M-channel protein(s) are presently unknown (but see Cloning resource, 53-10 for candidates). From studies conducted in native cells (this entry), there are some grounds for expecting some structural distinctions between presently known K+ channels and those of M-channels. For example, several conduction properties of M-channels (summarized under Selectivity, 53-40) predict some novel structural features of M-channels that are not seen in the Kvl to Kv4 gene subfamilies. For similarities of KM to novel 'non-inactivating' channels cloned from Aplysia, see Cloning resource, 53-10. Further distinctions between 'classical' 1M , I K and IA

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components are outlined/cross-referenced under Activation, 53-33. For example, there is a notable pattern of resistance to 'classical' K+ channel blockers (see Blockers, 53-43). M-channels also exhibit 'hallmark' patterns of modal gating (see Protein phosphorylation, 53-32 and Single-channel data, 53-41) and receptor-coupled modulation (see Receptor/transducer interactions, 53-49). Many other examples of native K+ channel inhibition coupled to ligand activation of 'separate' receptors exist, including that of ATP-inhibited K+ channels (KATP , described under INR K ATP-i [native], entry 30). In this case, functional 'reconstitution' of the native I KATP has been achieved following co-expression of an inward rectifier subunit (Kir 6.2, see INR K [subunits], entry 33) and the sulphonylurea receptor4 . Note that extracellular ATP has been reported to suppress (i) macroscopic McurrentS and (ii) the I KM .ng component in NCI08-IS cells following P2 receptor activation (see Receptor/transducer interactions, 53-49). Intracellular ATP can augment K M activity by promoting mode 2 gating behaviour (e.g. see Fig. 1 under Protein phosphorylation, 53-32).

Trivial names Generic tM' nomenclature denoting a wide range of specific receptor-channel interactions 53-07-01: The terms 'M-current' or 'M-channel' (used on the basis of suppression following muscarinic (M) receptor stimulation) has also been used to describe channel effectors coupled to receptors other than muscarinic types (for examples of these, see Receptor/transducer interactions, 53-49).

EXPRESSION

Cell-type expression index Original description and twell-characterized' preparations 53-08-01: The 'M-current' was originally identified in bullfrog sympathetic neurones 6 as a potassium current that is suppressed by the cholinergic t agonist muscarine. Although it has been described as 'widely distributed' in mammalian sympathetic t and central neurones, M-channels do not appear to be ubiquitous. As described elsewhere in the entry, M-current is particularly well-characterized in sympathetic and parasympathetic 7 ganglia of frog, rat superior cervical ganglion (SeC) neurones, guinea-pig coeliac neurones, rat hippocampus (CAl pyramidal neurones), olfactory cortical neurones8 , cultured spinal cord neurones 9 , NG108-15 differentiated mouse neuroblastoma x rat glioma hybrid cells1 (a 'neurone-like' cell line model expressing the '1M -like' conductance designated as IKM,ng 1 - see Channel modulation, 53-44), toad gastric smooth muscle cells ('1M -like'), pituitary lactotrophs, rat ('non-neuronal, M-like current')10 and rod photoreceptor inner segments (I KX , '1M -like', see Phenotypic expression, 53-14). For more detailed tissue distributions, see the references in Related sources and reviews, 53-56 (see also next paragraph).

II

_ entry 53 - - - - - - -

Comparative note on K+ channel mRNA analyses in sympathetic ganglia 53-08-02: A quantitative expression analysis of 18 different KVQ and Kv{3 subunit mRNAs in rat sympathetic ganglia has been made using an RNAase protection assayll. Eleven Q subunit genes and two {3 subunit genes were found to be expressed in sympathetic ganglia by this method, with evidence for differential expression (between the superior cervical, coeliac and superior mesenteric ganglia) being obtained for Kv{31, KVQl.2, KVQl.4 and KVQ2.2 11 . Notably, none of these distributions 'matched' those of the M-current, which is prominent in sympathetic neurones (this entry).

Cloning resource 53-10-01: Two candidates for M-channel-related cDNAs have appeared: First, when expressed in Xenopus oocytes, the 'non-inactivating' K+ channel aKv5.1 from Aplysia 12 (briefly described in VLG K Kvx, entry 52) has a similar activation slope to KM, activates from -60mV and can therefore contribute to the resting potential and firing patterns of neurones. Secondly, the rat eag-candidacy for an M-channel was mentioned in the perspective by Hille13 and discussed in the original paper14 . r-eag produces a non-inactivating K+ current that is suppressed by muscarine in HEK293 cells (see note). Further to this, two commentaries148,149 have specifically discussed the resemblance between the Drosophila eag current and the mammalian M-current. Note: Initial comparisons between r-eag and M-current (e.g. selectivity, channell current tissue distributions, pharmacology) did not show exact concordance at the time of compilation, and hence have been omitted. Further properties of r-eag are described under VLG K eag/elk/erg, entry 46. 53-10-02: Predicted 'close associations' of 'the K M channel' and kinase, phosphatase or anchor proteins of known sequence (for example, see Protein interactions, 53-31 and Protein phosphorylation, 53-32) could conceivably offer a potential cloning route from eNS cDNA libraries, either by employment of protein immunoaffinityt methods or yeast 2-hybridl technology (for technical background, see Resource D - Diagnostic tests).

Developmental regulation tDevelopmental consequences' of receptor-regulated KM channel activities are unknown 53-11-01: Although largely undocumented, receptor control of neuronal excitabilityt by M-current modulation may be expected to have regulatory roles in 'long-term' developmental expression phenotypes (e.g. involving patterns of de novo gene expression/suppression) in addition to 'short-term' adaptive effects (see Phenotypic expression, 53-14 and Receptor/transducer interactions, 53-49). The 'developmental consequences' of neuropeptide receptor stimulation affecting KM channel gating are largely unknown, but several of these affect neurosecretory and hormone-release phenotypes of significance in brain development {for further background, see the Developmental regulation fields of several ILG series entries (Volume II) in particular those of ILG Ca InsPJ, 19-11 and ILG K Ca, 27-11.

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Growth conditions affecting neuroblastoma cell morphology and selective expression of 1M 53-11-02: Differentiatedt mouse neuroblastoma x rat glioma (NGI08-15) hybrid cells exhibit both a 'classical' delayed rectifier potassium current (IK ) and an M-current-like component (IKM .ng ). Experimentally defined growth conditions have been shown to markedly affect cell morphology and electrical phenotype of these cells15 . NGI08-IS cells transfected with Ml muscarinic receptors grown with I % foetal bovine serum in the absence of PGE 1 (prostaglandin El ) and IBMX (isobutylmethylxanthine - see note 1) show abolition of the usual pleomorphism, leaving two populations of small cells with (i) stellate morphology and (ii) spherically symmetrical geometries. Whole-cell patch-clamp studies indicate that the two cell morphologies have identical electrophysiological properties, displaying I K , a small current through a 'T-like' Ca2 + channel, but no detectable M-current. Stimulation with the muscarinic agonist carbachol can shift the distribution of cells to a 'more stellate morphology' (within 24 hours) and later 'downregulate' 80K/MARCKSt by 22 ± 70/0 (after 48 hours, see note 2)15. Notes: 1. PGEl and IBMX are usually added in preparation for electrophysiological studies of NGI08-IS cells. 2. MARCKS is an acronym for myristoylated, alanine-rich C-kinase substrate (also known as 87K, pp80). MARCKS is a prominent calmodulin-binding PKC substrate. K+ -induced depolarization of rat hippocampal slices results in significant phosphorylation of this and other PKC substrates (see, for example ref16. and Resource G - Consensus sites and motifs).

Phenotypic expression General phenotypic functions/characteristics of M-channels 53-14-01: Open M-channels provide an outward, K+ -selective (hyperpolarizing) current under 'resting' conditions (i.e. in the absence of receptor agonists that induce its suppression) and thus plays an important role in stabilization of cell excitabilityf (see below). Suppresion of M-current following activation of several G protein-linked receptors (see Receptor/transducer interactions, 53-49) results in membrane depolarization and an increase in membrane input resistance (i.e. making it more likely that a cell will fire action potentialst). When neurones are depolarized (i.e. towards the 'threshold for firing' action potentials) 'slow activation' of M-channels tend to hyperpolarize the cell membrane back towards rest (see also Activation, 53-33). In the absence of agonist-induced inhibition, M-channels do not inactivatet under maintained depolarization, and thus are often described as being 'tonically active', remaining 'steadily activated' or 'persistent' at potentials positive to -70mV6 . Upon membrane hyperpolarization, M-channels are deactivatedt ('turned off'), exhibiting slow relaxationsf in response to voltage jumps (see also legend to Fig. 3, under Blockers, 53-43).

tClassical' observations defining M-current function in sympathetic neUlones 53-14-02: M-current plays a major role in spike adaptationt phenotypes. Synaptic suppression of M-current following application of muscarinic-cholinergic

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agonists underlies 'slow' and 'late slow' excitatory post-synaptic potentials (slow EPPs) in sympathetic neurones17. Induction of slow EPPs reduces adaptationt and permits prolonged, repetitive firing of action potentials, for example in sympathetic ganglia18.

tOver-recovery' phenotypes in sympathetic neurones 53-14-03: M-current is transiently augmented following removal of receptor agonist in frog sympathetic neurones, a phenomenon termed 'over-recovery,t19. 'Over-recovery' is [Ca2+ h-dependent and a similar phenomenon has been shown following removal of agonists that induce suppression of voltage-gated calcium currents (for details, see Protein phosphorylation, 53-32 and Single-channel data, 53-41).

Phenotypic roles of M-like K+ channels in rod photoreceptor inner segments 53-14-04: An 'M-channel-like' activity designated as Kx in has been characterized within the inner segment of salamander rod photoreceptors20- 22 (for background, see Fig. 2 under Phenotypic expression of ILG CAT cGMp, 22-14). IKx has likely roles in (i) setting the dark resting potential; (ii) shaping small photovoltages (i.e. 'accelerating' rod responses to light of low intensity)21 and (iii) sensory adaptation: In rod cells, I Kx has been described as a 'standing' outward current (of about 40 pA at -30mV in the dark) that deactivates t slowly (Tmax == 0.25 S)21 following light-induced hyperpolarization (due to cGMP-gated channel closure - (see ILG CAT cGMp, entry 22). As IKx falls, Ih channels (described under INR K/Na IfhQ, entry 34) tend to open (particularly with large hyperpolarizations). Thus both Kx (closures) and hchannel (openings) tend to oppose or 'damp-out' the strong hyperpolarizing effects of light. Furthermore, since Kx and h-channels have relaxation times in the order of 100-200ms (at 22°C) they reduce the photoreceptor voltage response over this time course. These and other mechanisms of neural processing in the photoreceptor inner segment (including the role of Kx channels) have been reviewed22 . Note: Although the voltage and time dependences of IKx are similar to M-current, IKx can be distinguished from M-current because it is not suppressed by acetylcholine and external Ba2+ lblock' shifts the activation range of I Kx strongly in the positive direction21 (for further details, see Blockers, 53-43).

M-channels in other cell types 53-14-05: 1. Suppression of M-current by synaptic activation may underlie seizure-generation activity in cortical neurones23 . 2. 'KM -like' channel responses have also been characterized in gastric smooth muscle cells (see Receptor/transducer interactions, 53-49). 3. Changes in 1M -like expression coincident with phenotypic changes in neuroblastoma cell morphology are described under Developmental regulation, 53-11.

Functional distinctions between receptor-coupled KM and KCa channels 53-14-06: The opening of any K+ channel with depolarization tends to limit the depolarization itself, and in this regard, 1M has been referred to as a

1'--_e_n_t_ry_5_3

_

lbraking' current, effectively bringing action potential firing frequency under 'receptor control' (see previous paragraphs and Receptor/transducer interactions, 53-49). This type of function is broadly similar to receptormediated reductions in calcium-activated K+ currents (for details, see ILG K Ca, entry 27). Although KCa inhibition may occur over a wide range of membrane potentials, M-current inhibition can only occur when the channels are open (i.e. by depolarization from about -60 m V). In bullfrog sympathetic ganglion cells, muscarine reduces both I K.M and IK.Ca, while in other cells (e.g. hippocampal pyramidal neurones) approx 10-fold greater concentrations of muscarinic agonist are required to suppress IK.ca than for reduction in I K.M • Differential transducer t /effector t coupling in different cell types may be able to account for these observations (see Receptor/ transducer interactions, 53-49).

Protein distribution 'Differential distribution' of M-channels by functional criteria 53-15-01: Based on their response to a maintained depolarizing current stimulus, neurones in rat pre-vertebral and paravertebral sympathetic ganglia can be classified as lphasic' or ltonic' neurones. Notably, M-current has been characterized as present in all phasic neurones, but is 'very weak' or absent in 'tonic' neurones24 . Computer models (based on voltage-clamp data), suggest that different firing properties of phasic and tonic neurones can be accounted for by differential expression of the M-current/channels24 .

STRUCTURE AND FUNCTIONS

Protein interactions Predicted close associations of K M channel and kinase/phosphatase proteins 53-31-01: Patterns of 'reciprocal' phosphomodulation of M-channel gating modes25 (see Protein phosphorylation, 53-32) predict native M-channels to be closely associated with enzymes regulating both channel dephosphorylation (e.g. calcineurin, protein phosphatase 2B) and phosphorylation (putatively MLCK, ibid.). Note: Approximately 50-700/0 of rat brain calcineurin is membrane associated26 and is known to bind to membraneassociated 'anchoring protein' which may 'target' calcineurin to its protein substrates, such as ion channels. Single classes of anchoring protein are known to associate with both calcineurin and protein kinase A27. Observations of K M activities in sympathetic neurone preparations (see Protein phosphorylation, 53-32) are consistent with both kinase and phosphatase activities remaining associated with membrane patches following excision.

Functional interactions involved in 'neural processing' within photoreceptor inner segments 53-31-02: In the photoreceptor inner segment, Kx channels (sharing kinetic properties of 'classical' M-channels) appear to shape responses to dim light

_'--

e_n_try_5_3_

and set the dark resting potential. For functional interactions of Kx channels with cation-selective, hyperpolarization-activated h-channels (described under JNR KINa JfhQ , entry 34) and cGMP-gated channels (described under JLG CATcGMp, entry 22) see ref.22 and Phenotypic expression, 53-14).

Protein phosphorylation Ca 2+ -dependent phosphorylation/dephosphorylation underlying K M gating mode transitions 53-32-01: Calcium-dependent phosphorylation/dephosphorylation cycles have been shown to determine macroscopic M-channel amplitudes in bullfrog sympathetic neurones by controlling transitions between gating modes 1 and 2 (for illustration, see Fig. 1, this field and Single-channel data, 53-41). Agonists such as muscarine decrease M-channel activity by selective reduction of the long open time, high open probability gating mode 2 (ibid., ref. 28, but see also note 3). At moderate [Ca2+h (50-150nM) calcium-dependent phosphorylation appears to promote gating mode 2 (ibid.), producing characteristic macroscopic M-current relaxations t. At higher [Ca2+Ji (>200nM) calcium-dependent dephosphorylation of Mchannels increases the short open time, low open probability gating mode 1 activity. The calcium/calmodulin-dependent phosphatase calcineurin (protein phosphatase 2B, see Related sources and reviews, 53-56) has been implicated as a candidate dephosphorylating activity in M-channel gating mode shifts25 (Fig. 1): Addition of a 'pre-activated' form of calcineurin (CaN420, see note 1) to whole-cell pipette solutions inhibits the macroscopic M-current in sympathetic neurones. In excised, inside-out patch recordings, this latter effect has been shown to be associated with a selective loss of mode 2 M-channel activitr5 (see note 2). Notes: 1. The pre-activated, calcium-independent form of rat brain calcineurin (CaN420 ) contains a stop codon at residue 420 which lacks an autoinhibitory domain t that normally binds both calcium and calmodulin29 " 2. The action of CaN420 can sometimes be reversed by the inclusion of ATP in whole-cell pipette solutions or by application to the intracellular face of excised patches. Long Popen states restored by ATP are, howeve.r, distinguishable from 'authentic' mode 2 behaviou~5. 3. Additional observations25 suggest that activation of calcineurin does not mediate receptor-coupled suppression of M-current. For example (i) cells dialysed with calcineurin autoinhibitory peptide (in the electrode solution) retain sensitivity to suppression by muscarine and (ii) the kinetic mechanism for effects of CaN420 appears different from that of muscarine (i.e. kinetic models predict effects of muscarine being due to selective reduction in mode 2 with no effect on mode 128, while CaN420 produces a large increase in transition rate between modes25 ). For further details on calcineurin affecting M-current 'rundown', see Rundown, 53-39.

MLCK: A candidate kinase promoting mode 2 gating behaviour of K M 53-32-02: The effects of ATP in 'restoring' CaN42o-inhibited M-current (see paragraph 53-32-01) take several minutes to plateau, consistent with the activation of an ATP-dependent kinase (of unknown identity, see Fig. 1, this field). This kinase does not appear to be (i) protein kinase A (as addition of

('l)

(b) Modal gating shifts by phosphorylation/dephosphorylation

(a) Suppression of M-current by agonists

(Comparative note: muscarine induces selective reduction of mode 2, see this field)

(diffusible messenger; presently unclear)

(mode 2) K

e.g for ACh and BK in rat ganglia

Pi

II

long open time high open probability

open time behaviour shows no apparent voltage-dependence

open time and voltage-dependence similar to macroscopic M-current deactivation

_--- (dephosphorylated) ----

. . -- I

Unknown 'inhibitory' messengers (for distinctions between possible mechanisms, see also Single-channel data, 53-41 and Channel modulation, 53-44).

short open time low open probability

K

diffusible

Elevated 'local' increases in intracellular [Ca]? For reported 'direct' inhibitory effects of calcium (independent of phosphoregulation) See Channel modulation, 53-44; see also ILG Ca InsP3, entry 19, ILG K Ca, entry 27 and VLG Ca, entry 42

Gating mode 2

Gating mode 1

\

~

'"

I

.

'Modest' elevations of Ca (50-150 nM) ~ M-current • 'Larger' increases (>200 nM) can inhibit M-current See Channel modulation, 53-44.

_

I?-

ATP- and Cadependent kinase

I

I

,

,/ Putatively, myosin

" II I I I I I I

",/

'1

Phosphorylation-dephosphorylation cycle

I·"

light-chain kinase (MLCK, see text)

controlling transition between gating modes and controlling M-current amplitUde

•

Figure 1. Proposed Ireciprocal' regulation of KM channel modal gating behaviour by Ca 2+ -dependent phosphorylationdephosphorylation cycles. For background, see paragraphs 53-32-01 and 33-32-02. Based on data from N.V. Marrion (1996) Neuron 16: 163-173.

::st"'t'" ~ CJ1 CJ.J

_ entry 53

- - - - - - cAMP analogues or forskolin do not affect macroscopic M-current in sympathetic neurones30,31) or (ii) protein kinase C (as activation of PKC by phorbol esters or diacylglycerol analogues inhibit the M-current31 - 35 (see this field, below). A candidate for this kinase is myosin light chain kinase (MLCK) and a 'pre-activated' form of MLCK enhances M-current in bullfrog sympathetic neurones36 . MLCK has been shown to be expressed in these neurones and application of MLCK inhibitory peptide or wortmannin suppresses both (i) agonist-induced 'over-recovery' (see Phenotypic expression, 53-14) and (ii) 1M when measured at a intracellular calcium concentration of 100 nM36,37. Mechanisms and calcium dependency for wortmannin inhibition of 1M were further refined in ref. 38 .

Comparative note: Protein phosphatase inhibitors without apparent effect on 1M 53-32-03: Inclusion of diphosphoglyceric acid (DPG, 1-2.5 mM, a phosphatase inhibitor) or alkaline phosphatase (100 Jlg/ml) fail to affect amplitude of muscarinic responses of 1M channels in frog sympathetic ganglion cells to muscarine39 (see also Channel modulation, 53-44 and Rundown, 53-39). The protein phosphatase 1 (PP1) inhibitors okadaic acid (1 JlM) and microcystin LR (200nM) also do not have any effect on macroscopic M-current in bullfrog sympathetic neurones25 .

The mechanisms mediating agonist-induced suppression of K M are presently unclear 53-32-04: Several receptors that couple to phospholipase C and the production of InsP3 and diacylglycerol may inhibit KM (see ILG Ca InsP3, entry 19). However the agonist-induced suppression of M-current in frog sympathetic neurones has been reported to be independent of the phospholipase C second messenger cascade32 . Furthermore, LHRH receptor activation has been shown to inhibit M-current in a PKC-independent manner19. In hippocampal pyramidal cells, agonists that stimulate phosphatidylinositol (PI) turnover (as well as direct injection of InsP3) reduce M-current. Protein kinase C activators have no effect however, indicating that modulation is Ca2+ independent in these cells40 (see also next paragraph}. A specific role for diacylglycerol (DAG) in M-channel regulation in toad gastric smooth muscle cells has been proposed by some authors 41 : In support of this, extracellular application of 1,2-dioctanoyl-sn-glycerol (DiCS, a synthetic DAG that is a potent activator of PKC), reversibly suppresses M-current in these cells41 . In this study, DiC8 suppressed endogenous and isoproterenol-induced M-current without altering the time course of M-current deactivation, suggesting that it acts by decreasing the number of channels available to be opened41 .

Inhibition of M-current by phorbol esters (as distinct from agonist pathways) 53-32-05: Non-selective activation of PKC by phorbol esters is known to suppress M-current in bullfrog sympathetic neurones. For example, PDBu (4-,B-phorbol 12,13-dibutyrate) irreversibly suppresses M-current in a concentration-dependent manner (Ki approx. 38 nM)34. Common treatments that induce PKC inhibition (e.g. pseudo-substrate peptides like PKCI 19-31),

l

e_n_t_ry_53

_

staurosporine and H-7 (1-(5-isoquinolinylsulphonyl)2-methylpiperazine, see note, below) all antagonize PDBu-mediated suppression of M-current34. Inhibitors such as these have variable effects in suppressing M-current coupled to endogenous receptor/transducer systems: For example, suppression of Mcurrent in bullfrog sympathetic neurones by muscarine and luteinizing hormone-releasing hormone (LHRH) is unaffected by PKCII9-31 and H-7, but is antagonized by staurosporine. Overall, this has suggested that suppression of M-current by agonists is probably not mediated by activation of PKC34 (see also previous paragraph). Notably, addition and subsequent removal of PDBu to M-current previously suppressed by muscarine prevents the action of PDBu (closing M-channels by voltage or Ba2+ block cannot prevent PDBu suppression). To reconcile these results, it has been further proposed that two 'interconvertible' populations of M-channels exist in bullfrog sympathetic neurones, one that is sensitive to both agonist and PDBu and another that can only be suppressed by agonist. This invokes a 'protective' effect of muscarine channel closure to subsequent effects of PDBu. Partial suppression of M-current (by low concentrations of muscarine) antagonize the response to PDBu, where the magnitude of 1M suppression is equivalent to that induced with PDBu alone34 . Notes: 1. Other studies have found that H-7 did not prevent responses to phorbol 12-myristate 13-acetate (PMA), suggesting a PKC-independent mechanism for PMA modulation42 . 2. PKCII9-31 peptide (an inhibitor of protein kinase C) inhibits phorbol ester and arachidonate-induced decreases of 1M .ng in NGI08-15 cells43 .

ELECTROPHYSIOLOGY

Activation M-current possesses a unique characteristic of sustained activation 53-33-01: M-current is voltage dependent and is slowly activated in the 'subthreshold' range for action potential initiation, Le. at potentials 'close to rest' or at hyperpolarized potentials (but generally, positive to -65 mV, see below). Due to its 'non-inactivating' nature, M-current has been frequently described as 'persistent' at slightly depolarized membrane potentials. Thus, hyperpolarization induced by 1M is frequently described as 'stabilizing' cell excitability (in the absence of receptor agonists that induce its suppression) by contributing most of the sustained membrane current in the 'subthreshold' range (for details, see Phenotypic expression, 53-14 and Receptor/transducer interactions, 53-49). In general, the rate of KM opening is relatively slow for small depolarizations (e.g. to -40 or -30mV) but may be faster for large depolarizations occurring during action potentials t or strong excitatory post-synaptic potentials t (see also Phenotypic expression, 53-14).

Distinctions between 1M, 1K and 1A 53-33-02: In addition to its sustained activation (see previous paragraph) Mcurrent in rat sympathetic neurones can be distinguished from other potassium currents by four features. (i) Its activation range (V 1/ 2 == -45 mV)44 is hyperpolarized compared to the delayed rectifier (V 1/ 2 == -6mV)45 and the

_'---

e_n_try_5_3----J

fast-inactivating A-current (V1/ 2 = -30mV)46 in the same preparation. (ii) Activation/deactivation kinetics are 'substantially slower' for KM than for other voltage-activated potassium currents (e.g. deactivation rate constants are approx. 10 times slower at equivalent potentials47). (iii) Although Mchannels have similar selectivity characteristics to other voltage-gated K+ channels, other pore properties, for example the lack of anomalous molefraction behaviourt and the effects of external Ba2+ ions are unusual (see ref. 48, summarized under Selectivity, 53-40 and Blockers1 53-43). (iv) Mchannels also exhibit 'hallmark' patterns of modal gating I proposed to be regulated in part by calcineurin (see Protein phosphorylation, 53-32 and Single-channel data, 53-41) and receptor-coupled modulation (see Receptor/ transducer interactions, 53-49). Other distinctions between M-channel and other K+ channel pharmacology are described under Blockers, 53-43. See also the Methodological note describing M-current 'isolation' protocols employed when 1M is co-expressed with 1K and 1A (Blockers, 53-43).

Different external cations do not affect deactivation kinetics of 1M 53-33-03: With the exception of Na+, the nature of the permeant ion appears to have little effect on the gating properties of M-channels, i.e. the voltagedependence of the deactivation time constant t is similar when recorded in 15 mM K+, TI+, Rb+ andNHt48 (see also Selectivity, 53-40). The time constant decreases e-fold for a 60-80mV hyperpolarization48,49. Comparative note: Deactivation kinetics of cloned Shaker channels50 and several native delayed rectifiers are slowed when Rb+ is the permeating ion, an effect proposed to be due to impedance of channel closing when the pore contains an ion51 . In M-channels, ions other than K+ apparently have longer pore residency times, but do not impede channel closing48 .

Current-voltage relation 53-35-01: 1M channels generally display non-rectifying 1- V relations.

Inactivation 53-37-01: M-current is characteristically non-inactivating, although it does undergo slow calcium-dependent 'rundown', possibly mediated by calcineurin (see Rundown, 53-39). Sustained M-current ultimately depends on the period of time that M-channels reside in the long open time, high open probability mode 2 (for details, see Protein phosphorylation, 53-32 and Single channel data, 53-41).

Kinetic model Open and shut states of the KM are voltage sensitive but only shut states are muscarine sensitive

53-38-01: A kinetic analysis identifying M-channel states t sensitive to muscarine and membrane potential has been presented for KM channels in the cell-attachedt, dissociated rat superior cervical ganglion neurone preparation52. In this analysis, M-channel activities recorded at 30 mV positive

1'--_e_n_t_ry_53

_

to the resting membrane potential level (-60 m V) show three shut times t (7s 1 == 8.0 ± 2.2ms; 7 s2 == 71.3 ± 8.6ms and 7s3 == 740 ± 220ms) and two open times t (70 1 == 10.6 ± 1.9ms and 7 0 2 == 59.3 ± 8.7ms). When bursts t of Mchannel openings were determined as those including 7 s 1, two exponential components are evident in burst duration distributions (7bl == 11.0 ± 0.9 ms and 7b2 == 80.4 ± 11.0 ms). Membrane hyperpolarization significantly lengthens all three shut times and shortens both open times. Hyperpolarization also enhances the relative contribution of lhigh-conductance M-channels' (see Single-channel data, 53-41) and decreases the relative contribution of low-conductance M-channels to overall activity. Application of 10 ~M muscarine outside the patch (see Receptor/transducer interactions, 53-49) lengthens all three M-channel shut times without significantly affecting their open times 52 . Note: A kinetic model which permits interpretation of how the distribution of M-channel modal gating may be affected by both phosphorylation and dephosphorylation is presented in ref. 25 (see also Fig. 1 and associated text under Protein phosphorylation, 53-32).

Models for 'over-recovery' phenomena 53-38-02: A model has appeared fitting observed relations between extent and rate of transient enhancement or 'over-recovery' of 1M responses following agonist removal in bullfrog sympathetic neurones 53 (see Channel modulation, 53-44). This equilibrium model can be extended to account for actions of arachidonic acid metabolism inhibitors in this preparation (ibid.).

M-current kinetics can be modelled to produce resonant behaviour in neurones 53-38-03: Neurones are able to generate voltage signals at a certain frequency (as a result of the subthreshold oscillations) and to 'preferentially respond' to inputs arriving at the same frequency (resonance behaviour characterized by a peak in impedance magnitude)54. To aid understanding of subthreshold behaviour and resonance, an isopotential membrane model of guinea-pig cortical neurones was developed and compared to experimental observations 54 . The model consists of a leak current, a fast 'persistent' sodium current and a slow non-inactivating potassium current. The kinetics of the M-current and membrane capacitance are sufficient to produce both voltage oscillations and resonant behaviour. The kinetics of the K+ current by itself is sufficient to produce resonance behaviour (typically observed at depolarized levels). Within the model, the Na+ current amplifies the peak impedance magnitude and is essential for the generation of subthreshold oscillation54.

Kinetics of G protein-mediated modulation of M-current 53-38-04: The time course of 1M suppression and recovery has been shown to 'closely follow' the agonist-dependent kinetics of nucleotide interaction with G proteins, thereby suggesting that subsequent messengers were unnecessary55. In this analysis, M-current inhibition rates displayed an exponential time course, 'hyperbolically dependent' on GTP analogue concentration with a limiting value of 0.53 min-I. Muscarine induces a 'concentration-dependent acceleration' of the rate of nucleotide-induced inhibition, with a plateau of about 20 min- 1 and an exponential time course.

_"--

e_nt_ry_5_3_

In control (untreated) neurones, 1M recovery rate (following agonist removal) is approx.3-7min- 1 •

Rundown M-current rundown resulting from activation of a Ca 2+ -dependent phosphatase 53-39-01: In voltage-clamped bullirog sympathetic neurones recorded using a deactivating protocol t (see Blockers, 53-43) M-current reactivation (within the first minute of clamping to -38 mV) is observed as an increase in current amplitude ('run_up/)25. This run-up is calcium dependent (see note 1). During 20 min continuous recording periods, this augmented M-current can be observed to run-down25 . Inclusion of CaN420, a truncated, pre-activated form of calcineurin in the whole-cell pipette solution (for background, see Protein phosphorylation, 53-32) inhibits M-current to a much greater extent (rv 55%) after 20 min recording than rundown observed in control cells. This effect of CaN420 can be blocked by adding the 25 aa calcineurin inhibitory peptide (CaN2S ) corresponding to the autoinhibitory domain of wild-type calcineurin. Furthermore, inclusion of CaN2S or the calcineurin inhibitor cyclosporin A results in (i) greater augmentation of M-current during the first minutes of recording and (ii) a reduction in the amount of current lost during rundown. These and other results 25 suggest that rundown results from activation of endogenous calcineurin. Notes: 1. 'Run-up' is absent in cells bathed in Ca2+free/Mn 2+ replacement Ringer solution25 . 2. Voltage-clamp of neurones at -38 mV causes an accumulation of intracellular calcium19,25 due to sustained calcium channel activation57 which may modulate M-current. 3. A mildly acidic intracellular pH reduces 'rundown' of M-current31 . For further details on Phosphorylation/dephosphorylation cycles affecting M-current activity, see Protein phosphorylation, 53-32.

ATP-,-S substantially reduces M-current 53-39-02: Under whole-cell recording conditions, the steady-state 1M in frog sympathetic ganglion cells is maintained 'for at least 20 min/ when the patch pipette contains neither ATP nor C~9 (compare ATP additions reversing the effect of CaN420 under Protein phosphorylation, 53-32). This study39 further reported that inclusion of ATP or cAMP or the ATP antagonist, ,8,,-methyleneATP (rv l-2nM) failed to alter the rate of 1M 'run down/. By contrast, inclusion of ATP..,-S (1 or 2lllM) resulted in a rv60% reduction of the current within 18 min. Despite the inability of ATP-,-S to maintain steady-state 1M , it had no effect on the ability of muscarine (2-100 IlM) to suppress a 'constant fraction' of the available current39. Independent studies of frog sympathetic neurones have shown that stimulation of G proteins with high GTP-,-S/GTP ratios (inducing a net accumulation of the active G* state) can produce faster rates of 1M inhibition (see also Channel modulation, 53-44).

ATP hydrolysis is required for desensitization of M-channel responses to substance P

53-39-03: Desensitizationt of M-channel responses to the agonist substance P (SP) has been studied in dissociated sympathetic neurones from bullfrog58 .

E

l_e_n_t_ry_53

_

When ATP (in the recording pipette) is replaced with AMP-PNP, SP still inhibits 1M , but no desensitization is observed, indicating that ATP hydrolysis is required for desensitization (see also notes 1 and 2). When low doses of muscarine (sufficient to inhibit 1M , but not to elicit desensitization), are applied simultaneously with a 'desensitizing dose' of SP, 1M remains depressed and does not desensitize 58. Notes: 1. Classical neurotransmitter receptor protein desensitization t may also account for 1M desensitization of inhibition. Desensitization mechanisms may thus be specific for different agonists. 2. Hypothetically, the enzyme(s) which mediate different forms of desensitization may be 'compartmentalized' in cells58 .

Selectivity Permeation versus conduction properties of M-channels 53-40-01: In rat superior cervical ganglion neurones, reversal potential t (Erev ) measurements for macroscopic 1M yield a relative ionic permeabilityt (Px/ PK ) series of Tl+ (1.5»K+ (1.0»Rb+ (0.8»Cs+ (0.2»NHt (O.l»>Na+ (0.008)48, under bi-ionic t conditions, with 150mM internal and 15mM external monovalent cation. Although the permeability sequence is very similar to other K+ -selective channels (implying a high degree of conservation in the selectivity filter) M-channels show a number of features atypical of potassium channels, as summarized in Table 1. Relative conductance t measurements estimated by the slope of instantaneous current-voltage relationships indicate a different selectivity sequence to that measured by Erev : gX/gK in 15 mM external cation was K+ (1.00) > Tl+ (0.28) > NHt (0.23) > Rb+ (0.11) > Cs+ (0.10). Collectively, these may be indicative of a distinct pore structure outside of the selectivity filter as compared to previously cloned K+ channels 48 (see also Table 1). Comparative note: In general terms, other K+ channels, both voltage-dependent and inward rectifiers ('native' and 'cloned') pass another ion (Tl+ or NHt) better than K+.

Single-channel data Single M-channels display non-stationary kinetic behaviour in sympathetic neurones 53-41-01: In bullfrog neurones bathed in isotonic K+ solutionst, KM displays conductance levels of approx. lOpS and 15pS where each level exhibits similar modal gating t (for details, see paragraph 53-41-02 and Table 2). Other estimates of KM channel conductances (reviewed in ref.28) exhibit some variance. For example (i) an M-channel conductance of 1-3pS was estimated by noise analysist using physiological Ringer'st solution5,47,66 and (ii) sustained, depolarization-activated M-current with higher conductances was reported in cell-attachedt recordings of dissociated rat superior cervical ganglion neurones (three main levels of open-channel conductance t approx. 7pS, 12pS and 19p5 in physiological Ringer's)52 (see also Kinetic model, 53-38). It has been noted, however, that under conditions where Goldman rectificationt is removed (i.e. by recording macroscopict M-current in isotonic K+ solutions) M-channel conductances are increased by four- to sixfold, yielding an estimated unitary conductance of between 8 and 18 pS

_'---

e_n_try_5_3_

Table 1. IAtypical' conduction properties of M-channels in rat sympathetic neurones48 (From 53-40-01) Feature

Summary/comparison with other K+ -selective channels (see note 1)

Conduction of monovalent cations relative to K+ is Ivery low'

53-40-02: All permeant monovalent cations are 'much less conductive' than K+ through the M-channel (see paragraph 53-40-01). These results appear consistent with the M-channel pore possessing a large and non-selective outer vestibule (Le. like other K+ channels). In M-channels, however, only K+ ions may be able to permeate quickly, with other ions being relatively 'retarded'48.

The nature of the permeant ion does not affect deactivation kinetics

53-40-03: The time course of M-current deactivationt induced by hyperpolarization is unaffected by different external cations. In comparison, several potassium currents exhibit a slowing of deactivation when Rb+ is used instead of K+ as the permeant ion (e.g. Shaker B ~6-4650, inward rectifiers in bovine pulmonary artery endothelial cells59 and other native K+ channels60 ).

M-current does not exhibit anomalous mole fraction behaviour

53-40-04: K+ channels exhibit an anomalous mole fraction t effect, suggesting that they are multi-ion pores t. In two independent studies of KM channels (in bullfrog sympathetic ganglion cells61 and rat sympathetic neurones 48 ) no evidence for anomalous mole fraction behaviour is apparent. In the latter study, titration of K+ with increasing fractions of Rb+ causes the relative conductance to decrease, but it does not exhibit a conductance minimum as expected for anomalous mole fraction behaviour. The absence of the expected increase in conductance (Le. upon complete replacement of one ion for another at some point in the titration) may be related to the low conduction of ions other than K+ thorugh KM channels (this table).

IPreferential' block of 53-40-05: M-current is blocked by low concentrations of Ba2 + ions 62,63 acting at external outward current by sites1 and exhibiting a 'preferential' block for external Ba 2+ ions (mechanisms unclear) outward current48 . The mechanism(s) of Ba2+ block of M-channels appears distinct from Ba2+ block in calcium-activated K+ channels, K+ -selective inward rectifiers and other voltage-gated K+ channels (for details see comparative notes under Blockers, 53-43).

entry53 I-

_ - - - - -

Table 1. Continued Feature

Summary/comparison with other K+ -selective channels (see note 1)

Cs+ and Na+ permeability (comparative note)

53-40-06: KM channels are 'moderately permeable' to

Predicted conservation of selectivity filter region (comparative note)

Cs+ ions and are 'slightly permeable' to Na+ ions (see paragraph 53-40-01) but are 'essentially impermeable' to NMDG. In comparison, Cs+ and Na+ are not permeant in (i) skeletal muscle BKca channels (although Cs+ is permeable to SKca channels in chromaffin cells - see ILC K Ca, entry 27); (ii) delayed rectifiers in myelinated nerve or T lymphocytes (see VLC K DR [native], entry 45) or (iii) starfish egg inward rectifier channels (see Selectivity under INR K [native], 32-40). 53-40-07: KM channels share the permeation sequence K+ > Rb+ > NHt > Cs+ with several cloned K+ channels belonging to distinct gene families (for example Shaker64, see VLC K Kv1-Shak, entry 48) and the renal K+ channel ROMK2 65 (see INR K [subunits], entry 33). Although these cloned channel types have different gating mechanisms and physiological functions, their conservation of the TXGYG motift (see Selectivity under VLC K Kv1-Shak, 48-40) might also predict that KM channels possess this motif.

Notes: 1. See also properties of novel 'non-inactivating' K+ channels cloned from Aplysia under Miscellaneous information, 53-55. (W. Gruner, cited in ref. 28). A 21 pS channel conductance with similar properties to KM recorded in bullfrog sympathetic neurones (ibid.) is consistent with conductance values extrapolated from noise analysis and the quantitative descriptions in ref. 28 . See also influence of [Ca 2+h on switching of gating modes (cross-referenced from paragraph 53-41-03, below) which may also help to account for the difficulties in resolving KM activities between studies.

Control of modal gating as a mechanism for M-current neuromodulation 53-41-02: Evidence for selective reduction of a single modet of M-channel gating following application of muscarine has been reported28 . M-channels in dissociated bullfrog sympathetic neurones can be resolved in the cellattachedt configuration into two conductance states, which exhibit appropriate voltage-dependent kinetics and two modes of gating: mode 1 and mode 2. 'Mode I' openings comprise short open time, low open probability events; open time behaviour of mode 1 has no apparent voltage dependence.

II

Table 2. Summary of properties derived from single-channel recordings of M-channels in bullfrog sympathetic neurones (From 53-41-01)

Property/characteristic/preparation

Description

Illustration

Amplitude classes NB Cell-attached configuration, symmetrical K+ (see notes 1 and 2)

Two amplitude classes of channels can be recorded with properties consistent with macroscopic M-current (see panel (a)). At hyperpolarized potentials (e.g. - 70 mV) fast channel events of two amplitudes are discernible. Membrane depolarization (e.g. to -30mV) evokes longer duration openings of both amplitude classes.

(a)

-30~

~.~~I,~~~r'J"

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,~' '~'Ir~~o

Il~ 40ms

Unitary 1- V relations and estimated conductances of the two amplitude classes in symmetrical K+ (see notes 1 and 4) Channel open probability versus membrane potential

Panel (b) is the 1-V relation for the same patch as in panel (a). For n == 10 records (mean ± SD), , == 14.7 ± 3.2 pS and 10 ± 1.5pS. Membrane depolarization increases the frequency of channel opening (Popen ) from 0.03 (at -70 m V) to 0.3 (at -30 m V).

(b) -1 ()()

V (mV) -80

·60

·40

·20

-200

~ Q)

·400

~

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~

a. E

g

-800

C Q)

~

-1000

~

CJ1

·600

ctS

(J

·1200

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Ensemble t current characteristics

Open state kinetics for M-channel conductance classes

Holding a cell-attached patch (exhibiting both conductance classes) at -80 mV and stepping to -50mV (500ms pulse duration every 5 s) produces a time-dependent increased inward current as the channel opens slowly during pulses (see note 3).

Open time analysis (all events, not shown) in amplitude and open duration histograms could be best described by the sum of two exponentials with time constants (TO of 9.2 and 3.1 ms)

("t)

I:' t'+

~ (Jl

UJ

~I

200 ms

Exclusion of either conductance class (lOpS or 16pS, see above) from this analysis results in distributions identical to those seen with all events, indicating the two conductance classes have identical open state kinetics (see also note 4).

Notes: 1. Pipette solution was 90mM KMeS04' 20mM KCI, 5mM MgCI2, 0.1 mM CaCI2, 5mM Na-HEPES (pH 7.2); cells perfused in this solution have an m.p. of OmV. 2. Openings are downward deflections in single-channel records. 3. Deactivation rates of these ensemble currents resemble M-current, increasing e-fold in 22mV of hyperpolarization, from 56.2± 15.8ms at -60mV to 8.7±3.4ms at -80mV (as compared with e-fold in 23mV for macroscopic M-current 5 ). 4. The lower conductance openings (lOpS, see above) and the higher-conductance openings (16pS, see above) are consistent with subconductance states of the M-channel (i.e they are unlikely to represent different channels28 ). 5. Data and figures reproduced with permission from Marrion (1993) Neuron 11: 77-84.

11

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_5_ 3

---l

'Mode 2' openings represent long open time, high open probability behaviour with open time and voltage dependence appropriate for that underlying Mcurrent deactivation5,28. Significantly, muscarine decreases M-channel activity by selective reduction of mode 2 through an unidentified Idiffusible' second messenger (see Protein phosphorylation, 53-32 but also Channel modulation, 53-44). Consistent with a diffusible messenger, KM channel activity can be reduced by application of agonists distal from patches of membrane containing K M channels. Notes: 1. In general, K M resides in both modes with the time spent in each varying between patches. When M-current is suppressed, its macroscopic kinetics do not change, consistent with the effect of muscarine being on the opening rate of the channel and not on the closing rate. The action of muscarine in reducing high Popen modal activity (by affecting the opening rate, see above) has the consequence that the closed times become longer. 2. Loss of mode 2 M-channel activity occurs within 1 min of patch excision25 (see also Rundown, 53-39). Application of ATP (lmM) can sometimes restore a long Popen state, but this state is distinFurther descriptions of M-channel guishable from mode 2 behaviou~5. unitary behaviour and gating modes are summarized and illustrated in Table 2. For the calcium-dependent phosphorylation/dephosphorylation mechanisms underlying transitions between gating modes, see Fig. 1 and associated text under Protein phosphorylation, 53-32.

Comparative notes: Modal gating in other channels

53-41-03: As discussed in ref. 28, several aspects of M-channel modal behaviour have similarities to both N- and L-type calcium currents (for further refs, see VLG Ca, entry 42). Reduction of mode 2 M-channel activity by muscarine (paragraph 53-41-02) is reminiscent of suppression of neuronal (N-)type Ca2+ current by noradrenaline, where similar reductions in long To, high Popen states have been described67. Modal gating changes can also account for the observed facilitation of N-type Ca2+ current that is evoked following removal of agonist 68 (compare M-channel lover-recovery,19 under Phenotypic expression, 53-14). Augmentation of cardiac (L-)type Ca2+ current by isoproterenol occurs by potentiation of high-activity (long To, high Popen ) gating modes of the L-channe169 . In comparison, augmentation of K M channel activities following removal of agonist19 can be mimicked by raising intracellular calcium levels 70 (since 'switching' of gating modes is influenced by [Ca2+h, 'low intracellular Ca2+' conditions may account for difficulties in maintaining the K M signal transduction pathway (for significance, see Fig. 1 under Protein phosphorylation, 53-32). Further examples of modal gating have been documented for (i) glutamate receptor-channels in locust muscle 71 (for background see the ELG CAT GLU series, entries 07 and DB); (ii) nicotinic acetylcholine receptorchannels 72 (see ELG CAT nAChR, entry 09); (iii) intracellular Ca2+-activated K+ channels (see ILG K Ca, entry 27); voltage-gated Na+ channels 74 (see VLG Na, entry 55) and possibly the S-channel l in sensory neurones of Aplysia 75 (see Miscellaneous information, 53-55). The functional diversity of these channel types suggests that modifications of modal gating behaviour by biological ligands is a significant mechanism for neuromodulation of channel function in vivo.

l"---e_n_t_ry_5_3

_

PHARMACOLOGY

Blockers M-channels are resistant to most tclassical' K+ channel blockers 53-43-01: There are no well-characterized, highly selective blockers available for M-channels. When applied to extracellular or electrode solutions at the stated concentrations, M-channel currents are 'relatively unaffected' by the blockers 4-aminopyridine (4-AP, 1 mM), caesium ions (2 mM), gadolinium ions (0.1 mM), 3,4-diaminopyridine (1 mM), zinc (0.1 mM) and d-tubocurarine (for further details, see ref.28 and Brown (1988) under Related sources and reviews, 53-56).

Effects of external Ba 2+ on M-current

53-43-02: M-channels are sensitive to block by external barium ions 62 (e.g. K i == 300 J.lM in sympathetic ganglion cells)63. Notably, external Ba2+ shows a preferential block of outward current48 : Although the muscarinic agonist oxotremorine-M (10 J.lM, see Receptor/transducer interactions, b3-49) has equal effects on both activation and deactivation relaxations (approx. 500/0), Ba2+ ions (1 mM) inhibit inward M-current by approx. 150/0 and outward M-current by approx. 500/0 48 (as illustrated in Fig. 2). Ba2+ block of M-channels does not appear to be voltage sensitive and does not significantly alter gating kinetics 48 . '1M -like' responses in some preparations are also weakly and non-selectively 'blocked' by Ba2+ ions (see the following paragraph).

Ba 2+ inhibition of the tIM-like' component I Kx in rod photoreceptor inner segments 53-43-03: The voltage-dependent K+ current I Kx in the inner segment of salamander rod photoreceptors (see Phenotypic expression, 53-14) is inhibited by external Ba2+ ions. Ba2 + ions are likely to interact with Kx channels at multiple sites, as indicated by distinguishable effects on (i) tail current voltage sensitivitYi (ii) voltage dependence and (iii) conductance properties2o . In (i), reductions in the voltage sensitivity of I Kx tail currents are induced by Ba2+ ions (with a Ko.s approx. 0.2 mM). In (ii), Ba2+ ions shift the voltage dependence over which I Kx appears (to more positive potentials, Ko. s approx. 2.4mM, see note 4). In high [K+]o (100mM) the voltage range of activation of I Kx is shifted 20 mV negative, as is the tau-voltage relation. In (iii), Ba2+ ions do reduce I Kx conductance but at low sensitivity (Ko. s approx. 76 mMi 'high [K+]o' does not prevent this effect, but does abolish barium's ability to affect voltage dependence and voltage sensitivity)2o. Notes: 1. Ca2+, Co 2+, Mn2+, Sr2+ and Zn2+ ions do not show comparable actions to Ba2+ on the voltage dependence or the voltage sensitivity of I Kx tail currents. 2. Ba2+ ions also alter apparent time courses of activation and deactivation of I Kx channels in a concentration-dependent manner, consistent with a slow but steeply voltage-dependent blocking and unblocking action. 3. These data can be accommodated in models which assume that Ba2+ has a voltageindependent and a voltage-dependent blocking action on open or closed I Kx , with the voltage-dependent component accounting for both (i) the reduction

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_5_3_

(a)

Ba

OxoM

=-------- .

---------. ~-------v

~~_-----o

1200 PA

1200 pA lOOms

(b)

lOOms

Outward current

Inward current 60

.g=

50

j

40

u

I

30

~

20

u

&

10

WOXOM

~OXOM

Ba

N4yJBa i

-120 -100

-80

-60

-40

i

i

-20

0

voltage (mY) Figure 2. Comparison of M-current suppression by external Ba 2+ ions and the muscarinic agonist oxotremorine M in rat sympathetic neurones. (a) Mcurrent deactivations were evoked by a 1 s voltage step from -70 to

-30mV Activation was elicited by 30mV depolarizations from -50mV (for full recording conditions suppressing IA and IK, see ref.48 and methodological note under Activation, 53-33). M-current suppression following application of external Ba2 + ions (1 mM, is larger for inward current (0) compared to outward current (e). Ba 2+ reduces the instantaneous current with relatively little effect on the time-dependent relaxations evoked by negative voltage steps. In comparison, oxotremorine M (10mM) has approximately equal effects on inward current (V) and outward current (~). (b) Effect of external Ba 2+ ions and oxotremorine M on M-current relaxations evoked by the protocol (avel'aged from 4 or 5 cells for each data point). (Reproduced with permission from Cloues and Marrion (1996) Biophys J 70: 806-12 (From 53-43-02).

in voltage sensitivity of IKx tail currents and (ii) the Ba2+ -induced shift in voltage dependence20 . 4. The strong 'positive shift' induced in the activation range of IKx by Ba2+ (and the absence of muscarinic suppression) distinguish IKx from I M 21 (for further comparison and roles of llO(, see Phenotypic expression, 53-14).

-----J_

lL....--e_n_t_ry_53

Inhibition properties of the 1M-like

IKM,ng

53-43-04: The current designated IKM,ng in differentiated NGI08-15 (mouse neuroblastoma x rat glioma hybrid) cells has been shown to be inhibited by external divalent cations, comprising both depression of the maximum conductance and a positive shift of the activation curve1. Inhibition by these ions persists in Ca2+-free solutions and addition of Ca2+ (10 J.1M free [Ca2+]) or Ba2 + (1 mM total [Ba2+]) to the pipette solution under whole-cell patchclamp does not significantly change IKM,ng (for significance, see Table 3 under Channel modulation, 53-44 and this field, above). In this study, cation inhibition of IKM,ng showed the following decreasing order of potency (with millimolar IC so values as denoted): Zn2+ (0.011) > Cu2+ (0.018) > Cd2+ (0.070) > Ni2+ (0.44) > Ba2+ (0.47) > Fe2+ (0.69) > Mn2+ (0.86) > Co2+ (0.92) > Ca2+ (5.6) > Mg2+ (16) > Sr2+ (33). La3+ ions do not inhibit IKM,ng at concentrations which inhibited lea in the same cells and organic Ca2+ channel blockers are ineffective. IKM,ng is reduced by 9-amino-1,2,3,4-tetrahydroacridine (ICso 8 J.1M) and quinine (30 J.1M) but is insensitive to tetraethylammonium (IC so > 30 mM), 4-aminopyridine (> 10 mM), apamin (>3 J.1M) or dendrotoxin (>100nM)1. See also Receptor/transducer interactions, 53-49.

Weak KM-inhibiting activity of an SKca blocker 53-43-05: Dequalinium, a bisquaternary compound used as a non-peptide SKca blocker (see Blockers under ILC K Ca, 27-43), produces a 18% inhibition (at 10 J.1M) of the M-current in rat sympathetic neurones 76 .

Methodological note: M-current tisolation' protocols 53-43-06: In native cells, M-current is frequently co-expressed with other voltage-dependent K+ currents, particularly the delayed rectifier and Acurrent types. Deactivating protocols in combination with blockers can be employed to isolate M-current components (see also legend to Fig. 3 and Rundown, 53-39). For example, whole-cell voltage-clampt can be applied at depolarized levels (e.g. -30 to -38 mY, where most other voltage-gated K+ currents are inactivated); M-current can then be revealed by its deactivation t (relaxation t ) following hyperpolarizing steps (typically in 10mV increments of 1 s duration) which produce slow increases in outward current. 'Contaminating' delayed rectifier components are often blocked with tetraethylammonium ions (TEA+, 10mM, see Blockers under VLC K DR [native], 45-43); except where TI+ is the external cation (Ki = 1 mM48 ). 4-Aminopyridine (4-AP, 10 mM, see Blockers under VLC K A-T [native], 44-43) may also be included to reduce both A-current46 and delayed rectifier components (Ki = 0.3 mM48 ). Notes: I. Tail current t through KM channels is sensitive to oxotremorine M (see Table 4 under Receptor/transducer interactions, 53-49). Generally, oxotremorine M-sensitive components should display the same deactivation kinetics t as non-subtracted tail currents. 2. Since M-current is suppressed by muscarinic agonists, most biophysical parameters are measured in their absence. 3. Preliminary evidence for expression of M-channels can be obtained by monitoring membrane potential (i.e. observing agonist-induced depolarizations that fail to reverse when the membrane is hyperpolarized). However it should be noted that this behaviour may also be expected from a decrease in a Kca conductance (in which the

II

_'---

.

e_n_try_5_3_

voltage-sensitivity results from the voltage sensitivity of the 'persistent' or 'activating' calcium current). Thus voltage recordings alone cannot distinguish between 1M suppression and general reductions in 'background' K+ conductances coupled with increases in cation conductance 77 (see also ILG K Ca, entry 27 and the KM/Kca comparative note under Phenotypic expression, 53-14).

Distinguishing

K1eak

and K M responses in pre-vertebral neurones

53-43-07: Muscarinic responses in guinea-pig coeliac neurones are produced by suppression of two K+ currents: the M-current and a muscarine-sensitive leak current. These components can be distinguished by their differential sensitivity to Cs+ and Ba2+ ions. Briefly, barium (2 mM) reduces the M-current and the leak K+ current, whereas caesium (2 mM) reduces the M-current but does not affect leak current (for further details, see ref, 78).

Channel modulation For collated evidence that M-current suppression involves diffusible messenger(s) see also Protein phosphorylation, 53-32, Rundown, 53-39 and Receptor/transducer interactions, 53-49.

[Ca 2+ Ji modulation of K M responses (cross-references)

53-44-01: Receptor agonists that suppress the M-current in bullfrog 7o,79 (but not rat80 ) sympathetic neurones simultaneously increase the intracellular calcium concentration (for background and further cross-references see Table 3, this field and Receptor/transducer interactions, 53-49). The effects of [Ca2 + h chelation of intracellular [Ca2 +- h following receptor stimulation can block the occurrence of 'over-recovery' (ref. 70, see Phenotypic expression, 53-14 and Fig. 3 and associated text under Protein phosphorylation, 53-32). Variable effects on 1M observed at different [Ca2+h may be able to be reconciled by considering alternative sites of protein modulation, e.g. (i) on the channel itself; (ii) on associated transducer or effector molecules; (iii) by coinvolvement of additional messenger molecules or (iv) a combination of these. Higher levels of [Ca2 +Ji may activate membrane-bound phosphatase activities associated with KM channels and responsible for selective reductions in mode 2 gating behaviour (ibid.). Low intracellular Ca2+ conditions may account for difficulties in maintaining the KM signal transduction pathway (see discussion

in paragraph 53-41-03).

Common

tIM

suppression-uncoupling' effects of decreased cellular

NAD+ 53-44-02: NGl08-l5 cells pre-treated for 5-15 h with 5 mM streptozotocin can prevent (or 'uncouple') suppression of M-current induced by several G proteinlinked receptor agonists 81 . This effect can be shown to prevent suppression of 1M following bath application of ATP (100 JlM), bradykinin (lOnM), angiotensin II (lOOnM), endothelin 1 (lOOnM) and acetylcholine (lOJlM, in an NGl08-l5 cell line stably expressing M1-muscarinic receptors). Notably, suppression properties through all receptor types are restored by simultaneous incubation with nicotinamide (5 mM), suggesting that signal transduction

II.-._e_n_t _ry_53

_

from these five different receptors to M-channels shares a common pathway requiring NAD+ 81 .

Arachidonate and its metabolites may non-selectively modulate K(M) responses 53-44-03: Modulation of M-current responses by arachidonic acid metabolites has been characterized in several preparations, including bullfrog sympathetic neurones, hippocampal neurones and NGI08-15 neuroblastoma x glioma hybrid cells (see paragraphs below). The multiplicity of proteins susceptible to lipophilic 'modulation' (described in ILG Ca AA-LTC4 [native), entry 15, and ILG K AA [native}, entry 26) can often 'confound' analysis of these responses. In consequence, some of the literature is confusing or inconsistent and descriptions below are limited to the 'overall' reported effects on KM-like responses (i.e. some observations may not have been 'reproducible' between independent studies and may even be considered 'epiphenomena' by some authors. Please use 'feedback' (field 57) to clarify and update 'consensus' properties wherever possible.

Evidence for leukotriene C4 augmentation of M-current in hippocampus 53-44-04: In rat hippocampal pyramidal neurones, somatostatins (SS, somatotatin-14 and -28) partly exert hyperpolarizing effects by augmentation of muscarinic-inhibited channels, a process that can be mimicked in slice preparations by exogenous application of arachidonate82. The action of both arachidonate and SS can be blocked by (i) the general lipoxygenase inhibitor nordihydroguaiaretic acid (see note 4) or (ii) the specific 5-lipoxygenase inhibitors 5,6-methanoleukotriene ~ methylester and 5,6-dehydroarachidonic acid. The 1M -augmentation response is insensitive to (i) the cyclooxygenase inhibitor indomethacin; (ii) PGE2; (iii) PGF2a; (iv) PGI2; (v) the 12-lipoxygenase product 12-hydroperoxyeicosatetraenoic acid or (vi) the 12-lipoxygenase inhibitor baicalein, suggesting the involvement of 5-lipoxygenase metabolite(s) (probably leukotriene C4 ) as a mediator in the process82,83 (see also ILG Ca LTC4 [native), entry 15). Notes: 1. The studies reported in ref. 82 also found evidence for a 'direct' activating effect of arachidonate on a hyperpolarizing K+ channel distinct from KM (see also ILG K AA [native), entry 26), which was most effective at at membrane potentials 'near rest' (as compared to LTC4 modulation of KM , which was most effective at 'slightly depolarized' potentials). 2. In ref. 83, the 1M -augmenting effects of somatostatin were abolished by the phospholipase A2 inhibitors quinacrine and 4-bromophenacyl bromide. 3. The augmenting/ inhibiting effects of somatostatin/acetylcholine have been taken as evidence for lreciprocal' regulation of KM by two different receptor/transducer systems in hippocampal neurones. 4. Nordihydroguaiaretic acid can also prevent 1M enhancement by arachidonate in bullfrog sympathetic neurones 53,54 (see also following paragraphs).

Prevention of somatostatin-induced 1M augmentation by a FLAP inhibitor 53-44-05: Comparative note: 5-Lipoxygenase-activating protein (FLAP, an

18 kDa integral membrane protein required, in peripheral cells, for the

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_5_3_

activation of 5-lipoxygenase, see paragraph 53-44-04) is expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus and brainstem. Highest levels of expression are observed in cerebellum and hippocampus, and co-localization of 5-lipoxygenase and FLAP has been shown in CAl pyramidal neurones 85 . Inhibition of FLAP with the compound MK-886 (0.25-1 JlM) can prevent the somatostatin-induced augmentation of the hippocampal K+ M-current85 .

Proposed roles of arachidonate in M-current tover-recovery'in bullfrog sympathetic neurones 53-44-06: Exogenous application of arachidonate enhances 1M of whole-cell voltage-clamped bullfrog sympathetic neurones in a 'dose-dependent and reversible manner' (ICso rv2.8 JlM)53. Application of muscarine or LHRH in this preparation shows an initial phase of M-current reduction, followed by a transient enhancement or lover-recovery' when agonists are removed. Notably, the extent of 'over-recovery' increases with the extent of the preceding inhibition, while the rate and degree of inhibition increases with the concentration of agonist. By contrast, the rate of recovery and the extent of 'over-recovery' are independent. Inhibitors of phospholipase A2 (quinacrine and bromophenacyl bromide, BPB) and a lipoxygenase inhibitor (nordihydroguaiaretic acid) prevent over-recovery without significantly affecting the rate or extent of 1M inhibition (compare previous paragraph and subsequent proposals for Ca 2 + -dependent mechanisms of tover-recovery' as described under Protein phosphorylation, 53-32). Notes: 1. The 'inhibitory' process (typical half-life rv21 s) and 'enhancement l process (typical half-life rv53 s) as described above required fast solution exchange techniques to distinguish them kinetically. 2. Application of muscarine can also inhibit A-type K+ channels in several preparations (e.g. ref. 84, see also Channel modulation under VLC K A-T, 44-44). In the bullfrog sympathetic neurone preparation53, the effect of PLA2 inhibitors (above) appears to prevent over-recovery by extending the inhibitory process half-life to >80 s (compare note 1), and is inconsistent with 'simultaneous' 1M enhancement and l A inhibition (see also Kinetic model, 53-38). 3. Further work86 in bullfrog sympathetic neurones has suggested that intracellular Ca2+ ions enhance 1M by stimulating the arachidonate metabolic pathway86 (see also Channel modulation, 53-44). In the latter studies, dephosphorylation was interpreted to upregulate 1M , while 'over-recovery' was interpreted as a result of stimulation of the lipoxygenase pathway and phosphatases by increased [Ca2+h 86 •

Membrane potential of NG10B-15 cells (set principally by 1M ) is modulated by arachidonate 53-44-07: Arachidonic acid (AA) affects membrane potentialt (EM) in NGI0815 neuroblastoma x glioma hybrid cells) in a dose-dependent manner87. At relatively high concentrations (25-50 JlM), AA first increases and later decreases a current (designated as Ih, see note below) flowing mainly through open M-channels (which holds EM at -30mV). At lower concentrations (5-10 JlM) AA only decreases Ih. AA also accelerates the gating t kinetics t of 1M (for further descriptions, see ref. 87). AA may exert its effects

l_e_n_t_ry_53

_

following internal accumulation or by depletion of eicosanoidt products (for background, see ILG K AA [native], entry 26). Non-specific increases in membrane fluidityt by AA may also explain some effects on 1M gating t kineticst seen in this preparation. Notes: 1. The designation Ih is most often used to describe certain members of the hyperpolarization-activated current family whose collective properties are listed under INR K/Na IfhQ, entry 34.

Other tmodulators' of M-channel responses 53-44-08: The specific mechanism(s) of M-current modulation may be dependent on cell type, or may be conditional due to 'indirect' effects (e.g. dependent on selective co-expression of specific receptor/transducer components (see Receptor/transducer interactions, 53-49). With these caveats, a number of other (mostly non-specific) putative 'modulatory' effects on KM-like responses have been noted, which are summarized in Table 3.

Receptor/transducer interactions For further data suggesting that diffusible messengers are involved in Mcurrent suppression, see also Protein phosphorylation, 53-32 and Channel modulation, 53-44.

Cell-type-selective receptor/transducer control mechanisms of 1M 53-49-01: M-current suppression patterns have mostly been described as consistent with involvement of a diffusible second messenger (i.e. channel activity can be reduced by application of receptor agonists outside nonexcised membrane patches (see also Table 3 under Channel modulation, 5344). For discussion of the formal possibility that G proteins interact directly with M-channels, see refs. ss,99 and Kinetic model, 53-38. 'Direct' G protein interactions with effector t channels are characterized with relatively fast agonist response times (see, for example, the entry INR K G/ACh [native], entry 31). In this regard, it is pertinent to note the relatively slow agonist response times observed for many native KM channels and to cite evidence for involvement of 'extrinsic' proteins which are likely to modulate KM channels in situ (see Protein interactions, 53-31 and cross-references therein). In bullfrog neurones, receptor agonists that suppress the M-current simultaneously increase the intracellular calcium concentration 70,79 (for further background, see ILG Ca Ca RyR-Caf, entry 17 and ILG Ca InsPJ, entry 19). Effects of elevated [Ca2 +h in control of M-current are summarized under Protein phosphorylation, 53-32 and Channel modulation, 53-44.

tObligate dependence' of K M regulation on co-expressed protein components 53-49-02: Receptor/transducer control of M-current is a significant pathway for regulation of excitability in sympathetic neurones 17,100. For example, suppression of 1M by neurotransmitters such as acetylcholine result in membrane depolarization and increased input resistance (making cells more likely to fire action potentials) (for further details, see Phenotypic expression, 53-14). Regulation of KM gating in sympathetic neurones is closely associated with kinase and phosphatase effectorst, which themselves could be under

II

Table 3. M-current response modulators (mostly non-specific, not necessarily proving direct effects on the KM protein or pertinent to a physiological regulation of the channel - see Protein phosphorylation, 53-32 and Receptor/transducer interactions, 53-49) (From 53-44-08) Putative modulators

Description/cross-references

Effects of intracellular calcium on KM (see also effects of Ca2+-dependent calcineurin and MLCK under Protein phosphorylation, 53-32).

53-44-09: M-current modulation by intracellular Ca2 + has been described as 'biphasic', where small increases in [Ca2+h enhance 1M (SO-lS0nM, comparable to [Ca2+h produced during action potentials). By contrast, larger, sustained increases in [Ca2+h (>200nM) tend to reduce the sensitivity of muscarinic response and inhibit 1M (for further details see

refs 37,7o,88-90).

ATP-dependent and ATP-independent effects

53-44-10: Maintenance of 1M at 'resting' calcium levels, and the enhancement of 1M by 'modest' rises in [Ca2 +Ji both require ATP in whole-cell pipette solutions 19,9o (for likely significance, see Protein phosphorylation, 53-32). Reductions in M-channel open probability following application of Ca2+ ions to excised inside-out patches of rat sympathetic neurones have been described91 . This effect occurs in the absence of ATP, suggesting independence from protein phosphorylation and proposes the role of Ca2+ ions as a 'direct' inhibitor of M-channels following receptor activation. Perspective on 'direct binding' Note: Whether (i) 'direct' Ca 2 + modulation; (ii) the alternative interpretation, of Ca 2 + acting by changing activity of modulatory enzymes (e.g. kinases/phosphatases, see Protein versus 'indirect Ca 2 + modulation' of M-channel phosphorylation, 53-32) or (iii) both was able to account for all M-current modulations was effectors unclear at the time of compilation. Certainly 'direct inhibitory' effects occur at {Ca 2 +h much greater than that seen following application of receptor agonists. Furthermore, no calcium rise is seen in rat superior cervical ganglion (SCG) sympathetic neurones with agonists. However, it may be argued (see ref.91) that the effects of Ca 2 + are local to the plasma membrane, and not measurable in the bulk of the cytoplasm. Induced {Ca 2+Ji rises that inhibit M-current also activate BKca channels (see ILG K Ca, entry 27) but BKca activation is not normally observed with receptor agonists that modulate M-current. As described elsewhere in this entry (e.g. field 32), small increases in internal calcium (50-150nM) appear to increase M-current by phosphorylation by an unknown kinase (possibly MLCK in bullfrog neurones) to promote high Popen channel activity. Larger {Ca 2 + h loads would be predicted to decrease M-current by activating calcineurin and promoting low Popen channel activity. Notably, however, these modulations may have little in common with the pathways that agonists use, which are still uncertain.

9 f""t

~ CJ1

(J.J

Proposed ttonic regulation' of M-channels by variations in resting intracellular [Ca 2+J

Conflicting reports on effects of Ca 2+ chelation tRequirement' for low concentrations of Ca 2+ for 1M suppression

II

53-44-11: In rat superior cervical sympathetic neurones, application of Ca2+ to the internal face of inside-out patches produces two forms of unitary M-channel inhibition151 : (i) a slow, all-or-nothing suppression of activity, enhanced by patch depolarization and (ii) a fast block associated with a concentration-dependent shortening of open times (Le. compatible with open-channel block, also enhanced by patch depolarization). In this study, both forms of block appeared independent of dephosphorylation events 151 (cf. Protein phosphorylation, 53-32). In cell-attached patch recordings; (i) M-channel activity increases during exposure of the cell to Ca2+ -free solution and (ii) is rapidly reduced on applying 2 mM Ca2+ to the extra-patch solution, observations which have been used to suggest a 'tonic' regulation of M-channels by [Ca2+Ji in these cells 151 . 53-44-12: Chelation of intracellular [Ca2 +] following receptor stimulation can block the occurrence of 'over-recovery,70 (see Phenotypic expression, 53-14 and Protein phosphorylation, 53-32). Some reports state that high concentrations of intracellular Ca2 + buffers such as BAPTA (e.g. 20 mM) block80,92 or do not block actions of muscarine19,70,90. In ref. 80, muscarinic stimulation of rat sympathetic neurones was able to suppress 1M without apparently raising [Ca2+Ji, however, 150/0). This was taken as injection of BAPTA (r-v11-12mM) reduced suppression of 1M (82~ evidence for a 'minimal [Ca 2+]j requirement' for 'continued operation' of the pathway coupling muscarinic receptors to M-type K+ channels in frog neurones.

Effects of Ca 2 + store releasing agents

53-44-13: Elevation of intracellular [Ca2 +] with caffeine (see ILG Ca Ca RyR-Caf, entry 17) reduces IKM,ng 1. Other methylxanthines (isobutyl-methylxanthine, theophylline) in the millimolar range also reduce 1M 23,93.

Ca 2+ -dependent potentiation Internal Ca 2+ and tover-recovery' phenomena

53-44-14: Other work 94 in cultured bullfrog sympathetic neurones has described a calcium-dependent potentiation of M-current, showing a hyperpolarizing displacement of the steady-statet activation curvet with high internal calcium (suggesting Ca2+ ions modulate kinetics of M-current, thereby regulating the number of M-channels being open at given membrane potentials 94 ). In a separate paper95, these authors proposed a similar shift in the M-current kinetics/activation curve 'caused' over-recovery phenomena (washout of 20 JlM muscarine inducing an hyperpolarizing shift of approx. 13 mY). For background to tover-recovery', see cross-references, this table, above.

(t)

='

t""I"

~ c.n

VJ

II

Table 3. Continued Putative modulators

Description/cross-references

Ca 2+ -dependent AHP Heparin prevents M-current over-recovery but not M-current suppression

53-44-15: A calcium-dependent after-hyperpolarization t (AHP) in dissociated bullfrog sympathetic neurones has been ascribed not only to an IAHP component (see ILG K Ca, entry 27) but also to a calcium-dependent potentiation of M-current150. Other studies have shown intracellular application of the inositol1,4,5-trisphosphate antagonist, heparin (150M (see ILG Ca InsPs, entry 19), has little or no effect on muscarine-induced M-current suppression whilst the 'over-recovery' phase of the response was markedly attenuated, supporting the interpretation that agonist-induced elevation of intracellular Ca2 + may account for M-current 'over-recovery'.

Effects of varying extracellular 53-44-16: Steady-state IKM,ng in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid calcium on IKM,ng cells is increased on removing external Ca2 + 1 . In the presence of external Ca2 +, reactivation of IM,ng (after a hyperpolarizing step) is delayed. The delay in reactivation is preceded by an inward Ca2+ current, and coincides with an increase in intracellular [Ca2+] as measured with indo-1 fluorescence. IKM,ng response inhibition by

calmodulin antagonists

53-44-17: In NG 108-15 neuroblastoma x glioma hybrid cells, I KM .ng responses are inhibited by the calmodulin antagonist compound W7 (N-( 6-aminohexyl)-5-chloro-l-naphthalenesulphonamide )93.

Alteration of KM responses by 53-44-18: For effects of intracellular ATP on M-current phosphomodulation, see Fig. 3 and ATP and nucleotide analogues associated text under Protein phosphorylation, 53-32 53-44-19: ATP-,-S and ,B,,-methylene-ATP increase the rise time and duration of the response of 1M channels to muscarine in frog sympathetic ganglion cells39 (see also Rundown, 53-39 and Protein Phosphorylation, 53-32).

Modulation of 1M by arachidonate and its metabolites

53-44-20: Several studies have described modulation of M-current responses by arachidonic acid and its metabolites (see separate paragraphs, this field; for general background, see also ILG Ca AA-LTC4 [native], entry 15 and ILG K AA [native], entry 26).

('l)

::s

~ CJ1

W

Comparative effects of ethanol on 1M related to electrical changes in hippocampus

53-44-21: Ethanol enhances muscarinic excitatory responses in rat hippocampal neurones in vivo and, like muscarinic agonists, reduces the M-current in these neurones in vitro. Although superfusion of relatively low concentrations of ethanol (11-22mM alone) has 'little effect' on stratum radiatum (SR)-evoked field potentials t in hippocampal slices (for background, see Receptor/transducer interactions, 53-49), these concentrations enhance (by 10-90%) both the depressions of evoked field potentials and depolarizations elicited by muscarinic agonists in this preparation (ibid., ref. 97). At higher concentrations (22-44 mM) ethanol also enhances amplitudes and duration of muscarinic slow excitatory post-synaptic potentials t (sEPSPs) recorded intracellularly in CAl and CA3 neurones 97. These latter effects require muscarinic receptor stimulation, and can be enhanced by eserine and blocked by atropine.

1M modulation by linopirdine,

53-44-22: It has been hypothesized that the augmentation of neurotransmitter release induced by linopirdine (DuP 996) in rat hipfocampal CAl pyramidal neurones in vitro may be due in part to 'block' of 1M in these neurones 9 . Note: There is no evidence for direct physical blockade (Le. pore occlusion) of M-channels by linopirdine.

a neurotransmitter release enhancer

'Modulation' of M-current 53-44-23: For references describing the modulatory/blocking effects of external Ba2+ ions on 1M and responses by external Ba2 + ions '1M -like' component l Kx (see Blockers, 53-43).

It

(t

::s

M'

~ CJ1

W

_ entry 53 '----------receptor regulation (see Protein phosphorylation, 53-32). Different 'combinations' of receptor/transducer/effector pathways may be able to account for reported differences in M-channel regulation. The 'precise' mechanisms involved in coupling neurotransmitter receptors to KM channels may have a cell type dependence, and several examples of these are described in Table 4.

Up-regulation of tKM-like' channels in gastric smooth muscle 53-49-28: In freshly dissociated gastric smooth muscle cells of the toad Bufo marinus, an '1M-like' current (see notes 1 and 2) can be activated ('upregulated') following isoproterenol (ISO) acting at ,a-adrenoceptors126. The M-current activation with ,a-adrenergic agonists can be mimicked by the cell-permeant analogue 8_bromo_cAMp126,127, suggesting that channel phosphorylation by protein kinase A may activate the M-channel. In keeping with a 'dual regulation' hypothesis126, muscarine or acetylcholine antagonizes the increase in 1M induced by adrenaline or CAMP128. Notes: 1. Some differences were noted126 between 'endogenous' M-current and the 'ISO-induced' M current, namely that the latter usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steadystate conductance/voltage relationship that was shifted (relatively) in the negative direction along the voltage axis. 2. The criteria used to identify the 'ISO-induced' component as 'M-current' included it being (i) outward and K+ -selective; (ii) 'suppressible' by muscarine or acetylcholine; (iii) remaining 'steadily activated' following depolarization and (iv) deactivatedt with hyperpolarization, exhibiting slow relaxations in response to voltage j umps126. 3. Substance P (see Table 4) also reduces the acetylcholine-sensitive KM-like conductance in freshly dissociated toad stomach smooth muscle cells 129,130.

Cell type selective effects of neuromodulators: 1M suppression in ganglia 53-49-29: The electrical effects of somatostatin-28 (SS-28 or 'whole somatostatin') and somatostatin-14 (SS-14 or 'cyclic somatostatin') on bullfrog paravertebral sympathetic (dorsal root) ganglion neurones are variable according to cell type 131,132. In C cells (relatively small cells of diameter ~20 JlM) muscarine produces hyperpolarization; SS-28 is also inhibitory and activates an inwardly entry 31); SS-14 is ineffective in rectifying K+ current, KG (see 1NR K G/A(~h, C cells. Note: In separate studies on C cells 133, substance P (0.1-1 JlM) was shown to inhibit an M-type potassiuln current while ATP (1-10 JlM) activated an Na+/K+ current (see 1h under 1NR K 1fhQ [native], entry 34 and also the comparative notes, below). In B cells (relatively large cells where muscarine produces excitatory effects), S8-14 is also excitatory and is more effective than SS-28 in suppressing 1M131 . Comparative notes: In A cells (~65 JlM in diameter) here ATP has been shown to inhibit M-current, substance P (0.1-1 JlM) also inhibited this potassium current without activating 1h133. A full analysis of the modulation and interactions between 1M and 1h in bullfrog sympathetic ganglia has appeared134, concluding that resting membrane conductance of these ganglion cells can be regulated by basal activities of calmodulin-dependent protein kinase (acting to increase amplitude of 1M, which was activated at potentials more positive than -65mV) and protein kinase A (acting to increase amplitude of 1h, which was

Table 4. Receptor/transducer and signalling pathways involved in M-channel regulation (From 53-49-02)

II

Receptor/transducer

Examples (non-exhaustive, notes and cross-references)

Receptors/ligands mediating 1M suppression (excitatory responses) Muscarinic Ml and M 3

53-49-03: In rat superior cervical ganglion (SCG) sympathetic neurones, muscarinic suppression of M current is slow, BAPTA sensitive, and mediated by receptors of the M 1 subtype (as distinct from the pertussis toxin-sensitive suppression of Ca2+ current in the same cells, mediated primarily by M 4 muscarinic receptors)101. For general effects of muscarinic agonists on hippocampal CAl field potentials, see separate paragraph, this field.

Note: 1M is sometimes designated 1KM .ng in NGI08-IS cells.

M 3 receptors couple 'most effectively' to 1M in transfected mouse NG108-15 models 53-49-04: Direct comparison of the ability of different muscarinic acetylcholine receptors (mAChR) to inhibit 1M , has been assayed in clones of NGI08-IS mouse neuroblastoma x rat glioma cells transfected cDNA encoding mAChRs M 1 -M4 using tight-seal, whole-cell patch clamp recordings. No significant inhibition of 1M is seen in non-transfected cells, or in M 2 or M 4 DNA-transfected cells 102 (for AChl mM; muscarine 100 J,1M). At maximally effective concentrations, ACh or muscarine produces complete inhibition of 1M in M 3 -transfected cells, but only partial (50-60%) inhibition in M 1 DNA-transfected cells 103 . --Comparative note: An earlier, detailed study of the mechanism of M-current inhibition by stably expressed muscarinic M 1 receptors in NGI08-IS cells suggested that activation of phospholipase C and inhibition of 1K .M represented parallel (Le. independent) pathway responses to acetylcholine104. This study also found that inhibitors of phospholipase A2 , lipoxygenase, cyclooxygenase or nitric oxide synthase (for example) had 'no significant effect' on ACh-induced inhibition of 1K .M (compare to other studies described under Channel modulation, 53-44). These findings may still be compatible with an interpretation that heterologously expressed receptors coupling to phospholipase C and the production of InsP3 and DAG can inhibit M-current. However, evidence that either InsP3 or DAG can mediate agonist-induced suppression of M-current is lacking.

P2U purinoceptors

53-49-05: Extracellular ATP suppresses macroscopic M-current100.

(l)

~

M'"

~ CJ1 ~

III

Table 4. Continued Receptor/transducer

Examples (non-exhaustive, notes and cross-references)

M-current inhibition following activation of P2 receptors in NG10B-15 cells

53-49-06: Phospholipase C-linked nucleotide receptors (probably of the P2U subtype, see below) activated by both UTP and ATP (100 JiM) inhibit I KM .ng by 44% (UTP) and 42% (ATP) in NGI08-15 cells 10s . Mean ICso values derived in these cells were: UTP: 0.77 ±0.27 JiM; ATP: 1.81 ±0.82J,1M. The order of nucleotide t and nucleoside t activities for M-current inhibition in NG108-1S cells (100 JiM agonist concentrations) was determined as UTP ==ATP > adenosine Sf -O-3-thiotriphosphate (ATP-,S)==inosine Sf-triphosphate (ITP) > 2-methylthio ATP (2-Me-S-ATP) > ADP==GTP» G:,,B-methylene ATP (AMP-CPP), adenosine 1 0 5 . Notes: 1. Inhibitory effects of the P2 agonists on M-current can not inhibited by (i) suramin (500 JiM) or (ii) pre-incubation with pertussis toxin (12 h in 500 ng/ml PTx). 2. M-current inhibition is frequently preceded by a Ca2+-activated transient outward current, responding to intracellular Ca 2+ release (see ILG K Ca, entry 27) but no effects on the delayed rectifier K+ current in this preparation were noted105 . 3. The nucleotide/nucleoside-sensitivity series for M-current inhibition is consistent with agonist sensitivities for PLC-coupled P2U receptors. cDNAs encoding P2U receptors have been isolated from from a NG108-1S cell cDNA library.

Substance P (SP, NK1)

53-49-07: Substance P (SP, 3 nM to 1 JiM) suppresses M-current in neurones of bullfrog dorsal root ganglia (DRG) by reducing the maximum M-conductance in the voltage range of -10 to -130mV106. Notes: 1. Neurokinin A (NKA) and neurokinin B (NKB) also produces the inward current in DRG cells with the rank order of agonist potency being NKA == SP » NKB. 2. SP also decreases a voltageindependent background K+ current (IKB , regulated by intracellular ATP, see also INR K ATP-i [native], entry 30) at a holding potential more negative than -60 m V. K+ current suppression/ decreased membrane conductance is associated with development of an inward current following application of Sp 106 . 53-49-08: In coeliac ganglion neurones, muscarine (ICso 3 JiM) and substance P (SP, ICso 100nM) cause depolarizations or inward currents (under voltage clamp) at the resting potential (-SSmV) associated with a decreased membrane conductance107. These changes have been correlated with 'block' of M-like current, slow IAHP and a K+ 'leak' component (but not an apamin-sensitive IAHP nor the IA 107.

Bradykinin

53-49-09: Bradykinin (BK) is a peptide mediator released during inflammatory processes that has a potent excitatory action in sympathetic neurones, partly due to its inhibitory effects on M-channels

(t)

l:j t'+

~ CJ1

(J.J

(EC so ~ 1.9 nM)108. The BK response is mediated by a G protein pathway similar to that activated by muscarinic acetylcholine receptors - Le. is selectively inhibited by microinjection of antibodies to Go (aq/ld and has been shown to involve the B2 receptor subtype108. In a separate study109, bradykinin (10 JlM) applied outside cell-attachedt patches of differentiated NGI08-15 cells was able to reversibly reduce steady-state ('in-patch') M-channel activity to 28.8 ± 6.1 % of control value (more potently than muscarine - cf. 38.4 ± 11.7% with muscarine)109. Inhibition in NGI08-1S cells was accompanied by a lengthening of channel shut times without significant change in open times or distribution of conductance levels 109. 53-49-10: Bradykinin produces 'dual' opposing but sequential effects on separate outward and inward (opposing) currents in voltage-clamped NG-I08 cells at clamp potentials between -60 and -30 mV (designated IBK(out) and IBK(in), respectively)110. IBK(in) results primarily from inhibition of the Ca2 +-independent 1M (IBK(out) results from activation of a Ca2 +-dependent, voltage-insensitive K+ conductance (see ILG K Ca, entry 27). The effect on IBK(in) is not replicated by a rise of intracellular Ca2 + and must therefore be generated by another mechanism 110. 53-49-11: In NGI08-1S cells, M current modulation by bradykinin appears to involve a PTx-sensitive G protein110-112. Protein kinase C (PKC) activation by phorbol esters causes suppression of I KM .ng in these cells l12 . Note that data compatible with PKC mediating part of the bradykinin effect in NGI08-1S have been published (focal application of O.I-S JlM bradykinin inhibits 1M by about 60%; SnM bradykinin inhibits by about 40%)113. Bradykinin can produce an additional suppression to that induced by phorbol dibutyrate (PDBu). 53-49-12: Inhibition of the M-current suppression in PC12 cells by bradykinin is mediated by a PTx-insensitive G protein; some steps in the second messenger cascade are modulated by Ca2 +152.

TRH

II

53-49-13: Thyrotrophin-releasing hormone (TRH) has been shown to suppress M-current in dissociated rat hippocampal CAl pyramidal neurone preparations l14 . Similarly, TRH inhibits an 'non-neuronal, M-like current' in normal rat pituitary lactotrophs; since this current begins to activate at ~ -60 m V it is likely to be active under the normal resting potentials of lactotrophs (-3S to -4S mV)10. Functional note: The TRH-induced increase in firing frequency is dependent on extracellular calcium and contributes to prolactin secretion.

(l)

=:s

M-

~ c.n VJ

II

Table 4. Continued Receptor/transducer

Examples (non-exhaustive, notes and cross-references)

LHRH

53-49-14: In rat superior cervical ganglion neurones, tLHRH (luteinizing-hormone releasing hormone) receptors activate a 'PLC/DAG/PKC' pathway (similar to that used by muscarinic M}/Ma- or substance Preceptor-mediated M-current suppression, see above). 53-49-15: In frog sympathetic neurones at concentrations that inhibit M-current, LHRH (and substance P) 'strongly reduce' N-type Ca2+ current but induce a leak conductance that may contribute to slow EPSPs (muscarine produces little reduction of Ca2+ current under these conditions, even in cells in which it strongly suppress the M-current)115.

Glutamergic agonists

Angiotensin II

See also endothelin-l under ICommon IIA1-suppressionuncoupling' effects of decreased cellular NAD+' under Channel modulation, 53-44.

Inhibition of 1M by metabotropic glutamatergic agonists 53-49-16: In addition to muscarinic cholinergic agonists, the metabotropic glutamatergic receptor agonist ACPD (I-aminocyclopentane-ls,3R-dicarboxylic acid) and appears to activate separate intracellular transduction pathways having 'convergent' inhibitory effects onto 1M and I AHP in basolateral amygdala pyramidal neurones of rat. These modulations result in membrane depolarization and reductions in the amplitude and duration of the slow AHpl16 (see also ILG K Ca, entry 27). 53-49-17: The octapeptide angiotensin II inhibits 'approx. SO%' of KM channels in cultured rat superior cervical ganglion (SCG) neurones without significant elevations in [Ca2 +Ji concentrationl17 (for earlier work first suggesting a common inhibition of KM by muscarine and angiotensin II in SCG, see refs 44,118). This inhibition appears to use a slow, second messenger-dependent, pertussis toxin-insensitive signalling pathway (as used by muscarinic agonists, possibly involving G q , see this table). By the same (apparent) pathway, angiotensin II also inhibits an N-type calcium channel via AT} receptors and this can be attenuated by inclusion of GDP-,B-S (2 mM) in the pipette. M-like current in NGIOS-IS cells is also suppressed by angiotensin II (see ref. 1, this table). Supplementary note: Angiotensin II is best characterized in the renin-angiotensin-aldosterone t system regulating blood pressure, but it also acts as a neurotransmitter in the central and peripheral nervous systems. For references to G protein coupling mechanisms and signalling functions of angiotensin II, see listings under Related sources and reviews, 53-56, and Receptor/transducer interactions under (for example) VLG Ca, 42-49 and INR K [native], 32-49.

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'Opposing' effects of opioids on 1M depending on receptor subtype coupling Receptors mediating 1M augmentation (inhibitory responses) Kappa opioid subtypes; {3-adrenoceptors (possibly, '1M-like')

For complex effects of somatostatin, see separate paragraphs, this field.

53-49-18: The opioid kappa receptor-selective agonist U-50,488H significantly augments 1M in most CA3 hippocampal neurones l19. Similarly, the kappa-selective agonists dynorphin A and dynorphin B (which exist in mossy fibre afferents to CA3 pyramidal neurones), also markedly augment 1M at low concentrations (20-100nM)119. Note: At relatively high concentrations (1-1.5 JlM) dynorphin A has an inhibitory effect on 1M • In contrast, the opioid delta receptor-selective opiate agonists DADL (D-Ala2,D-Leus-enkephalin) and DPDPE ([D-Pen(2,5)]-enkephalin) reduce the hippocampal CA3 M-current, with partial reversal of this effect by naloxone. 1M is 'not consistently altered' by mu-opioid receptor agonists such as DAMGO ([D-Ala2,NMe-Phe4,Gly-ol]enkephalin) or non-opioid fragments of dynorphin (e.g. des-Tyr-dynorphin)119. These and other results help to explain (i) the mixed effects of opiates seen in other studies and (ii) a potential post-synaptic function for the endogenous opiates contained in the CA3 mossy fibres l19. 53-49-19: In gastric smooth muscle cells of the toad, an '1M -like' current can be activated ('up-regulated') by activation of {3-adrenoceptors (see paragraph 53-49-29).

G protein transducers mediating 1M inhibition

PTx-insensitive (compare PTx-sensitive coupling in NC108 cells; this table, below) Analysis of G protein selectivity using anti-C protein antibodies

II

53-49-20: M-current inhibition mediated by G protein(s) 'coupling to' receptors for muscarinic agonists, LHRH, substance P and UTP (this table) have been described in sympathetic neurones 19,3l,129. In cultured rat sympathetic neurones, 1M modulation is independent of the cyclic nucleotides cAMP and cGMP (but appears to involve activation of a pertussis-toxin {PTx)-insensitive G protein, generation of diacylglycerol (DAG) and activation of protein kinase C 3l (see other paragraphs, this table). The 'precise' roles of DAG and PKC in KM modulation are unclear and may exhibit marked cell type dependence (see also Protein phosphorylation, 53-32). 53-49-21: Following their microinjection in rat superior cervical ganglion (SCG) sympathetic neurones, antibodies specific for Gag or Gall (but not those for Gao) reduce M-current inhibition by the muscarinic agonist oxotremorine-M (acting at M I muscarinic receptors, see this table). Immunoblotting experiments demonstrated the presence of both Gaq and Gall while GaOl (but virtually no G a02 ) was present in the preparation12l . Note: This study also established that anti-Gao antibodies (but not those specific for Gaqjll or Gall-3) reduce a-adrenoceptor-mediated inhibition of lea in SCG neurones (see also Receptor/transducer interactions under VLC Ca, 53-49).

(b

=:s

M-

~ CJ1 VJ

III

Table 4. Continued Receptor/transducer

Examples (non-exhaustive, notes and cross-references)

Signal transduction mechanisms

53-49-22: Generally, in the cell-attachedt recording configuration, sustained depolarization-activated

Muscarinic receptor agonists M-current can be inhibited by application of muscarine or muscarinic receptor agonists outside the-patch. In dissociated rat superior cervical sympathetic neurones, M-channel closure by muscarinic acetylcholine receptor stimulants is consistent with a requirement for a diffusible messenger (i.e. the inhibition is not due to a local direct 'membrane-delimited' interaction between the receptor, transducer and channel proteins)122 (compare Receptor/transducer interactions under 1NR K G/ACh, 31-49; for Ca 2 + as an candidate for a diffusible messenger, see Channel modulation, 53-44). 53-49-23: Comparative note: Models based on agonist-dependent kinetics of nucleotide interaction with G proteins (accounting for time course of slow synaptic potentials caused by 1M inhibition in sympathetic neurones) without requirement for subsequent messengers are briefly described under Kinetic model, 53-38. 53-49-24: Note: Involvement of multiple messengers may be implicated in M-current suppression in

view of the multiple receptor types with an 'appropriately' slow time course123.

Time course of M-current modulation by muscarinic agonists

53-49-25: The time course and latency of M-current inhibition has been studied in whole-cell dissociated bullfrog sympathetic neurones under conditions where fast inward currents (through nAChR, see entry 09) are abolished by voltage-clamping to -39mV124. Under these conditions; (i) ACh or muscarine induces a slower inward current developed after a latency of ~200 ms, due to M-current inhibition; (ii) the time elapsed from the termination of maximal agonist ~plication (10 JlM muscarine) to the initiation of recovery was 4.5 s ('tr - a '); (iii) the t r - a value is independent of the duration of the stimulus between 0.5 and 10 s, (iv) applications >4 s are necessary to reach ~95% of the maximal inhibition during agonist application and (v) saturating concentrations of the second messenger are produced in <500 ms in response to maximal concentrations of agonist124.

s

~

CJl

(J.J

II

Receptor agonist insensitivities of the IIM like'IKM,ng in NGI08-15 cells

53-49-26: 1KM ,ng in differentiated NGI08-15 cells can not be inhibited by the following receptor agonists (at the concentrations denoted in brackets): acetylcholine (10mM, i.e. without co-transfected M 1 or M 3 receptors), muscarine (10JJM, ditto), noradrenaline (100JJM), adrenaline (100JJM), dopamine (100JJM), histamine (lOOJJM), 5-hydroxytryptamine (10JJM), Met-enkephalin (1 JJM), glycine (100JJM), ,-aminobutyric acid (100 JJM) or baclofen (500 JJM)l. Compare inhibition by bradykinin (1-10 JlM, this table) or angiotensin II (1-10 JlM, this table).

Other responses See also separate paragraphs, this field

53-49-27: A study examining activation of various cation currents (including 1M ) following stimulation of subtypes of cholecystokinin (CCK) receptors in neurones of the rat nucleus tractus solitarius (NTS) has appeared125.

('b

I:'

c-t-

~ CJ1 UJ

_'--

,

e_nt_ry_5_3_

activated at potentials more negative than -50mV)134 (see also INR K IfhQ [native], entry 34). Note: The augmenting/inhibiting effects of somatostatin/ acetylcholine respectively has provided evidence for 'reciprocal' regulation of KM by two different receptor/transducer systems in hippocampal neurones (see arachidonate under Channel modulation, 53-44).

Excitatory mechanisms of the cognition enhancer KST-5452 53-49-30: Application of the novel cognition enhancer and muscarinic Mlbinding compound KST-5452 [3-(m-phenoxybenzylidene)-quinuclidine] to NGPMl-27 cells (NG10S-15 cells stably expressing an Ml muscarinic receptor) elicits a sustained inward current associated with decreased conductance and reduced M-current relaxations (h.p. -20mV)135. KST-5452induced responses are blocked by the Ml antagonist pirenzepine, consistent with its effects on cognition being due in part to excitation following M 1 muscarinic receptor activation and M-current suppression in brain neurones 135.

Comparative note: General effects of muscarinic agonists on CAl field potentials 53-49-31: Superfusion or local application of low concentrations of acetylcholine (ACh), carbachol (CCh) or muscarine in hippocampal slices reduces the ampli-

tudes of CAl field potentialst evoked by stratum radiatum (SR) stimulation97. This effect can be blocked by 1 JlM atropine (independently of the method of agonist application, the site of application or the SR stimulus paradigm) 97. In intracellular and extracellular (single unitt) recordings, cholinergict depressions of field fotentialst were correlated with: (i) depolarization of pyramidal neurones i (ii) spike discharge increase:i (iii) reduction of amplitudes of post-synaptic potentialst and (iv) reduction of late after-hyperpolarizations t (AHPs) (for complex (Idose-dependent') effects of ethanol on M-current reduction coupled to (i) enhanced depressions of evoked field potentials; (ii) depolarizations elicited by muscarinic agonists, and (iii) enhanced amplitudes and duration of muscarinic slow EPSPs in hippocampal preparations, see Channel modulation, 53-44).

Adenosine-induced hyperpolarizations opposing muscarinic-induced depolarizations 53-49-32: A role for adenosine acting selectively to oppose mechanisms of depolarization of the rat superior cervical ganglion (SCG) neurones that are

due to the action of muscarinic agonists (acting via M1-receptors) and by other M-current inhibitors has been described136. Application of adenosine (100 JlM) depresses depolarizations in response to the muscarinic agonists carbachol, muscarine and methylfurmethide. In contrast, adenosine can not alter depolarizations in response to 1,1-dimethyl-4-phenylpiperazinium, 2methyl-5-hydroxytryptamine and potassium and enhanced depolarizations to 5-hydroxytryptamine and ,-aminobutyric acid. Interestingly, adenosineinduced depressions of the depolarizations to carbachol, muscarine and methylfurmethide tended to be increaseq in the presence of a partially M2 receptor selective agonist methoctramine (0.3 JlM). Notes: 1. Adenosineinduced hyperpolarizations are not affected by n-Ala(6)-luteinizing hormonereleasing-hormone or uridine 5'-triphosphate-induced depolarizations (which

l_e_n_t_ry_53

_

are relatively small in the SCG preparation)136. 2. Earlier studies137 on postganglionic neurones of avian ciliary ganglia found that currents with the collective properties of 1M were 'little affected' by adenosine (10 flM).

INFORMATION RETRIEVAL

Related sources and reviews 53-56-01: Reviews of basic properties of KM-type currents and tissue distributions 138,139; G proteins and neuronal K+ currentsl40; M-current update99; K+ and Ca2+ current inhibition in symapthetic ganglial15; neural processing in the photoreceptor inner segment (including the role of KX )22; modulation of ion channels by muscarinic receptor subtypes141; reviews on regulation of protein phosphorylation and dephosphorylation by calmodulin142 and calcineurin26 (see Protein phosphorylation, 53-32); reviews on angiotensin II receptor diversity and physiological roles in the CNS and PNSl43-145 (see Receptor/transducer interactions, 53-49); role of 1M and IAHP modulation in the spatial and temporal pattern of activity generated in cortical circuits l46; review of methods for studying neurotransmitter transduction mechanisms, pertinent to studies of M-current147.

Book reference: Brown, D.A. (1988) M-currents. In Ion Channels (ed. T. Narahashi), pp. 55-94. Plenum Publishing, New York.

Feedback Error-corrections, enhancements and extensions 53-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource J, Search criteria). For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 53-14-03). Please feedback on only one specified paragraph or figure per message, normally by sending a corrected replacement according to the guidelines in Feedback etJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

REFERENCES 1 2 3 4 5 6

Robbins, TPhysiol (1992) 451: 159-85. Carrier, Brain Res (1995) 701: 1-12. Block, J Gen Physiol (1996) 107: 473-88. Inagaki, Science (1995) 270: 1166-70. Marrion, Proc R Soc Lond B (1992) 248: 207-14. Brown, Nature (1980) 283: 673-6.

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56 57

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60 61 62 63

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67

68 69

70 71 72 73

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78 79 80

81

82 83 84

85 86 87

88 89 90

91 92 93

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96 97 98 99

100 101 102 103 104

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e_n_t_ry_53_1

VLG (K) minK

'Minimal' protein subunits (minK, IsK) eliciting 'slow-activating' voltage-gated currents in oocytes

Edward C. Conley

Entry 54

NOMENCLATURES

Abstract/general description 54-01-01: 'Minimal K' or 'lsK' protein subunits (minK/lsK in this entry) are type III membrane glycoproteins with no structural relationship to ion channel proteins with intrinsic pore-forming domains. minK/lsK's activities were first characterized in 1988 using an expression cloning protocol involving injection of rat kidney cRNA pools into Xenopus oocytes coupled to electrophysiological screening for 'elicited' currents. Thus IsK/minK was first characterized as inducing a slowly activating, non-inactivating, voltagedependent, K+ -selective current. Examples of various IsK/minK nomenclatures in use are compared in the fields Category/sortcode, 54-02, Channel designation, 54-03 and Current designation, 54-04. 54-01-02: The robustness of the Xenopus oocyte for heterologous expression of minK/lsK cRNAs led to its extensive use for characterization of the elicited IsK/minK currents, which became synonymous with IsK/minK channels in much of the literature. It has since become clear that this 'equivalence' is an oversimplification, as direct evidence now exists for well-defined channel regulatory (activating) function(s) associated with extracellular and intracellular minK/lsK peptides which omit transmembrane (putative channelforming) domains. These results (summarized in Selectivity, 54-40) can also account for the independent activation of K+ - and CI- -selective currents in untreated oocytes. Other studies have begun to assign biological roles for minK/lsK by postulating specific protein interactions in cell types other than Xenopus oocytes (e.g. minK/KvLQTl proteins underlying the native current IK,s, see below and Protein interactions, 54-31). There remains a need for studies in mammalian heterologous expression systems to clarify minK/lsK's action on specified channel effectors. 54-01-03: No closely related gene family (analogous to the Kv channel genes) has been described beyond the prototype IsK. Alternative splice variants that exist (within a species, see Transcript size, 54-17) do not affect the protein-coding region, such that all tissues that express the IsK gene possess an identical protein (see below); alternative splicing appears to regulate tissue-specific or developmental expression properties of the minK/lsK locus. Native currents resemblinf those associated with minK have been been described in both excitable and non-excitable t cells (e.g. various cardiac myocyte types and secretory epithelial cells). Cell-type 'expression' of the minK gene has been extended through mRNA and protein distribution patterns (without electrophysiological expression data) as exemplified in the listings under Cell-type expression index, 54-08, mRNA distribution, 54-13 and Protein distribution, 54-15. A detailed literature describes the developmental ontogeny of minK/lsK gene expression in heart, uterus and kidney. Transcriptional effects of oestrogens appear to be an important mechanism for regulation of excitability,

_'--

e_n_try_5_4_ _

particularly in uterus and heart (see Developmental regulation, 54-11 and Phenotypic expression, 54-14). 54-01-04: Generation of an isk-/- mousel, disrupted in both copies of the IsK/ minK-encoding locus kcne1, has begun to provide some direct 'phenotype' data associated with in vivo expression of the minK gene (Table 2 under Phenotypic expression, 54-14). The gross behavioural changes characteristic of isk-/- mice suggested compromised inner ear function following isk gene knockout, and as a result of this, the initial phenotypic descriptions l detailed the anatomical structure and physiological deficits of the hearing system. Initial descriptions of the isk-/- mice phenotype included profound deafness, developmentally sensitive locomotor deficits, hyperactivity (e.g. a classic 'shaker/waltzer' behaviour), little response in reflex/startle tests, fluid imbalances in the inner ear vestibules (leading to irreversible changes in morphology), positionselective hair cell degeneration and loss of transepithelial potassium secretion (e.g. across strial marginal cell and vestibular dark cells). 54-01-05: Several primary papers and reviews have discussed evidence that minK proteins contribute to channels that underlie the native 'slow see Phenotypic outward' current IK,s in heart (for refs and overvie~ expression, 54-14). In late 1996, two papers appeared simultaneouslr,,3 supporting a hypothesis that IsK/minK and KvLQTl subunits co-assemble to form channels conducting current 'near-identical' to native cardiac [K,s.. The minK-KvLQT1 data support a role for minK as a regulator (activator) of otherwise 'latent' ion channels as introduced above. Earlier data supporting the hypothesis that a minK-like protein contributes to [K,s in heart together with summaries of data supporting the 'channel regulator' hypothesis appear in Table 4 under Protein interactions, 54-31. minK/lsK proteins and mRNA are widely distributed in apical t membranes of epithelial cell types (see previous paragraph, Cell-type expression index, 54-08, mRNA distribution, 54-13, Protein distribution,. 54-15 and Subcellular locations, 54-16). Roles for minK/lsK as a potassium permeation pathway activated in response to the depolarizing effects of sodium ion entry or cAMP-mediated CI- secretion from epithelial cells are illustrated and discussed under Phenotypic expression, 54-14. Currents resembling [Ks have been demonstrated in rat pituitary nerve terminals, suggesting that IsK/minK plays a more general role, inhibiting excitability in peptidergic nerve terminals (compare neurological/locomotor deficits of isk - /- mice under Table 2 in Phenotypic expression, 54-14). 54-01-06: Comparison of the IsK/minK gene structure across different species has identified multiple mRNA species to arise through a number of distinct mechanisms including (i) transcription initiation at different sites; (ii) alternative RNA splicing t; and (iii) polyadenylation at least three alternative sites (for details, see Gene organization, 54-20). Low-stringencyt Southernt blots of genomic DNA suggests that the minK/lsK locust is in single copy within the genomet. A number of independent studies have mapped the human IsK gene (HMGW name KCNE1) to human chromosome 21q22.1q22.2; this region also contains a putative Down's syndrome t (trisomy 21) marker (see Chromosomal location, 54-18).

l_e_n_try_5_4

_

54-01-07: MinK/lsK proteins are also relatively small (f'J129 or 130 amino acids), forming a single predicted membrane-spanning domain per monomer (for species homologue alignments, see Encoding, 54-17). Independent studies have concluded the minK/lsK transmembrane peptide to adopt (i) an a-helical or (ii) a tilted ,a-strand secondary structure (using different techniques and defined conditions - see Predicted protein topography, 5430). MinK/lsK proteins typically migrate as a band of approximately 15 kDa (e.g. from rat kidney) following denaturing PAGE. A role for N-glycosylation of minK/lsK N-termini in 'polarized' expression of minK has been described, while a single cysteine in the C-terminus of the human/mouse IsK proteins appears to act as an oxidizable site. Several other single-transmembrane domain proteins (unrelated to minK at the amino acid level) that can 'induce' transmembrane ion flux are described under Miscellaneous

information, 54-55. 54-01-08: Numerous point t and deletion/truncationt mutants of minK subunits have been expressed, some of which give rise to dominant-Iethalt phenotypes (see Predicted protein topography, 54-30), changes in I-V relationships (see Current-voltage relation, 54-35), subunit association and 'activator' functions on 'silent' channel activities (see Protein interactions, 54-31 and Selectivity, 54-40), susceptibility to modulation by protein kinase C (see Protein phosphorylation, 54-32), ionic selectivity functions (see Selectivity, 54-40), Hg2+ ion chelation patterns or sensitivity to oxidants (see Table 7 under Channel modulation, 54-44). The interpretation of many of these studies has been complicated by uncertainties surrounding the effector channel when expressed in oocytes (see paragraph 54-01-02) but the data are of value, with this caveat, for comparison of within the oocyte system. The mechanism of effector 'channel formation' remains unclear in any system; assembly of the constituent subunits plays a role in channel activation, which may partially explain the very slow activation kinetics of minK channels. 54-01-09: A number of distinct mechanisms for second messenger-dependent phosphoregulation of minK proteins have been proposed; several of these collective properties show remarkable similarity to those observed for native IK,s components in heart (see listings under Protein phosphorylation, 54-32 and Channel modulation, 54-44; see also general significance of PLC-linked receptor/transducer systems under Receptor/transducer interactions, 54-49). Channel complexes induced by minK appear to be 'directly' modulated by [Ca2+]il chromanols, cAMP, H+ ions, Hg2+ ions, nitric oxide donors, diacylglycerol and peroxides (see Channel modulation, 54-44) although the same caveat on effector channel specificity (previous paragraph) applies to all studies conducted in oocytes. MinK channels expressed in Xenopus oocytes (ibid.) are weakly but reversibly inhibited by some 'classical' K+ channel blockers, as summarized in Table 6 under Blockers, 54-43. Indirect evidence for 'positive regulation' ('opening') by stabilization of open IsK channels by chloride channel blockers has been presented (see Openers, 54-48). 54-01-10: In summary, the literature has carried much debate on how the IsK/ minK proteins elicit various ion currents in heterologous cells, and not all of

_ entry 54

- - - - - - - the data can be easily combined in a single model of minK/lsK action. In that a small portion of published data on minK/lsK is conflicting or inconsistent, the protein remains an enigma; however, much confusion has arisen because the 'molecular' nature of the channel effector was undefined. Recent results have begun to define physiological roles of minK/lsK by selective expression of channel effectors in cells other than oocytes, in which its activities were first discovered by expression cloning. Future work, including determination/analysis of molecular stmcture, protein-protein interaction specificities and gene disruption phenotypes are likely to clarify minK/lsK's functional roles.

Category (sortcode) 54-02-01: VLG (K) minK, i.e. 'minimal' protein subunits (type III membrane glycoproteins t according to the nomenclature of von Heijne4 ) inducing slowly activating voltage-gated K+ currents. The brackets () acknowledge that the criteria for classification as a protein with 'intrinsic' selectivity functions have not been satisfied or universally accepted (for discussion and references, see Protein interactions, 54-31 and Selectivity, 54-40). MinK protein sequences do not exhibit the main 'hallmark' conserved in all other known K+ -selective channels (i.e. an H5 K+ -selective pore domain associated with other membrane-spanning domains - see other entries covering Icloned' K+ channels). Evidence that minK protein alone cannot form a K+ channel, and/or that its cellular role is as a regulator/activator of 'latent' endogenous K+ and CI- channels (as demonstrated for other singletransmembrane proteins like phospholemman, Mat-8 and CHIF) is summarized within this entry (e.g. see Protein interactions, 54-31, Selectivity, 54-40 and Miscellaneous information, 54-55). Note, however, papers have been published describing 'direct' effects on K+ selectivity by site-directed mutagenesis of IsK/minK transmembrane regions (consistent with a channel-forming model).

Information sorting/retrieval aided by designated gene product nomenclatures 54-02-02: The gene product prefix (used as a 'unique embedded identifier' or VEl) for 'tagging' and retrieving information relevant to this entry on the CSN website will be of the form VEl: minK. If a systematic nomenclature for minK/lsK gene products is proposed, then it will be used in 'preference (see this field in other entries).

Channel designation Note: For interpretation of minK expressio.n data in Xenopus oocytes, see also Protein interactions, 54-31. 54-03-01: 'Minimal' K subunits are variously referred to in the literature as minK proteins, minK channels (see Abstract/general introduction, 54-01), l-sK channels (commonly, ibid.) and IsK channels (ibid.). In this entry, the term 'minK' and 'lsK' are used interchangeably or as 'minK/lsK' to

II..-_e_n_t_ry_54

_

designate the same protein (compare Current designation, 54-04). In articles that have proposed a role for minK proteins as activators of 'latent' potassium- or chloride-selective channels' (e.g. ref. s ) the designation IsK, CI was suggested, although later results were able to define peptide domains involved in the independent activation of these currents (see Selectivity, 54-40).

Current designation Conventions for native versus heterologous, minK-associated currents 54-04-01: Because of the uncertainties regarding their origin, no systematic designation exists for 'minK-associated' current(s). Current (italicized 'I') is mostly conjoined with' sK' (subscripted) denoting a slow K+ current, as in I sK (compare to IsK, which with 'minK' is generally used to designate the protein subunit). To avoid confusion with the native ~low-activating delayed rectifier K+ current component in heart, the designation IK,s (with a comma) can be used to specifically denote the latter (as in this entry). Note that prior to findings relating native IK,s to co-assembly of the minK and KvLQTl proteins (see Protein interactions, 54-31), IK,s was proposed by some authors to be 'equivalent' to minK current in Xenopus oocytesj these studies did not anticipate the Xenopus KvLQTl homologue subsequently characterized and cloned from oocyte cDNA pools3 (for further clarification, see Phenotypic expression, 54-14 and Protein interactions, 54-31). Comparative note: The IK,s designation should be distinguished from that used for the native !apidly activating delayed rectifier K+ current component in heart (IK,r), which exhibits a distinct pharmacology (see Blockers, 54-43). For further details on human IK,r, probably encoded by HERC, see VLC K eag/elk/erg, entry 46.

Gene family 54-05-01: The gene encoding minK variants (see Encoding, 54-19) represents a distinct (unrelated) lineage from the 'classical' voltage-gated ion channels, and, beyond the prototype member, no gene family has been described (for known species homologues, see Database listings, 54-53). Note: Several other single-transmembrane domain proteins (unrelated to minK at the amino acid level) which can induce transmembrane ion flux are described under Miscellaneous information, 54-55. For interpretation of minK expression data in Xenopus oocytes, see also Protein interactions, 54-31.

Trivial names 54-07-01: Minimal K channels, mini K+ channels (see Abstract/general description, 54-01), minK proteins, IsK proteins, slow-activating K+ channels. Note: Use of the term 'channel' is generally reserved for proteins with intrinsic channel-forming properties (i.e. polypeptides which can fold

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e_n_t_ry_54_

and/or associate to form ion-selective pores with gating functions - see also Introduction eiJ layoutof entries, entry 02).

EXPRESSION

Cell-type expression index 54-08-01: Native currents resembling those associated with minK have been been described in both excitable t and non-excitable t cells (Table 1). Cell-type 'expression' of the minK gene has usually been based on retrieval of cDNAs, as exemplified in Table 1 (for other source references, e.g. genomic and synthetic minK sequences, see Database listings, 54-53). Note that by (low sensitivity) Northern analyses, the tissue-specific expression patterns of mRNA encoding minK/lsK shows 'complete parallelism' with that of KvLQTl (cited in ref.2), with both transcripts abundant in mouse heart, kidney (see Cloning resource, 54-10) and salivary glands, but are 'almost undetectable' in brain, skeletal muscle and liver.

Expression of human IsK (RIsK) in Jurkat T cells 54-08-02: RT-PCRt has been employed to retrieve a eDNA encoding the 'RIsK

channel' from Jurkat cDNA21 . This cDNA has the same sequence as that previously isolated from genomic DNA23 and encodes a slowly activating, non-inactivating K+ channel as seen in other preparations. HIsK transcripts are detected at similar levels before and after activation of purified human T lymphocytes and Jurkat cells, suggesting a constitutivet mode of expression. Notably, however, channels with the properties of RIsK have not been reported in native Jurkat cells by conventional patch-clamp techniques (at the time of compilation).

Channel density Variations in IK,s density and 'electrical heterogeneity' of heart 54-09-01: Regional differences in native delayed rectifier K+ current in canine

left ventricular epicardium and mid-myocardium24 have been suggested to be due to differential expression of an IsK channel-associated current component: On the basis of tail current fitting, sensitivity to £-4031 and rectification properties, the greater tail current density in epicardial myocytes correlated with a greater IK,s with 'no discernible difference' in IK,r (for details, see ref. 24; for background to IK,r, see VLC K eag/elk/erg, entry 46). In separate studies, a detailed comparison of guinea-pig cardiac ventricular and sinoatrial node (SAN) native delayed reetifier currents (IK ) to the current associated with the minK channel clone has appeared25 • This analysis further suggested that native guinea-pig cardiac I K channels may be related to each other and also to minK25 • Non-stationary state fluctuationt analyses predict a large number of functional channels (308) associated with wholecell SAN I K. Note: For interpretation of .minK expression data in Xenopus oocytes, see also Protein interactions, 54-81.

I

entry54

L...----

_ _

Table 1. Summary of cell-type expression for mink or 'mink-like' channels/currents (From 54-08-01) Heart (atria and ventricles)

Rat, neonatal6 ; mouse heart, neonatal and adult 7,s. Native IK,s components (for comparison with 1sK/ minK, see Phenotypic expression, 54-14) have been well-characterized in several cardiac ventricular myocyte and sinoatrial node preparations 9. Quantitatively similar currents have been recorded in heart from guinea-pig, rabbit, chick and frog. Note: In atrial tumour myocyte lines (AT-l cells) minK mRNA is detectable, but no 1K ,s can be recorded (see also Cloning resource, 54-10). In rabbit cardiac ventricle there is now evidence for both IK,r and IK,s 11. Previously I K of rabbit ventricle was interpreted with only the single, rapidly activating, dofetilide-sensitive current, 1K ,r (as described under VLG K eag/elg/erg, entry 46).

Secretory epithelia

Some electrophysiological data in situ; expression also determined by mRNA/cDNA retrieval and/or protein/ mRNA distribution patterns. Examples include rat kidney (apical membrane of proximal tubule cells)12; rat duodenum 13; rat stomach13; rat submandibular gland13; rat uterus 6,14; endometrium (protein and mRNA) and myometrium (from immunoblotting; M. Boyle, cited in ref.15); rat pancreas 13; gerbil inner earl cochlea, secretory epithelial tissue (stria vascularis; 'lsK-like' currents - see Phenotypic expression, 541416 ); corneal epithelia17 (see mRNA distribution, 5413). The IsK protein has been immunolocalized to the endolymphatic surface of the marginal cell in rat stria vascularis1s; basolateral membrane of rat tracheal epithelia19 (functional data plus RT-PCR signals); mouse skeletal muscle20 (low abundance; detectable by RT-PCRt only). Human21 - based on mRNA/cDNA retrieval; no

T lymphocytes

electrophysiological data in situ (see next paragraph). Peptidergic nerve terminals

Rat. A current similar to the slow current 1Ks , has been demonstrated rat pituitary nerve terminals22 . This could suggest that IsK/minK plays a more general role, inhibiting excitability in peptidergic nerve terminals (compare neurological/locomotor deficits of isk - /mice under Table 2 in Phenotypic expression, 54-14).

Retinal ganglion neurones 17

Based on in situ hybridization signals; no electrophysiological data (see mRNA distribution, 54-13).

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_ 4

----l

Cloning resource Utility of the atrial tumour cell line AT-l 54-10-01: MinK mRNA is readily detected in atrial tumour myocytes derived from transgenic mice (AT-I cells)26. For applications ofAT-1 cells for studies of cardiac ion channel gene expression, functional regulation and biochemistry, see Cloning resource under VLG K eag/elk/erg, 46-10.

Developmental regulation Effects of oestrogens on minK gene transcriptional regulation

54-11-01: The rapid induction of minK RNA by oestrogen t has been reported in rat uterus 14,27. The minK RNA species is not detectable in uterine preparations from oestrogen-deprived rats, but hybridization t signals are 'substantially induced' after 3 hours of oestrogen treatment (see Fig. 1). minK mRNA is abundant in pools of mRNA derived from ovariectomized but diethylstilbestrol-primedl rat uterine preparations 6. Supplementary note.' In addition to these effects, a demonstration of 'direct' inhibitory effects of oestrogen on IsK protein (i.e. not apparently mediated by the oestrogen receptor) have been noted28 . These effects are additive to moderate inhibitory effects of the anti-oestrogen t tamoxifenTM.

(a) Oestrogen-deprived

(b) Oestrogen-implanted

125nAL 2s

1m

_"bi_"__"'~

Figure 1. MinK mRNA induction by oestrogen implantation. Figure shows Xenopus oocyte currents following injection with uterine poly(A)+ mRNA from ovariectomized rats (a) or 3 days following oestrogen capsule implantation (b). (Reproduced with permission from Boyle et a!. (1987) Nature 330: 373-5). Note.' For interpretation of minK expression data in Xenopus oocytes, see also Protein interactions, 54-31.

1l..-_e_n_t_ry_S4

_

Differential expression of 1sK mRNAs in mouse tissues during development and pregnancy 54-11-02: In the developing mouse heart, IsK mRNA displays a fourfold 'upregulation' during the perinatal period followed by a 20-fold decrease between birth and the adult state29. Notably, the 0.9 and 3.4 kb IsK transcripts (see Transcript size, 54-17) are differentially regulated following birth. In the developing mouse kidney, IsK mRNA 'progressively' increases by approximately 10-fold, reaching steady-state (adult) values at 21 days. In the developing mouse uterus, IsK mRNA increases threefold in late pregnancy and is 'rapidly decreased sixfold' following birth. These changes suggest that IsK plays a significant role in myometrium during late pregnancy and delivery29 (for the mechanistic basis of differential (tissuespecific) expression, see Gene organization, 54-20}.

Sex hormone transcriptional regulation correlated with effects on cardiac repolarization 54-11-03: A study evaluating effects of ovariectomy (OVX) together with

oestradiol (£2) or dihydrotestosterone (DHT) treatment on IsK mRNA levels in rabbit has appeared30 • In this study, both a 0.7kb IsK and HK2 (Kvl.S, see entry 48) mRNAs were down-regulated in cardiac ventricular tissue from OVX rabbits treated with either £2 or DHT. The Q- T interval (an index of cardiac ventricular repolarization (see also h-ERG under Phenotypic expression in VLG K eag/elk/erg, 46-14) was prolonged in £2- and DHT treated animals (OVX + vehicle, 223 ± 6 ms; OVX + DHT, 236 ± 10 ms; and OVX + DHT, 24S ± 6 ms; p < O.OS). In other experiments, quinidine-infused animals responded similarly to controls30 • See also minK/KvLQT1 interactions under Phenotypic expression, 54-14 and Protein interactions, 54-31.

Isolation probe 1sK/minK was discovered using an expression cloning protocol in oocytes 54-12-01: A cDNA representing the 'prototype' minK protein was originally isolated by an expression cloning t protocol using rat kidney cRNA pools injected into Xenopus oocytes 13 (for significance of minK expression data in Xenopus oocytes, see Protein interactions, 54-31, Selectivity, 54-40 and Database listings, 54-53). Relatively low molecular weight size fractions from mRNA sucrose density gradients are associated with minK activity (of average size rv 700 nt - for significance, see Fig. 3 under Transcript size, 54-17). Comparative note: Kv family channel expression other than KvLQTI has been reported to be augmented by 'low molecular weight' fractions from mRNA sucrose density gradients; in most cases, however, these appear most likely due to the presence of Kv(3 subunit mRNA in the fraction (e.g. the 2-4kb fraction described under Protein interactions in VLG K Kv4-Shal, 51-31).

mRNA distribution For further references, see Cell-type expression index, 54-08, Protein distribution, 54-15, and Subcellular locations, 54-16.

II

_'---

e_n_try_S_4___

Tissues expressing minK/18K mRNA detectable with relatively lowsensitivity assays 54-13-01: Using coding region probes (see Transcript size, 54-17), signals corre-

sponding to minK/lsK have been detected by Northernt assays using poly(A)+ mRNA pools from rat kidney, duodenum, stomach, neonatal rat and mouse heart (with homogeneous distribution in atria and ventricles); adult canine atrial and ventricular myocytes3l; atrial tumour myocytes derived from transgenic mice (AT-l cells)26, pancreas, uterine muscular preparations (i.e. including endometrial epithelium) and submandibular gland. In mouse skeletal muscle, minK mRNA expression is of low abundance20 and is only detectable by RT-PCRt. IsK signals have not been found in liver or brain (but see 'retinal neurones', paragraph 54-13-02), and signals in adult rodent hearts are 'low' or 'undetectable' (see Developmental regulation, 54-11). IsK mRNA distributions generally 'compare well' with immunolocalizations of IsK protein (see Protein distribution, 54-15). By Northern blots, the tissuespecific expression patterns of mRNA encoding minK/lsK has been noted to show 'complete parallelism' with that of KvLQTl (cited in ref.2, with both transcripts abundant in mouse heart, kidney and salivary glands, but being 'almost undetectable' in brain, skeletal muscle and liver).

1sK mRNA in retinal ganglion neurones and corneal epithelia 54-13-02: ISH: The ganglion cell layer of the retina expresses IsK mRNA in a subpopulation of ganglion cells composed of large cell bodies 17 distributed ('scattered') throughout the retina. Positive hybridization signals have also been localized to the epithelial cells throughout the cornea17.

Phenotypic expression Defective transepithelial K+ secretion/inner ear defects in minK/1sK gene knockout mice 54-14-01: Generation of an isk-/- mousel, disrupted in both copies of the

IsK/minK-encoding locus kcne1 has begun to provide some direct 'phenotype' data associated with in vivo expression of the minK gene (Table 2). The gross behavioural changes characteristie of isk-/- mice suggested compromised inner ear function following isk gene knockout, and as a result of this, the initial phenotypic descriptions l detailed the anatomical structure and physiological deficits of the hearing system. As itemized and further annotated in Table 2, isk-/- mice exhibit profound deafness, developmentally sensitive locomotor deficits, hyperactivity (e.g. a elassic 'shaker/waltzer' behaviour), little response in reflex/startle tests, fluid imbalances in the inner ear vestibules (leading to irreversible changes in morphology), position-selective hair cell degeneration, strial marginal cell and the vestibular dark cell dysfunction (i.e. lack of transepithelial potassium secretion). Note: Other phenotypic features of the isk - /- mice (e.g. in physiological systems other than the inner ear where IsK/minK has been suggested to play a role) were not documented at the time of compilation and this work is ongoing.

Earlier proposed functions of 18K proteins in secretory epithelia 54-14-19: minK/lsK proteins and mRNA, are widely distributed in apical t

_

1~_e_n_t_ry_54

Table 2. Behavioural phenotypes of isk-/- mutant mice. Unless otherwise stated, phenotypes of isk-/- mice are listed where clear differences have been reported between them and age-matched heterozygote (isk-/+ and wild-type (isk+/+) counterparts. The 'age-matching' is significant as the isk mutation phenotypes are generally developmentally sensitive. (From 54-14-01) Phenotype/ feature

Brief description (all data from ref. 1 )

General appearance

54-14-02: Heterozygote (isk+/-): normal; homozygous null (isk-/-): normal.

Locomotor deficits

54-14-03: isk-/- pups have difficulty righting themselves

(this phenotype disappears as animals mature). 54-14-04: isk-/- mice exhibit an 'awkward un-coordinated

movement' when age-matched isk+/- and isk+/+ animals walk normally. Profound deafness (see below)

54-14-05: Although some of the cochlear pathology associated with isk-/- animals (below) resembles other mouse models of human deafness (e.g. Scheibe type or cochleosaccular deafness), other aspects support isk knockout-induced deafness being a new model of deafness (see also note 4).

Hyperactivity: Shaker/waltzer phenotype

54-14-06: isk-/- mice show rapid head bobbing and

occassionally a head tilt. More mature mice display an intermittent bidirectional circling behaviour (displayed throughout life). Collectively, these behaviours are typical of mouse mutants with inner ear dysfunction, and are commonly referred to as a Shaker/waltzer phenotype. Generally, these signs have been interpreted as causing the animals to lose orientation with respect to gravity when tactile cues (such as feet touching the bottom of the cage) are removed32.

Preyer's reflex t performance

54-14-07: isk-/- mice fail to show any pinnea reflex or

Swimming ability

54-14-08: When placed in deep tanks filled with ambient temperature water, isk-/-mice begin rotating along their long axis and sink underwater. While underwater, they begin somersaulting (while still rotating along their body length). isk-/- mice are mostly unable to resurface within 30 s without assistance. isk+/+ and isk+/- mice swim and resurface normally.

Reissner's membrane collapse

54-14-09: In the cochlea of isk-/- mice (between ages PO and P3, note 1) Reissner's membrane (note 2) undergoes an irreversible collapse onto the surface of the spiral limbus (along the tectorial membrane and reticular lamina, and along the lateral wall of the cochlea in close apposition to

signs of startle (isk+/+ and isk+/- mice are normal).

_L...-

en_t_ry_54_

Table 2. Continued Phenotype/ feature

Cochlear hair cell death

Spiral ganglion cell death

Stria vascularis; intermediate cell morphology changes

III

Brief description (all data from ref. l ) the stria vascularis). All other ages examined (P7, P190, P20, P42, 3, 5 and 7 months old) exhibit the same pathology. Reissner's membrane collapse parallels the time when most strial cells have become post-mitotic t. These results imply that no volume can be added to the endolymphatic space without K+ permeating the IsK/minK-associated channels (i.e. once the apical membranes of the strial marginal cells express their tight junctions t); without a functioning IsK/minK, the volume of endolymph cannot increase significantly. 54-14-10: Hair cells of isk - /- mice cochlear duct differentiate normally, but then degenerate post-natally: At P3 (note 1), isk-/- mice exhibit degeneration of the organ of Corti in all turns of the cochlea. The degeneration did not appear to include the supporting cells outside the organ of Corti or within the developing spiral sulcus or spiral limbus. At P20, Gochleas of isk-/- mice do not contain an organ of Corti and some supporting cells (normally located laterally) have also undergone degeneration. Hair cell death may be attributable to calcium toxicity, particularly in the absence of K+ fluxes, as discussedl . Furthermore, differential rates of hair cell death in situ may be attributable to the different repertoire of ion channels each hair cell possesses to facilitate filtering of the transduction event in their particular frequency range in order to augment any response at a critical frequency or frequency range33 . 54-14-11: Spiral ganglion cells in isk-/- mice degenerate, a phenotype first observable at P20 as occasional vacuoles present in the ganglion. At P42, the majority of spiral ganglion cells in the base and middle turns of the cochlea degenerate. IsK/minK proteins are not expressed in spiral ganglia, so the mechanism of cell death may be indirect. The greatest loss of cells occ.urs in the basal turn of the cochlea (i.e. less loss in the middle turns and 'no significant loss' of ganglion cells in the apex (degeneration of hair cells within the cochlea was complete throughout all turns of the cochlea). Note: No loss of apical ganglion cells can be observed even in seven-month-old isk-/- mice. 54-14-12: Under electron microscopy, isk-/- exhibit an expansion of the intercellular space between marginal and intermediate cell processes, and between marginal cell processes and blood vessels; intermediate cells also appear vacuolated without cellular debris.

l_e_n_t_ry_5_4

_

Table 2. Continued Phenotype/ feature

Brief description (all data from ref. l )

Vestibular wall collapse

54-14-13: In the vestibular labyrinth, isk-/- exhibit an irreversible collapse of the vestibular wall, suggesting a decrease in endolymph volume (analogous to cochlear damage, see note 2)

Macula and cristae hair cell degeneration

54-14-14: In five-month-old isk-/- mice, the macula of the sacculus displays slight degeneration. At seven months, the macula displaY8COmplete degeneration of both types I and n hair cells (and in consequence, contains only support cells). In the cristae, hair cell death occurs relatively early but the time course of degeneration is different from that in cochlea. At P42 the cristae are undergoing 'massive degeneration'.

Vestibular dark 54-14-15: Vestibular dark cell degeneration is observed at cell degeneration P3 in isk-/- mice, with a less well-defined nuclear structure, and 'very little' cytoplasm near the apical surface, which appears jagged compared to isk-/+ mice or isk+/+ mice. No cell death is observed in the dark cell epithelial layer of the vestibular labyrinth. Lack of transepithelial voltage/short circuit current/ K+ transport

54-14-16: Strial marginal cells and vestibular dark cells from wild-type mice or isk-/+ mice develop a transepithelial voltage (and thus a significant equivalent short circuit current, Isc, a measure of transepithelial K+ transport). In contrast, isk-/- do not develop a transepithelial voltage (exceeding a lower detection limit of 70.5 mY). These and other datal suggest that constitutive K+ secretion in wild-type marginal cells and dark cells is dependent upon IsK/minK, consistent with earlier work16 showing that dark cell apical membranes display IsK current.

54-14-17: Functional note: As determined in wild-type animals, vestibular dark cells adjust the rate of K+ secretion into endolymph according to the perilymphatic K+ concentration34 • 'Physiologically relevant' increases in the perilymphatic [K+] from endolymph (incurred during sensory transduction) increase the rate of transepithelial K+ secretion (TK) by increasing KCI uptake across the basolateral membrane and activation of K+ release via IsK-associated channels in the apical membrane34 • Lack of K+ secretory responses to K+ or DillS

54-14-18: K+-stimulated K+ secretion is absent across strial marginal cells and vestibular dark cells from isk - /mice (elevation of the basolateral K+ concentration from 3.6 to 25mM induces K+ secretion in isk+/+ and isk-/+

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_S_4_

Table 2. Continued Phenotype/ feature

Brief description (all data from ref. l ) mice. Additionally, application of the minK/lsK 'agonist' DillS (at ImM - see Openers, 54-48) does not induce K+ secretion across these cell types in isk - /- mice.

Notes: 1. PO is defined as the first 24 h after birth. 2. Within the inner ear, the scala media (Le. the endolymphatic space) normally contains endolymphatic fluid and is enclosed (and separated from scala vestibule and scala tympani) by an epithelial lining contributed by Reissner's membrane, the reticular lamina, and supporting epithelial cells. The position of Reissner's membrane can indicate intracochlear fluid pressure; the 'collapsed' membrane may be due to fluid pressure build-up: It is hypothesized l that (in the absence of endolymph production in isk-/mice) (i) K+ and associated water does not diffuse passively into the endolymphatic space from the marginal cells of the stria vascularis (normally via IsK channels)3S,36; (ii) fluid in the endolymphatic space would slowly reduce in volume due to an active reabsorption process in the inner ear and (iii) Reissner's membrane would finally 'collapse' under the influence of the fluid pressure in the scale: vestibule. 3. Lack of significant differences in cell volume responses to K+ -induced and osmotically induced cell swelling amongst isk+/+, +/-, and -/- mice suggest that IsK/minK is not essential for volume regulation after osmotically induced cell swelling (compare shrinking of vestibular dark cells following K+induced and osmotically induced cell s~velling involving activation of the 1sK current in gerbils 37 and Channel modulation, 54-44). 4. In separate studies, KCNE1 was specifically excluded as a candidate gene for Jervell and Lange-Nielsen (JLN) syndrome38, which is of significance since minK's relationship to KvLQTl was defined2,3 (see Protein interactions, 54-31; Selectivity, 54-40 and Chromoso1T.lallocation under VLC K eag/elk/ erg, 46-18). However, Vetter et a1. l noted a 'striking similarity' in pathology of post-mortem JLN syndrome subjects to that of the isk-/- phenotype. 5. In humans, the minK/lsK gene KCl\lE1 has been localized to human chromosome 21q22.1-q22.2, which also contains a putative Down's syndrome t (trisomy 21) region39 (see Chromosomal location, 54-18). membranes of epithelial cell types 12 (see also Cell-type expression index, 5408; mRNA distribution, 54-13; Protein distribution, 54-15 and Subcellular locations, 54-16). In epithelial cells, the Na+/K+-ATPaset pump in the basolateralt membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane (e.g. via co-transportert activation, see Fig. 2). Taking into account the cellular localizations (ibid.) and electrophysiological properties (see other fields) it has been suggested that minK/lsK may function as a K+ permeation pathway activated in response to the depolarizing effects of sodium ion entry12. For

l_e_n_t_ry_54

----"_

I

III

f·····. CD Epithelial cell layer

e.g. in proximal tubules of the kidney

~-@~s:-

~

-\

\ K+~K+

.. \

/

1\

5 Other K+-permeation channels 2

Na+, K+ ATPase

\

.,

\

.

Na+

x

\ '

\ \

3

\ \

\ \

Na+/sugar or amino acid co-transporters

\ \ \ \ \

\

\t~~==~

Figure 2. Early model for IsK function in epithelial cell apical membrane K+ permeation. The IsK protein has been immunolocalized to apical faces of epithelial cell layers such as proximal tubules of the kidney (1) and excretory ducts of the salivary gland (see also Subcellular locations, 54-16). In these and other epithelial cell-types, K+ ions permeate into both the lumen and interstitial spaces when the basolateral Na+ /K+ -ATPase (2) generates an Na+ gradient across the epithelial cell (i.e. lower intracellular [Na+] coupled to a higher intracellular [K+ i). The Na+ gradient can drive entry of Na+ ions into the cell from the lumen through Na+ /sugar or amino acid co-transport systems (3) inducing depolarization of 10-15 m V over the stable apical membrane potential (approx. -70 m V at rest). According to the model, this change in membrane potential may activate apical IsK protein-containing channel activities in situ (4) resulting in K+ permeation from the epithelial cells into the lumen. K+ -permeation pathways other than IsK are implicated in movement of K+ ions from epithelial cells into the interstitial space (5). (After Sugimoto et al. (1990) J Membr BioI 113: 39-47). (From 54-14-19)

further details, see the sequence of events described in the legend to Fig. 2. A physiological role for IsK channels in cAMP-mediated Cl- secretion from epithelial cells was originally suggested in ref. 40; this model has been strengthened by further indirect pharmacological evidence (see Chromanols under Channel modulation, 54-44); direct evidence for IsK/minK mediating transepithelial K+ secretion into the endolymph in the inner ear has been obtained in minK gene-null mice (see previous paragraph and associated Table 2). A slowly activating K+ current in basolateral membranes of rat

_ _ _ _ _ _ _ _ _ _ _ _,

e_n_try_5_4-----1

tracheal epithelium has properties consistent with the biophysical characteristics of minK/lsK and appears to be important for cAMP-induced chloride secretion in these cells19.

Dark cell apical membrane K+ secretion through 'IsK-like , channels 54-14-20: Within epithelia, MinK proteins are thought to mediate ionic permeation from the cytoplasmic to the extracellular (luminal) sides, consistent with roles in ion secretion12 (as in Fig. 2). Within the inner ear of vertebrates, the vestibular dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by an osmo-sensitive apical transport mechanism in which the minK product is implicated37. Single-channel patch-clamp studies have demonstrated non-selective cation channels and maxi-K+ channels (see ILC K Ca, entry 27) in the apical membrane, but at densities which do not account for known rates of transepithelial K+ transport. Studies employing cell-attached macropatch-clampt methods have identified an outward apical membrane current at 0 mV pipette voltage, which can be stimulated by elevating bath K+ concentration from 3.6 to 25 mM16 . This pathway has several electrophysiological and pharmacological features similar to those described for IsK activities in other preparations16 (see also immunolocalization of IsK in stria vascularis under Subcellular locations, 54-16; Selectivity; 54-40 and H+ ions under Channel modulation, 54-44).

Potentiation of KvLQTl current by co-expression with minK 54-14-21: Several primary papers and reviews have discussed evidence that minK proteins contribute to channels that underlie the native 'slow outward' current IK,s in heart (e.g. refs41 - 44, see also VLC K DR [native], entry 45) and references under Related sources and reviews, 54-56). Furthermore, two papers appeared simultaneouslr,3 supporting a hypothesis that IsK/minK and KvLQTl subunits co-assemble to form channels conducting current 'near-identical' to native cardiac IK,s. The minK-KvLQTl data support a role for minK as a regulator (activator) of otherwise 'latent' ion channels, an interpretation that is supported by small peptides derived from the cytoplasmic and extracellular domains of minK being 'sufficient' to activate, respectively, K+ - and CI- -selective channels in oocytes (for details, see Protein interactions, 54-31, particularly Table 4, which summarizes most

of the data supporting the channel regulator hypothesis, and Selectivity, 54-40). Earlier studies linking KvLQT1 mutations with the most common form of inherited long QT syndrome (LQTl) therefore implicates dysfunction of native IK,s in LQT1, and provides a model for native IK,s as an important 'target' for class mt antiarrhythmic drugs. For further background, see Table 3 under Chromosomal location in VLC K eag/elk/ erg, 46-18.

Earlier data supporting the hypothesis that a minK-like protein contributes to 1K s in heart 54-14-22: Prior to the findings on minK/KvLQTl (see previous paragraph),

minK/lsK activity in (i) Xenopus oocytes6,8,21,42,44-46 and (ii) transiently

entry54

I-

_

- - - - - -

transfected mammalian (HEK-293) cells 9 showed activation kinetics and voltage dependence of the expressed (or endogenously activated) minK current to be similar to those of IK,s recorded from rodent cardiac myocytes under similar experimental conditions. Furthermore, antibodies directed against the minK channel protein were shown to react with a surface antigen on myocytes from adult guinea-pig ventricle and sinoatrial node, where IKs is the dominant outward K+ current44 • Other similarities related to partial block of both IsK/IK,s by the class ill antiarrhythmic compound clofilium but not by agents that block the rapidly activating component IK,r (e.g. sotalol derivative E4031 or low concentrations of lanthanum (for details, see Blockers, 54-43; for IK,r see HERC under VLC K eag/elk/erg, entry 46)). IsK/IK,s showed similar patterns of modulation by protein kinase C and possibly protein kinase A when certain 'species-specific' effects were taken into account (for details, see Protein phosphorylation, 54-33). Minor differences in ionic selectivity between IsK and the native IK,s component were reported, but these were attributed to 'species differences' in selectivity determinants (see Selectivity, 54-40). An alternative 'secretory' function in heart (as established for IsK proteins in epithelial cell types, this field, below) has not been proposed. Other factors apparently supporting an 'intrinsic channel protein' hypothesis included patterns of La3 + blockade (see Blockers, 54-43) and activation by DillS (see Openers, 54-48). Note: Data largely supporting the hypothesis that minK is an activator of 'latent' endogenous channels are summarized under Protein interactions, 54-31.

Possible physiological roles of minK in regulating uterine excitability 54-14-23: The marked changes observed in uterine IsK mRNA transcript levels during development and following birth (described under Developmental regulation, 54-11) suggest that IsK plays a significant role in myometrium during late pregnancy and deliverr9. In relation to these roles, the known patterns of receptor/second messenger/phosphoregulation of IsK may be of significance (for details see Protein phosphorylation, 54-33 and Channel modulation, 54-44 and Receptor/transducer interactions, 54-49). In particular, uterine myocytes express a number of receptor/transducer systems known to be involved in processes of labour induction. Notably, oxytocin receptors can couple to phospholipase C activation which appears to be an established mechanism for 'positive regulation' of IsK47 (for background, see also Resource A-C protein-linked receptors). Note: A current similar to the slow current IKs, has been demonstrated in rat pituitary nerve terminals22 . This could suggest IsK/minK plays a more general role, inhibiting excitability in peptidergic nerve terminals (compare neurological/locomotor deficits of isk-/- mice under Table 2).

Indirect evidence for involvement of RIsK channels in T cell activation 54-14-24: The HIsK protein derived from Jurkat T cell cDNA21 (see Cell-type expression index, 54-08) has been suggested to be involved in the T cell activation process by mitogens t (partly based on the supressive effects of

_'---

e_n_t_ry_54----'

clofilium on interleukin 2 mRNA induction in activated T cells - for significance, see Blockers, 54-43). For putative roles of other channels in T cell maturation, see ILG Ca InsPJ, entry 19; ILG K Ca, entry 27; Kv1.3 under VLG K Kv1-Shak, entry 48 and Kv3.1 under VLG K Kv3-Shaw, entry 50.

tCarrier/transporter' hypotheses 54-14-25: Comparative notes: 1. The kinetics of IsK activation (see Activation, 54-33) have been noted as 'similar' to an H+ current recorded from alveolar epithelial cells (for comparative 'surveys' of H+ and K+ currents in this preparation, see refs48- s2 ). However, the hypothesis that the minK channel protein constitutes a 'carrier-type' transporter has been countered (for example, see refs s3,s4). 2. Examples of other single-transmembrane domain proteins mediating transmembrane ion flux are briefly described under

Miscellaneous information, 54-55.

Protein distribution 1sK immunolocalizations within kidney 54-15-01: Within the kidney, minK (lsK) is localized in the tubular cells located in both outer and juxtamedullary cortical regions, as well as the subcortical region. Immunoreactive complexes can be further localized to the apical (luminal) membrane portion of the epithelial cells forming the proximal convoluted tubule and the early proximal straight tubules of the nephron. Signal is 'low or absent' in glomeruli}, blood vessels, thin descending/ ascending limbs of the loop of Henle and collecting tubules 12 (see also Phenotypic expression, 54-14). For other tissues, see also Cell-type expression index, 54-08, mRNA distribution, 54-13 and Subcellular

locations, 54-16.

Subcellular locations See also Cell-type expression index, 54-08 and Protein distribution, 54-15.

1sK proteins are localized to the apical portions of secretory --epithelia 54-16-01: Within epithelial cells (such as proximal tubules of the kidney and excretory ducts of the salivary gland), the IsK protein is located solely in apicalt membranes12. For example, within the submandibular gland, minK protein is restricted to the apical membrane portion encircling the salivary passage (epithelial cells in the striated duct and small excretory duct, located between the striated and large excretory duct)12 (see also Fig. 2 under Phenotypic expression, 54-14). A slowly activating K+ current in basolateral membranes of rat tracheal epithelium has properties consistent with the biophysical characteristics of minK/lsK and appears to be important for cAMP-induced chloride secretion in these cells 19. Within the utems, minK protein is limited to the periluminal membrane portion of endometrial epithelial cells located around the bicomicated uterine cavity (the apical cytoplasmic domain underneath the membrane of epithelial cells expresses minK to a lesser extent). Signal is absent from the myometrium12

(D

Table 3. Patterns of hybridization detected by the different genomic fragments used for probes 2-12 in Fig. 3 (From 54-17-01) Transcript Probe 2 size (knt)

Probes 4-8

Probe 9

10.0

No signal

++ in SMG

7.0

No signal

5.2 3.7

Detectable

Detectable

Probe 3

No signal +++++ inSMG + in kidney + in SMG No signal

Probe 10

Probe 11

Probe 12

+ in kidney + in kidney ++ in SMG

++ in SMG

II

Detectable

kidney Detectable

No signal

1.4

Detectable

Detectable

No signal

0.8 (see note 2)

inSMG '+ in kidney

+++++

No signal

SMG

+++ in

+++ in kidney

Detectable

+ in SMG

++ in SMG

++ in kidney

Detectable

Detectable

+ in SMG

+ in kidney

+++++ 2.6

Probe Probe (eDNA) (eDNA) (see note 1)

Detectable SMG, 0.3knt, uncharacterized

+++++ in ++ in SMG

kidney

Notes: 1. The stated sizes and signal intensities are not 'absolute' but are illustrative for mechanisms generating IsK transcript heterogeneity; see also original Northern hybridization assays. 2. Common band similar to those detected with coding region probes in other studies (e.g. average size rv700nt) representing a mixture of IsK-A and IsK-B mRNA species (see Gene organization, 54-20). For examples of differential mRNA transcript formation during development, see also Developmental regulation, 54-11.

=:s t"1"

~ CJ1 ~

_L.-

e_n_try_S_4_

(inconsistent with other mRNA/protein localization studies), the vascular system, the uterine covering and the cytoplasmic stroma portion. The IsK protein has been immunolocalized to the endolymphatic surface of the marginal cell in rat stria vascularis secretory epithelial tissue of the inner earlS (see also 'lsK-like' currents recorded in gerbil cochlea 16 under Phenotypic expression, 54-14).

Transcript size Characterization of multiple mRNA species arising from the 1sK locus 54-17-01: As illustrated under Gene organization, 54-20, the IsK genet is involved in the generation of a large number of mRNA species through a variety of cellular mechanisms 7,12,55. The actual sizes of the transcripts detected in mRNA pools derived from different tissues are generally dependent upon the region of the IsK gene used to make the hybridization t probe (for further technical background, see Resource D - Diagnostic tests). An example of the range of transcript sizes detected using probes from different regions of the rat IsK gene55 is shown in Fig. 3 (for relationship to gene organization, see legend to Fig. 3).

3

A Probe ~H~ 1A 18 B Exon---:ItdrK-~~I----.p.;..--+-

c

ABg

5

9

7

I

I

H

H

.•

81--nO

16~ ~

H

H

. . . .-+-.-...._.... I

I

I

B A B

2

B

~

, 1

12

H

r-----i

',

B

RR

I

•

E

_____- - - - . - - - - - - - - - - - - 5.2 knt 3.7 knt --~ 2.6 knt --------~ 1.4 knt 0.8 knt

--------_.-----

Figure 3. mRNA transcript sizes derived from the 1sK gene as detected by Northern t hybridization assays. Data an~ derived for poly(A}+ mRNA populations purified from (i) rat submandibular gland (SMG) and (ii) kidney preparations. (A) Regions of the 1sK gene used to construct numbered probes (2-12) are indicated, mapped to the 1sK gene structure shown in (B) as a partial restriction map (further described in C;ene organization, 54-20). Table 3 summarizes the patterns of hybridization detected by the different genomic fragments used for probes. Where tissues ajre indicated, there was clear evidence for differential regulation (otherwise, 'detectable' indicates other signals representing large transcripts, as reported55). The figure (C) also shows tentative structures for these RNA transcripts (included to illustrate transcript heterogeneity arising from differential transcript initiations, alternative RNA splicing and differential polyadenylations (see also Developmental regulation, 54-11 and Gene organization, 54-20). (Reproduced with permission from 1wai et al. (1990) J Biochem Tokyo 108: 200-6.) (From 54-17-01).

entry54

_

I' - - - - - - - - - - SEQUENCE ANALYSES

II

The symbol {PDTM} denotes an illustrated feature on the protein domain topography model (Fig. 6).

Chromosomal location Independent studies map the human IsK gene to chromosome 21q22.1-q22.2 54-18-01: A number of studies have mapped the human IsK gene (HMGW name KCNE1) to human chromosome 21 39,56,57. The earliest assignment 56, using panels of somatic cell hybridstwas confirmed57 by somatic cell hybridization and the assigpment was regionalized to 21q22.1-q22.2 by isotopic in situ hybridizationt. By PCR analysis of two complete panels of human/rodent hybrid DNA the KCNE1 gene was mapped to chromosome 21 with 1000/0 concordance . By performing PCR on DNA of a human chromosome 21 regional mapping panelt , KCNE 1 was again localized to 21 q22.1-q22.2, which also contains a putative Down's syndrome t (trisomy 21) marker39. Comparative note: Studies identifying a polymorphism in the minK gene46,58 (see Encoding, 54-19) could not establish any definite relationship with loci associated with congenital long QT syndrome (see Table 3 under VLC K eag/ elk/erg, entry 46; see also data on minK-KvLQT1 co-assembly under Protein

t

interactions, 54-31).

Encoding The minK gene encodes an identical protein sequence in all tissues where it is expressed 54-19-01: In rat, the gene for minK encodes a 130 amino acid residue protein with a single predicted membrane-spanning domain; similar sequences have been isolated from neonatal mouse heart (129 residues, 920/0 amino acid identity to rat) and humans (129 residues, 76% amino acid identity to rat). The deduced amino acid sequence of the guinea-pig IsK protein44 is rv780/0 identical to the rat, mouse and human variants. The structure of the minK gene (see Cene organization, 54-20) predicts identical minK amino acid sequences within a given species in all tissues where it is expressed (ibid.). Figure 4 shows an amino acid alignment of rat, mouse and human IsK sequences.

Polymorphisms in the human minK gene KCNEl 54-19-02: Sequencing of RT-PCRt products amplified from human genomic DNA and post-mortem cardiac ventricular tissue46,58 has indicated expression of Ser38 and Gly38 allelest in human heart (resulting from an A-to-G substitution at position 112 (AGT-GGT), creating a new MspAI restrictiont site). Of 32 alleles from 16 subjects studied58, 25 had a Gl12 and seven had an Al12 (see also Chromosomal location, 54-18). A G-to-A substitution polymorphism at position 253 has been described in the KCNE1 coding sequence and is detectable by sscpt or RFLPt analysis38 .

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_4-----.1

Rat Mouse Human

·

·

N-34

br--

MALSNSTTVL PFLASLWOET DEPGGNMSAD LARRSQLRDD SKLEALYILM MSLPNSTTVL PFLARLWQET AEQGGNVSG. LARKSQLADD SKLEALYILM MILSNTTAVT PFLTKLWQET VQQGGNMSG. LARRSPRSSD GKLEALYVLM - - Hl

i

C-27

Rat Mouse Human

VLGFFGFFTL GIMLSYIRSK KLEHSHDPFN VYIESDAWQEIKGKALFQARV VLGFFGFFTL GIMLSYIRSK KLEHSHDPFN VYIESDAWQE KGKAVFQARV VLGFFGFFTL GIMLSYIRSK KLEHSNDPFN VYIESDAWQE KDKAYVQARV

Rat Mouse Human

LESFRACYVI ENQAAVEQPA THLPELKPLS LESFRACYVI ENQAAVEQPA THLPELKPLS LESYRSCYVV ENHLAIEQPN THLPETKPSP

o

50 49 49

•

100 99 99 130 129 129

Figure 4. Predicted amino acid sequences for three 1sK (minK) proteins isolated from rat, mouse and humans. Locations of conserved N-glycosylation sites in the N-terminal domain (see Sequence motifs, 54-24) are indicated by asterisks. The predicted Hl domain (see Domain arrangement, 54-27 and the [PDTMj, Fig. 6) is shown. The position of conserved cytoplasmic serine residues phosphorylated by protein kinase C are indicated by an open arrow (see Protein phosphorylation, 54-32 and the [PDTMj, Fig. 6). The position of the conserved cysteine residue in the C-terminus (Cys107 residue in the C-terminus of the rat 1sK protein, Cys106 in mouse and human) which appears to act as an oxidizable site (for details, see Table 7 under Channel modulation, 54-44 and the [PDTMj, Fig. 6) is indicated by a black arrow. Underlined sequences marked N-34 and C-27 represent minK/lsK peptides that have been shown sufficient to induce Cl- or K+ currents (respectively) following superfusion or microinjection into untreated Xenopus oocytes (for details, see Selectivity, 5440). (Reproduced with permission from Swanson et al. (1993) Semin Neurosci (1993) 5: 117-24.) (From 54-19-·01)

Gene organization Generation of heterogeneous mRNA species from the rat 1sK gene 54-20-01: The entire IsK gene consists of three exons spanning approximately ",10 kb of genomic DNA. Analyses of the organization and expression pattern of the IsK gene from rat55 and mouse 7 have shown (i) mRNA initiation to occur from two different upstream exons t followed by (ii) an uninterrupted downstream exon covering the protein coding and 3' untranslated regions of the mRNA. The single gene is involved in the production of multiple mRNA species through a number of distinct mechanisms including (i) transcription initiation at different sites; (ii) alternative RNA splicing and (iii) polyadenylation at least three alternative sites55 " Thus RNA splicing occurs outside the protein-coding region giving rise to different mRNAs which vary in their 3' untranslated region. Figure 5 further delineates the structural organization of the rat IsK gene, as described in ref. 55 .

Cross-species conservation of 1sK gene organization 54-20-02: minK/lsK gene structural characteristics appear similar in rat, mouse, guinea-pig and human species44 • These shared features extend to (i)

1

e_n_t_ry_54

_

being present as a single-copy gene within the haploidt genome; (ii) the protein-coding sequence being present on a single uninterrupted exont; (iii) the presence of a 5' untranslatedt region intront and (iv) usage of multiple alternative polyadenylylationt sites for transcript processingr. For species differences in 1sK voltage dependence, kinetics and sensitivity to external La3+, see Activation, 54-33; Current-voltage relation, 54-35 and Blockers, 54-43.

Putative role of alternative mRNA transcription in the absence of minK protein variants 54-20-03: The mRNA variants arising from the IsK gene locus (this field, above, and Transcript size, 54-17) are predicted to encode identical minK protein sequences in those tissues where it is expressed, suggesting that the alternative forms play a role in expression control (i.e. by employing the different promoters t, enhancers t and regulatory elementsr, as opposed to producing structural variants)55. IsK gene transcripts are differentially expressed during development in a number of tissues (e.g. uterus and heart - see Developmental regulation, 54-11). IsK transcriptional control is known to be coupled to endogenous receptor/transducer signalling systems (see, for example, regulation of minK gene expression by oestrogen under Developmental expression, 54-11).

Homologous isoforms 'Species' sequence variations correlated with functional differences 54-21-01: IsK gene structure and gene regulatory mechanisms appear 'broadly conserved' across vertebrate species (see Gene organization, 54-20). At the amino acid level, species variants display high percentage identities (typically > 700/0 in vertebrates, see below) although 'species-dependent' sequence variations 441'59 have been specifically correlated with functional differences in a number of cases (see Protein phosphorylation, 54-32; Activation, 54-33; Current-voltage relation, 54-35 and Blockers, 54-43). Mouse and rat IsK share 920/0 sequence identity; human isoforms are 76-770/0 identical to the rodent sequences.

Protein molecular weight (purified) 54-22-01: MinK proteins typically migrate as a band of approximately 15 kDa (e.g. from rat kidney) following denaturing PAGE.

Protein molecular weight (calc.) 54-23-01: The rat kidney minK ORFt predicts a protein molecular weight of 14.7kDa (14698Da).

Sequence motifs 54-24-01: minK proteins contain two conserved consensus sites for Nglycosylation in their N-termini (see Fig. 4 under Encoding, 54-19 and the

II

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H

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IJ

K pK127,28 pKI 29, 30

E:~t

K

GTIIC

... actggattctgCCaCCCaCagagCaCagctccccatctgctttt9tcaaca99agtattgtct~gttgcaggtaggcccacg9a99gcct

Spl

tcagttacccccagaagcacacaggagctgtggtccttagaagtgaccagatgaggactaccatggtga99g999t99gggg999C99cagtgacccttg APl

gctgtgtcttttagctgtagctcagaagcctgtctctgtatcttatctactaggetgacggagccctttcaccagaactgcccctgctgiCtCicacctg tcaagcgqgtgggcaacgctatgaaattgaccaggaagtcacctgggccacaccagcgcttgctcctgctgggagagaaaggcggttcatacttgcctaa -143 Exon1A gaact~GGAGGTGGCGCCAGGGCTGGAGTTCCTCCAGCAACTGACTGCctGTAGCAGAGCCCCGACTGTTTAGCCACCTCTGCAC¢GTCCATCCAGGTC -40 ~~CCGT~C~taa9taacagctcaatatacccttgctgaata9ctgc9999ccacaggggtccagcaggatgtgaagacaaag9gagct9gagaagttc

tgaaccatctctgaeagtgtttggaactatttaacaaggtgtttgtccagtgctgacccagtcatctgtcctctttattctcccacagactgtctgccag GTI~

Sph

GTI

ccctaagcccacccgctggagtcaaagtgcttcctgccctt9999ct999tgag9catgcagcatgcagg9t99999tgcagggtgggttgggtaacctc ~ p U 1 Sp1 1 ~ 2 ~ -.. 2 cgagacctgggctgactgagccaagccctgcctgctggaagccccagggctctgaggctccgcccatccaaagctgcattgcttaggtgcctctgggatt

I

J .--.tIOF

I

- 9 109

. ~

.

Exon 18

-40

gtctgtcagtcttgtctg~1CtTAGAC¢CCAACACGGGCTCCAAGGATCCCGCTGCACACCAGGCTCCCTTGGCTTCTAGAC¢¢AOgtaagtcaggg 3 ~ 3 ~

l.

-39 agggc t cc tg t e t e ta ggaca gg t ace· •..•••• • ( I ntron - 9 • 5 kbp) .• • •.•..•. t tea t t t ca cG ....A. ,.G..T..T..T~T".G. ,.Cl"l lT. ,.C~C:"': A.,.C~A

-~F

..T~C:":"A.,.G.,.G.,.G-rAAA-r-r~Cr7l c~&r'IfI'f

Exon 2

GAAGCCCCAGGATGGCCCTGTCCAATTCC·······(366bp)·······CTGTCATGAACCCCATAGTTAATTAATAGACAAGTGATAAGTGGGTCTTT ~etAlaLeuSerAsnSer • • . (122aa) . . . LeuSerl 522 CTAGTCAAATGCCTGCCAGTCTTTATTGTAGAGGTACCCTTGAG~TAAGGGG~-~AATAACACCAGTTTTCTGAAATTG~ttctttctata

gtaatcaatttatt···3'

c

g ~ CJ1 ~

Figure 5. Organization of the gene encoding the rat IsK (minK) protein. Boxed text comprises exon lA (complete sequence, exon lB (complete sequence), exon 2 (beginning and ending of sequence, omitting 366 bp/122 aa as shown). In the upper part of the figure, the segment filled in black represents the protein-coding region, while the white segments represent the 5' and 3' untranslated regions. A B

C

D

E

G H I

J K L

II

Partial restriction map and derived gene structure produced from overlapping phage lambda genomic clones spanning the rat IsK region. Region common to two mRNA species designated IsK-A and IsK-B and encoded by a single exon (exon 2, filled black) consisting of 561 bp. Exon 2 thus contains the entire protein-coding region; this region and the 3' untranslated region are uninterrupted by intron insertion. The sequence AATAAC (double underlined) is located at 20 nucleotides upstream from the polyadenylation site. This AAUAAC variant of the Itypical' AAUAAA sequence can serve as a signal for polyadenylation of mRNA (for details on specificity, see Wickens and Stephenson (1984) Science 226: 1045-51). The 5' sequence specific for IsK-A and IsK-B mRNAs are encoded by two separate exons (exon lA, located rvl0.0kb upstream from exon 2 and exon lB, located rvl0.0kb upstream from exon 2). Nucleotide sequences of exons and flanking regions, with nucleotide numbering relative to the first residue of the translation initiation codon at F. Approximate nucleotide position denoting a 5' terminus deduced from primer extension analysis t Approximate nucleotide positions denoting 5' termini deduced from RNAase protection analysis t. The different locations predicted in G and H may be due to the presence of a palindromic structure in this region (see J). Single-headed numbered arrows indicate short direct repeat sequences Double-headed arrow indicating the location of a palindromic structure. Predicted alternative splice patterns as deduced from composite sequences of eDNA clones pK127 to pK130 as indicated. Ends of large intron of 9.5kb (internal sequence omitted). Other symbols Neither a TATA boxt nor a CAAT boxt is present in the 5' flanking regions of exon lA or exon lB. There are, however many potential promotert , enhancert and cis-regulatoryt element motifs present in the 5' flanking regions of both exon lA or exon lB (indicated by GTHC, Spl, APl, GT1, and Sph (for details on specificity, see Wingender, E. (1988) Nucleic Acids Res 16: 1879-902).

(Reproduced with permission from Iwai et al. (1990)

J Biochem Tokyo 108: 200-6.)

(From 54-20-01)

g t""t

~ c.n ~

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_t_ry_54---'

[PDTM}, Fig. 6). The role of N-glycosylation in 'polarized' expression of minK has been described60 . A single cysteine residue in the C-terminus (Cys106 residue in the C-terminus of the human/mouse IsK proteins, Cysl07 in rat) appears to act as an oxidizable site (ibid.; for details, see Table 7 under Channel modulation, 54-44 and the [PDTM), Fig. 6). There are no consensus N-terminal signals predicted from minK cDNA sequences, suggesting that they are not proteolyticallyt processed upon insertion in the membrane. For sequence motifs associated with modifications by protein kinases, see Protein phosphorylation, 54-32.

Southerns 54-25-01: Low-stringencyt hybridization of minK probes to rat genomic DNA digested with panels of restrictiont enzymes have shown 'not more than two bands,6. Such banding patterns are consistent with a single-copy gene (or limited number of minK-related sequences) being represented in the genome.

STRUCTURE AND FUNCTIONS

Domain arrangement Membrane orientation of minK subunits 54-27-01: MinK subunits possess a single hydrophobic transmembrane domain (MI, sometimes designated HI) of 23 amino acids (see Fig. 4 under Encoding, 54-19 and the [PDTM), Fig. 6). Immunolocalization of a nine amino acid 'epitope tag' fused to the N-terminus of minK have confirmed the N-terminal domain to be extracellular61 . Effects of Serl03Ala mutations in the rat kidney minK (which prevent inhibition by protein kinase C - see Protein phosphorylation, 54-32) indicate the residue to be intracellular (see

[PDTM), Fig. 6).

Alpha helical arrangement and functional effects of Cys substitutions 54-27-02: A spacing of 'critical' phenylalanines at every third residue have led to predictions of an a-helical conformation for the membrane-spanning domain of IsK (but see Protein topography, 54-30). For functional effects of MI Cys substitutions on activation and subunit association properties, see

Domain functions, 54-29 and Protein interactions, 54-31 respectively.

Domain conservation 54-28-01: The single predicted transmembrane domain (see [PDTM), Fig. 6, uncharged residues ",44-66 in the neonatal mouse heart sequence} shows 96% identity between mouse and human (residues 41-90). In common with ion channel proteins, the N- and C-termini show greater divergence, with the C-terminal being relatively 'more conserved': N-terminal domains share only 60-78 % identity amongst known isolates.

'-.--e_n_t_ry_5_4

_

For predicted N-terminal topography,

N H2

(aa 1) Approx position of 'extra' a.partate

~

se. legend.

~::~~~g(~~)

N- and C-terminal 'double· deletion' mu1ants (OAF's of 63 aa

1~O :~ ~~~~al: ~~~:red

are~(e.g.~10-39:~94-130.

to 129 aa for mouse, human and guinea-pig)

delineated by

~

symbols on figure)

Extracellular

Monomeric domains

Intracellular

(' ~

Cy.107

PKC-Inhlblton

_':!='~~.~-'~~--~ SH

I

PltC: • 10110210310.105106107108 Leu Qlu .IIJlX Phe Arg Ala eys Tyr

Rat

(S103 mutants are 'non-phosphorylatable' by PKC)

l

lIouse

Leu Qlu .IIJlX Phe ArIiJ Ala eys 'ryr Leu Qlu .IIJlX Tyr Arg Ser CYI Tyr

B\DII&D

", I

Note: IsK proteins also appear to form aggregate. within phospholipid membranes

COOH

Guinea-pig (ha.ologous reliJion) Leu alu Asn eys ArIiJ Ser Cys eys (Guinea-pig native IK,5 & recombinant 15K are 'PKC-enhanced') See Protein phosphoryfation. 44-32

(aa 130)

(

.

(C""'~lIn"n9

R

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could Includ"

c-s-sij . other minK subunits

C-SH ~

- cytoskeletal proteins ) Suggested minK subunit stoichiometry by experiments of Wang and Goldstein (1995) Neuron 14: 1303-1309. See Predicted protein topography, 54-30.

~

Key • •

-~

- other membrane proteins

Likely site for minK chelation by mercury ions See Channel modulation, 54-44.

Dlsulphlde bond formation at the cysteine 'oxidizable site' (Cys 107)

1 - - - - - - See Channel modulation. 44-30.

to protein kinase C substrate Arg-Ser-Lys-Lys (aa 68-71)

- Posltlon.of CamKII

l' - Positions

)

m.o.1I! (extracellular, non-functional; aa 35-41)

of motifs lor N-glyc05ylation (ae 5 and ae 26)

NOTE: All relative positions of motifs. domain shapes and sizes are diagrammatic and are subject to re-interpretation.

Protein symbol (K)

· ·:· :,.'~ O

";:-:":::'::1.

: 1,~,

.J. . · · · ·

+..•....

1:'"

Figure 6. Protein domain topography model (PDTM) for MinK (lsK) protein monomers. Amino acid residue positions apply to the rat kidney isoform (Takumi et al. (1988) Science 242: 1042-45). Note: The subunit topography is shown tunfolded'. Some studies (see Predicted protein topography, 54-30) have suggested that both the N-terminal and transmembrane segments of 1sK adopt a-helical structures (with both regions being located within the lipid bilayer while their linking segment lies on the surface of the membrane - see also later paper predicting tilted f3-strand structures in transmembrane domain, ibid.). Note: For interpretation of minK expression data in Xenopus oocytes, see Protein interactions, 54-31 and Selectivity, 54-40. (From 54-28-01)

Domain functions (predicted) Note: For interpretation of minK expression data in Xenopus oocytes, see Protein interactions, 54-31 and Selectivity, 54-40.

1sK transmembrane domain Cys substitutions affecting activation properties 54-29-01: A series of six mutants of the rat kidney IsK protein have been constructed where consecutive individual amino acids (residues 53-58) of the transmembrane region have been substituted by cysteine62 . Oocyte expression of one mutant (Phe55Cys) displayed activation curves that shift in a hyperpolarizing direction, while for another mutant (Phe58Cys) activation curves shift in a depolarizing direction, suggesting that the (hydrophobic) phenylalanine residues may play a critical role in IsK activation gating. The spacing of 'critical' phenylalanines at every third residue may indicate

_ entry 54 ~-------

(a)

500

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300 t=~ 0Coo

u:i

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o

100

~g~2~~~~~~~~~~~~~~g~~g2~~~~~~~g~~~~ ~~~:

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(b)

300......-------------------------------,

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(c)

200

100

o ~o~oo~~~N~~~~~~~~~o~~g2~~~~g~~g~~g~

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~bbffi~ zQ)

c(

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Cl

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EXTRACELLULAR

(d) Rat Human

CYTOPLASMIC 100

120

130

ARVLESFrr~I~A~V~THLPEL~LS ARVLESY~S~V~L~I~NTHLPET~SP

AT OR 00 01< N E

Figure 7. Summary of functional effects following amino acid substitutions in 1sK sequences. In (a) to (c), the columns show the mean percentage channel activity' for specified site-directed mutant 1sK proteins (as shown) relative to wild-type 1sK (WT) expressed in Xenopus oocytes*. The different histogram sets compare channel activities for the same set of mutants (measured 90 s after depolarization from a holding potential of -60mV stepped to -20mV (in (a)), to OmV (in (b)) I

l_e_n_t_ry_54

_

an a-helical conformation for the membrane-spanning domain of IsK. These results also indicate that one face of the helix may represent a region of subunit association (see also Protein interactions, 54-31). Note: Expression of the remaining four eys mutants (positions 53, 54, 56, 57) in Xenopus oocytes resulted in currents which were similar to wild-type62 . Mutations in the hydrophobic domain which do not significantly alter expression of the voltage-gated currents are described in refs S3,S4 • See also Fig. 7, Protein phosphorylation, 54-32; Selectivity, 54-40 and Blockers, 54-43.

Functional N- and C-terminal tdouble-deletion mutants' 54-29-02: Following oocyte expression, an N- and C-terminal deletion mutant designated ~10-39:~94-130 (see [PDTMj, Fig. 6) containing only 63 of the 130 residues of the rat IsK protein supports K+ currents that are indistinguishable from those expressed from wild-type proteins63 • This demonstrates that the internal (most highly conserved) sequences are 'sufficient' for the formation of functional channels (or, alternatively, do not impair putative 'activator' functions - see Protein interactions, 54-31). Comparative note: These conclusions should be compared with those describing the ability of the N34 and C-27 minK peptides alone to activate el- and K+ channel activities in oocytes (for details, see Selectivity, 54-40). The N-34 and C-27 peptide sequences are underlined on the sequence alignment (Fig. 4) shown under Encoding, 54-19.

Cross-references to other minK structure-function analyses 54-29-03: Numerous other point t and deletion/truncation t mutants of minK subunits have been expressed, some of which give rise to dominant-Iethal t phenotypes (see Predicted protein topography, 54-30), changes in I-V relationships (see Current-voltage relation, 54-35), subunit association and putative 'activator' functions on 'silent' channel activities (see Protein interactions, 54-31 and Selectivity, 54-40), susceptibility to modulation by

and to +20mV (in (c)). Unshaded columns denote substitutions outside the transmembrane domain M1, with shaded columns indicating mutants within the M1 region. In (d), the aligned sequences of rat and human IsK are annotated with positions of potential N-glycosylation sites (CHO), the domain positions, and the one-residue tdeletion'in the human IsK sequence (hyphen, see also the [PDTMj, Fig. 6). Locations of amino acid substitutions are mapped by reference to their tgross' effects: 'J, mutants showing tmoderate' reductions in channel activity; ., mutants showing tdrastic' reductions in channel activity; 0, tsilent' mutants showing no significant effects on IsK activity; e, positions of mutants which tenhance' IsK activity. The thick arrow denotes the direction of K+ current permeation. Note: The results shown here summarize effects within a single comparative study (Takumi eta al. (1991) J BioI Chern 266: 22192-8 from which the figures are taken with permission); other structure/function studies are indicated in the text. *Note: These and other results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40. (From 54-29-03)

III

_'--

e_n_t_ry_54_

protein kinase C (see Protein phosphorylation, 54-32)J ionic selectivity functions (see Selectivity, 54-40), Hg2+ ion chelation patterns or sensitivity to oxidants (see Table 7 under Channel modulation, 54-44). In one extensive study 63J a series of 31 amino acid mutations were made in the IsK protein (mostly in the 'internal! regionJ see previous paragraph) which produced a range of effects on IsK channel activity (including 'silene or 'neutral! effects! through 'moderate! to 'drastic! reductions in activity! to those which conferred IsK current enhancement). A summary of these 'variable effects J by reference to specific mutations is shown in Fig. 7 (for further details, see ref. 63).

Predicted protein topography Note: For interpretation of minK expression in Xenopus oocytes, especially minK-KvLQTl co-assembly data, see Protein interactions, 54-31.

Subunit topography/stoichiometry/assembly patterns of t.junctional' minK protein complexes 54-30-01: Subunit stoichiometry of minK channels in Xenopus oocytes has been investigated by co-expression of wild-type t (wt) minK subunits and a point mutant t D77N of minK subunits64 . In this study! evidence in support of three key assumptions was reported! namely (i) wild-type and D77N monomers are expressed in an equal and independent manner (see note 1); (ii) they do not rreferentially self- or transassociate and (iii) D77N has a 'dominant lethal! effect on minK functional expression (see note 2)64. By means of kinetic and binomial distribution analyses (but not single-channel analyses)! these observations supported a model for 'complete! minK potassium channels in which just two minK monomers are co-assembled with other (unidentified) non-MinK subunits (see the PDTM, Fig. 6). Notes: 1. MinK D77N mutants pass no current but have been shown to be expressed in the plasma membrane (as determined by immunolocalization of an incorporated c-myc epitope-tag t QKLISEEDL between minK aa residues 22 and 23). 2. When wild-type and D77N minK cRNAs are coinjected in a 1: 1 ratio J only a 'small fraction! of the current seen with wildtype cRNA alone is observed; following co-injection of 2 ng wild-type plus 0.5 ng D77N cRNA! a fraction of 0.63 ± 0.04 of wild-type current is obtained. Since the latter currents have pharmacological! selectivity and gating properties very similar to those of wild-type alone! it suggests that only 'fully wild-type! channels can conduct current64 . Note: The 'heterotetramer! model suggested by the D77N/wild-type study is incompatible with 'homomeric! models (see next paragraph).

'Homomeric' models for minK oligomers 54-30-02: According to an independent study 65 minK channels were predicted to form 'by assembly of at least 14 monomeric subunits!65. These authors applied a binomial distribution t to the reduction in current amplitudes (at -20mV) observed in oocytes (see note 1) following co-injection of a constant amount of wild-type minK mRNA with increasing amounts of mutant S69A mRNA (see note 2). On the basis of the stoichiometric J

entry54

_

I" - - - - - - - - - -

analysis, a model was proposed for channel formation. Notes: 1. These and other results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions, 5431 and Selectivity, 54-40. 2. Mutant S69A (see also Fig. 7) was employed in these experiments to induce a I discernible shift' in the I-V relationship at -20mV (i.e. to more depolarized potentials. minK currents are not normally observed at potentials negative to OmV with this mutant alone. The relationship of these findings to those showing minK acting as a channel regulator (see Protein interactions, 54-31) is unclear.

Membrane-bound secondary structure prediction of a-helical conformation 54-30-03: Synthesis, fluorescent-labelling, structural and functional characterization using circular dichroism t of five polypeptides (comprising 20-63 amino acids within the rat IsK protein) have suggested that both the Nterminal and transmembrane segments of IsK adopt a-helical structures66 (compare next paragraph). This approach has also suggested that (i) both the a-helical transmembrane segment and the N-terminal of IsK are located within the lipid bilayerj (ii) the linking segment between the two segments lies on the surface of the membranej (iii) IsK proteins form aggregates within phospholipid membranes and (iv) certain truncated forms of IsK protein are unable to form functional K+ channels in planar lipid membranes 66 . Note: The latter observations were taken as evidence to support the view that the IsK protein alone cannot form K+ channels (see Protein interactions, 54-31).

Membrane-bound secondary structure prediction of {3-strand conformation 54-30-04: Twenty-seven residue peptides corresponding to the transmembrane domain of minK/lsK have been incorporated into synthetic saturated-chain phospholipid membranes prior to structural characterization by a number of spectroscopic techniques 67. Under defined conditions, the minK/lsK transmembrane peptide has been determined to adopt a predominantly ,a-strand secondary structure (compare previous paragraph). In this case, the peptide backbone is oriented at an approx. 56° angle relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film (in the gel phase) does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes transition to the fluid phase67. ESR and NMR spectroscopic data indicate (i) that the incorporated peptide restricts rotational motion of lipids in a similar manner to that found for integral membrane proteins and (ii) a selectivity in the interaction of anionic phospholipids with the peptide. Other data indicate that (i) approximately two to three phospholipid molecules interact directly with each peptide monomer (consistent with a limited degree of aggregation of the ,B-sheet structuresh (ii) lipid-peptide complexes have a lamellar structure and (iii) the surface areas occupied by lipid molecules are "'-J30A2 per chain in the peptide complexes, while the additional membrane surface area contributed by the peptide is "'-J112A2 per monomer, consistent with the strong (56°) tilt in the long axis of the ,Bstrand in the dry film (as above)67.

II

II

Table 4. Summary of factors supporting the hypothesis that minK/1sK proteins act as activators of endogenous channel activities. The table lists factors which have been put forward which counter the proposition that minK forms independent channels. Features of minK regulation that are described in other fields of the entry are largely omitted; in consequence, the listing is not exhaustive, and is mainly intended to provide some perspective on the ongoing debate (see also paragraph 54-31-01 and Fig. 8). (From 54-31-01) Factor/effect/observation

Description, conditions, references (see note 1)

Sufficiency of synthetic hydrophilic peptides to induce Cl- or K+ channel activities (direct evidence for regulatory function)

54-31-02: Addition of synthetic IsK hydrophilic peptides derived from C- and N-termini (see next row) are sufficient to activate slow K+ and Cl- channels in untreated Xenopus oocytes. The peptide-induced biophysical and pharmacological characteristics are similar to those exhibited by the native IsK protein68 . For definition of peptides and selectivities of induced currents, see Fig. 10 under Selectivity, 54-40.

Designation of Icritical domains' for induction of Cl- or K+ channel activities

54-31-03: IsK mutagenesis has identified N- and C-terminal domains as critical for the induction of Cl- and K+ channel activities, respectively: The S68T mutant shows a 'complete loss' of K+ current activity with 'no alteration' of Cl- channel activityS (see also mutant S69A under Fig. 7). Truncation of the IsK C-terminus (in the Tr80 mutant) resulted in a 'complete loss' of the K+ channel activity with a 'striking increase' of Cl- current amplitudeS (compare to the reportedly functional 'double-deletion' mutant ~10-39:~94 130 described under Domain functions, 54-29). 54-31-04: Deletion of 28 residues of the IsK N-terminus (in the ~11,38 mutant) 'abolished' expression of the Cl- current and 'doubled' the amplitude of K+ currents.

KvLQT1/minK co-expression

See paragraph 54-31-01, Fig. 8 and 'lack of minK expression in mammalian expression systems' (this table, below).

Noted lack of K+ channel selectivity determinants

54-31-05: IsK/minK proteins do not exhibit 'signature sequences' associated with all other 'cloned' channels that are K+ -selective, Le. a conserved K+ -selective pore (P) region (latterly designated H5) flanked by either six or two membrane-spanning regions (compare entries such as 1NR K [subunits], entry 33; VLC K eag/elk/erg, entry 46 or VLC K Kv1-Shak, entry 48). The difficulty in accounting for a pore structure with both K+ and Cl- selectivity properties led to the suggestion that the IsK protein itself was an 'activator of endogenous and 'dormant' K+ and Cl- channels/so

B M-

~ CJl ~

54-31-06: Note: No structural model for K+ channel pores incorporating minK subunits has been suggested; existing models for Kv channel pores (e.g. see entries 48 and 49) do not appear directly applicable. Observation of CI- selective currents following heterologous expression in Xenopus oocytes

54-31-07: IsK/minK expression in Xenopus oocytes may induce a Cl- -selective current 5 'very similar to' or 'the same as' a Cl- current produced by phospholemman (PLM) expression in oocytes (for comparison, see below and description of phospholemman under Miscellaneous information, 54-55). These Cl- -selective currents were described as exhibiting biophysical, pharmacological and regulatory characteristics 'very different' from those of IsK-induced K+ channel activities 5 (see below).

Differential Ipermissive' expression conditions

54-31-08: IsK cRNA at low concentrations (threshold at 0.03 ng/Jll) induces slowly activating, K+ -selective currents upon depolarization5 . 54-31-09: IsK cRNA at high concentrations (typically 1 Jlg/Jll) induces slowly activating, Cl--selective currents upon hyperpolarization (amplitude -0.S±0.3JlA at -130mV; threshold potential -80mV)5. See also Activation, 54-33.

Differential pharmacology

54-31-10: The compound DIDS (see Openers, 54-48) was reported to 'potently inhibit' both IsK- and PLM-induced Cl- currents without affecting the K+ current (compare conflicting result, ibid.). 54-31-11: Ba2 + ions were reported to be more potent blockers of both IsK- and PLM-induced Cl- currents (IC so 0.4mM and 0.3mM respectively) than the K+ current (ICso 3mM) (compare Blockers, 54-43). 54-31-12: Clofilium was an activator of both IsK- and PLM-induced Cl- currents5 (compare Blockers, 54-43).

Differential modulation

II

54-31-13: While IsK-associated K+ levels were enhanced by elevating internal Ca2+, IsK-associated Cl- currents were enhanced by lowering external Ca2+S (see also Channel modulation, 54-44).

g t""I"

~ c.n ~

II

Table 4. Continued Factor/effect/observation

Description, conditions, references (see note 1)

54-31-14: Increasing osmolarity of the extracellular medium by 40 mOsM using saccharose or sorbitol69 decreases K+ channel activity by rv69% ±3% (the same treatment was 'without effect' on CI- channel activity following IsK or PLM expressions - compare Channel modulation, 54-44). Differential phosphoregulatory properties

54-31-15: The phorbol ester PMA decreased the IsK-associated K+ current (see Protein phosphorylation, 54-32) but strongly increased the IsK-associated CI- current and notably, did not affect PLM-induced CI- currentss. 54-31-16: The Serl02Ala mutant suppressed PKC inhibition of the K+ current (as in Protein phosphorylation, 54-32) but had no effect on PKC enhancement of the CI- current. This was taken as evidence for the existence of two different sites for PKC regulation on IsK, in support of a model in which IsK activates two distinct endogenous channels in oocytes S (see 'Alternative models', below).

Lack of minK expression in mammalian expression systems and IsK reconstitution studies in bilayers

54-31-17: Prior to the successful co-expression of minK and KvLQTl, numerous unsuccessful attempts were made to express minK/lsK alone in several eukaryotic cell types including the skeletal muscle cell line C2C12, the T lymphocyte cell line Jurkat, the vaccinia/T7, the fibroblastic cell line CHO and Sf9/baculovirus systems 70. In each case, although the transfection procedure appeared successful, no current with IsK characteristics could be recorded from these cells. Bilayer reconstitution experiments using membranes highly enriched in IsK protein were reported as inconsistent with IsK being a channel located in membranes of intracellular organelles 70. Other attempts to express the IsK protein as a K+ channel in lipid bilayers have also failed 71 . Note, however that recombinant phospholemman proteins can support lindependent' anion channel formation in lipid bilayers 72 (see Miscellaneous information, 54-55). Note: minK/KvLQTl co-expression yields large current in CHO, Sf9 and COS cells (see paragraph 54-31-01 and Fig 8).

B t""t'"

~ CJ1 ~

Co-existence of lfunctional' and Inon-functional' forms of IsK in the plasma membrane of oocytes

54-31-18: When Xenopus oocytes are injected with up to 50ng amounts of 'epitope-tagged' minK mRNA (see Predicted protein topography, 54-30 and the [PDTM], Fig. 6), the levels of surface protein are proportional to the amount of injected mRNA. Notably, however, the amplitude of the minK current recorded in the oocyte system saturates at 1 ng of injected mRNA. At mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA (activation is slower with higher levels of minK protein in the plasma membrane). Collectively, these and other results were taken to support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte, with non-functional forms of minK also being present61 (see also Activation, 54-33, Kinetic model, 54-38 and Channel modulation, 54-44).

Alternative models

54-31-19: Essential features of alternative models in which the IsK protein (designated as IsK, Cl) acts as 'a potent activator of endogenous and otherwise silent K+ or Cl- channels' are illustrated in ref. 5 . These models invoke a low-affinity interaction between IsK, Cl and phospholemman (i.e dependent on high IsK cRNA concentrations - but note evidence for 'independent' phospholemman channel formation, this table, above). Conversely, these models invoke a high-affinity interaction between IsK subunits and endogenous K+ channels (i.e. sustainable with low IsK cRNA concentrations).

Note: 1. For comparative purposes, see also Phenotypic expression, 54-14.

II

~

= t '+

~

CJ1 ~

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_4_ _

Protein interactions minK as a regulatory protein underlying IK,s in association with KvLQTl protein 54-31-01: There has been much debate regarding the ability or inability of minK-like proteins to form integral, ion-selective channels when expressed alone. Evidence for the hypothesis stating that minK/lsK is a regulator (activator) of endogenous channels through protein interactions5 is summarized in Table 4. Significantly, it has been shown that short synthetic peptides from the cytoplasmic and extracellular domains of minK protein are sufficient to induce K+ and CI- channel activities in Xenopus oocytes 68 • Furthermore, the type of heterologous cell used for minK expression appears to be critical for 'latent channel' activation. To date, no mammalian heterologous cell type has been able to support expression of minK alone (ibid.), although Xenopus oocytes produce robust current when injected with minK cRNA (see various fields of this entry). In late 1996, two papers appeared simultaneouslr,3 supporting a hypothesis that minK and KvLQTl protein subunits co-assemble to form channels conducting current 'nearidentical' to native cardiac IK,s.. In keeping with previous observations (see above) minK could be expressed 'alone' in Xenopus oocytes but not in the mammalian cell lines COS2 or CH03. Furthermore, in keeping with the 'latent channel' hypothesis; (i) a eDNA encoding a Xenopus homologue of KvLQTl was retrieved from an oocyte c:DNA library3; (ii) minK produced a large potentiation in KvLQTl current following mammalian cell expression and (iii) co-expression of FLAG epitope-taggedt versions of KvLQTl coexpressed with minK in the Sf9 (insect epithelial) cellline2 showed associated molecular species consistent with minK-KvLQTl protein interactions in vivo. Together with earlier observations linking KvLQT1 mutations with the most common form of inherited long QT syndrome (LQT1, see Chromosomal location under VLC K eag/elk/erg, 46-18) the minK/LQTl studies2,,3 therefore implicated dysfunction of native IK,s in LQT1, and provided a model for IK,s as an important 'target' for class mt antiarrhythmic drugs. Representative traces showing the effect of minK/KvLQT1 co-

expression in mammalian cells (i.e. in th.e absence of tendogenous or latent' subunits) are shown in Fig. 8.

Functional interactions of minK with ion pump/transporter proteins 54-31-20: Xenopus oocytes expressing slowly activating IsK channels

superfused with the nitroso-donort S-nitrosocysteine (SNaC, see Resource C) result in an increase of IsK which is greatly enhanced when the amino acid exchanger rBAT 73 is co-expressed (for further details on SNaC modulation and the role of co-expressed rBAT facilitating SNac transport into the oocyte, see Channel modulation, 54-44). In kidney epithelial cells, possible functional interactions with the Na+/K+-ATPase and Na+-coupled transport systems have been described12 (see also Fig. 2 under Phenotypic expression, 54-14). Notes: 1. MinK expression in non-excitable epithelia is associated with permeation of K+ ions13 into both the interstitial and luminal spaces (see Fig. 2 under Phenotypic expression, 54-14 and Protein

l_e_n_try_5_4

_

(b) __ K V LQT1 + hminK C 1.0l-.-KvLQT1

~

o.sl

B 0.6

.

Q)

:E; 0.4 co

~ 0.2 04J=lJ:-+.......--.,-------r--~---.

-60 -40 -20 0 mV

20

40

(e) Kv LQT1 + hminK hminK

K V LQT1

~] 2s

Figure 8. KvLQTl and human minK co-expression in CHO cells induces a current lnearly identical' to cardiac IK,s. (a) Currents recorded from CHO

cells transfected with KvLQTl alone. (b) Normalized isochronal activation curves for KvLQTl and KvLQTl + minK. (c)-(e) Currents from cells transfected as shown, illustrating potentiating effect of minK + KvLQTl interaction. (Reproduced with permission from Sanguinetti (1996) Nature 384: 80-3.) (From 54-31-01)

distribution, 54-15). 2. Structure-function studies of the single putative (0helical) membrane-spanning domain of IsK have indicated that one face of the helix may represent a region of subunit association62 (see Domain functions, 54-29).

minK antisense oligonucleotides reduce I KR (rapid component) amplitudes in AT-l cells -54-31-21: In atrial tumour myocyte lines (AT-I cells) minK mRNA is detectable, but no IK,s can be recorded10 (see also Cloning resource, 54-10). AT-l cells exposed to antisense oligonucleotides (targeting the 5' translation start site of the minK cDNA cloned from an AT-l library) exhibit a Isignificantly reduced' I Kr amplitude (= rapid component) compared with 'sense' and 'medium-only' controls. I Kr activation, rectification, deactivation and sensitivity to the blocker dofetilide were reported as 'unaffected'. Note: 1. Two different antisense oligonucleotides produced the same effect on I Kr amplitude without effect on cell size or on L- or T-type Ca2+ currents native to AT-l cells10. 2. Compare 'equivalence' of I Kr to erg-subfamily currents (see VLC K eag/elk/erg, entry 46) and see effect of minK/LQTl co-expression, described under Phenotypic expression, 54-14 and this field, above.

II

Table 5. Regulatory properties of heterologously expressed minK (IsK) proteins potentially involving phosphorylation. For comparative purposes, phosphorylation properties of the native cardiac current IK,s are also listed, together with reported species differences, and notable effects in selected mutants (all denoted by underlined prefix:). MinK/IsK data are applicable to heterologous expression in Xenopus oocytes, unless otherwise indicated. (From 54-32-01) Effect/regulator/mechanism

Characteristics, species dependence, references (see note 1)

Inhibitory effects of PKC and PKC activators e.g PDD, PMA. (inhibition by protein phosphorylation)

Rat, kidney minK wild-type: Heterologously expressed minK protein activities are markedly inhibited following microinjection of purified protein kinase C (PKC). The PKC-activators PDD (phorboI12,13-didecanoate, a phorbol ester, rvSOnM) and OAG (1-oleoyl-2-acetyl-rac-glycerol, a diacylglycerol analogue, rv 10 JlM) also inhibit the current 74 . Protein kinase C phosphorylation of the minK channel shifts its voltage-dependence of activation 74 (see also Voltage sensitivity, 54-42). Mouse, cardiac minK wild-type: Inhibitory effects have been observed for PMA (12-phorbol myristate 13-acetate, a phorbol ester t ) showing a 70% inhibition (at 0.1 JlM) with OAG also inhibiting minKs. Rat, kidney minK Ser103Ala mutant: Inhibition of the current is not seen in S103A mutants (a marginal enhancement occurs with PDD, although other properties of the current remain unchanged). This has been taken to indicate that inhibition results from direct phosphorylation of the rat kidney protein at Ser103 74 (see the {PDTM}, Fig. 6). In the rat kidney minK, Ser103 lies within a sequence that resembles the PKC consensus site Arg-Val-Leu-Glu-Ser-Phe-Arg. Substitution of Ser103Ala also prevents the inhibition of minK by PDD and OAG, whereas Ser7SAla has no significant effect upon the IsK current. Guinea-pig, cardiac rninK/IK,s: In 'sharp contrast' to the rat and mouse IsK currents (above), activation of protein kinase C (PKC) with phorbol esters moderately increases the amplitude of the guinea-pig IsK current44, analogous to its effects on the endogenous IK,s current in guinea-pig cardiac myocytes. Guinea-pig, cardiac minK mutant: Mutagenesis of the guinea-pig IsK sequence at four residues in the cytoplasmic tail can alter the phenotype of the current response (i.e. from a small PKC-dependent enhancement to PKC inhibition, the latter being characteristic of rat and mouse IsK (e.g. PKC phosphorylation at Ser102 decreases IsK current amplitude)44.

Species difference: Wild-type guinea-pig cardiac IsK/IK,s enhanced by PKC activators. For an illustration of species sequence differences at this site, see the {PDTM}, Fig. 6.

('l)

= ~ c.n ~

Preliminary evidence for protein kinase C mediation of minK channel down-regulation following activation of apical P2U purinoceptors in K+ -secretory epithelial cells of the inner ear has appeared 75 • Stimulatory effects of PKC inhibitors, Rat, kidney minK: The protein kinase C inhibitor staurosporine (rv3 JlM) prevents minK e.g staurosporine inhibition under conditions similar to those described above (e.g. see refs8 47 74). 7

Stimulatory effects of PKA and PKA activators (mechanism unclear)

Single PKA consensus site eliminations have no apparent effect.

Small stimulatory effects of microinjected Ca2 +

(see Channel modulation, 54-44)

7

Rat, uterine minK wild-type: In a heterologous expression system, the amplitude of minK currents can be substantially increased by agents that raise cAMP levels and decreased by treatments that lower cAMP levels 76 (see also Channel modulation, 54-44). Pre-injection of a protein inhibitor of the cAMP-dependent protein kinase blocks the effect of increased cAMP levels, without any change in voltage dependence or kinetics of the channel 76. A form of parathyroid hormone (PTH) regulation in oocytes can be mimicked by activators of protein kinase A (PKA) (see Receptor/transducer interactions, 54-49). Mutations that eliminate the only PKA consensus site on the minK protein do not block the effects of kinase activation. The basic mechanism of PKA regulation of minK proteins thus remains unclear. Hypothetically, these effects might be due to actions at other proteins that regulate minK current amplitude; alternatively, minK proteins may be inserted into or removed from the membrane in 'minK-loaded' vesicles in response to changes in PKA activity 76. Note: PKA has been shown to increase the membrane surface area of oocytes, consistent with this hypothesis. Guinea-pig, cardiac 11K,s: 'Positively regulated' by PKA in response to ,a-adrenergic stimulation 77 (for a review, see ref.78, see also Receptor/transducer interactions, 54-48). Mouse, cardiac minK: minK cloned from neonatal mouse heart is enhanced twofold by microinjected Ca2 + and Ca2 +Icalmodulin-dependent protein kinase II; this stimulation is reversed by the calmodulin antagonist W_7 8 (but see possible blocking effects of

calmodulin antagonists, this table, below).

II

Elevation of minK current amplitude by raised [Ca2 +h possibly through the activation of an associated calcium-activated protein kinase21,47.

(D

l:S

M-

~ tTl ~

II

Table 5. Continued Effect/regulator/mechanism

Characteristics, species dependence, references (see note 1)

Supplementary note on CamKII motifs

The CAM-kinase II motif region in mouse and rat (residues 35-41, SQLRDDSK) differ from the human sequence (SPRSSDGK) and later analyses have indicated an extracellular location for the N-terminal. The apparent functional modulation by CaM kinase II would require its modulatory site to be intracellular which is incompatible with the established single-transmembrane model shown in the PDTM (Fig. 6).

Inhibitory effects of calmodulin antagonists, e.g trifluoperazine, chlorpromazine, W-7: hypothetical, probably by direct channel blockade (see ref. 79 and Blockers, 54-43)

Inhibition of human IsK channels in oocytes by calmodulin antagonists The calmodulint antagonists trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2(trifluomethyl)-lOH-phenothiazine), chlorpromazine (2-chloro-1 0-(dimethylaminopropyl)phenothiazine) and W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalen-sufonamide) inhibit depolarization-activated IsK channels (ECso between 70 and 100 JlM). Notes: (i) These calmodulin antagonists inhibit IsK at both physiological and enhanced [Ca2+Ji (see Channel modulation, 54-44). (ii) None of the antagonists abolish the inhibitory effects of A23187 (calcimycin) or hypotonic t extracellular fluid on human IsK (see Channel modulation, 54-44).

Note: 1. For potential phosphorylation-independent effects, see Channel modulation, 54-44.

g t'+

~ CI1 ~

1'--_e_n_t_ry_S4

_

Protein phosphorylation 'Down-regulation' of minK activities by PKC-dependent protein phosphorylation 54-32-01: A number of distinct mechanisms for second messenger-dependent phosphoregulation of minK proteins have been proposed (summarized in Table 5). Several of these collective properties show remarkable similarity to those observed for native IK,s components in heart (see Phenotypic expression, 54-14). While minK regulation (inhibition) by protein kinase C isoforms is established, the mechanisms underlying protein kinase A and Ca2+ modulation are still unclear. Analysis of phosphoregulatory patterns for minK proteins are complicated by various 'species-specific' effects, although a number of these have been explained by specific differences in primary structure amongst homologous isoforms (see the [PDTM), Fig. 6}. A further 'complicating' factor for analysis of minK phosphoregulatory properties is its proposed functional interactions with 'latent' channel proteins (summarized under Protein interactions, 54-31).

ELECTROPHYSIOLOGY

Activation Note: All results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40.

Depolarization activates minK (IsK) channels very slowly, possibly by subunit aggregation 54-33-01: minK protein expression in Xenopus oocytes gives rise to K+ currents which slowly activate when the membrane is depolarized to potentials greater than -SOmV (activation occurs at least two orders of magnitude slower than any other ion channel, see example in Fig. 9). Activation thus takes place in the order of 'seconds-to-minutes' and often does not reach a steady state (even after several minutes, hence isochronal t currents, rather than steady-statet currents are generally stated in comparative studies42 ). Activation is sigmoidal in response to voltage steps, with a noticable delay occurring at higher depolarizations (see also next paragraph). Studies employing chemical cross-linking during activation80 (see also Table 7 under Channel modulation, 54-44) have suggested that a major conformational change occurs during minK channel gating (which can be stabilized by cross-linking agents). These findings are also consistent with models in which minK channels activate by voltage-dependent subunit aggregation (see also Protein interactions, 54-31). Alterations of gating parameters by neutral substitutions of Leu52 within the transmembrane domain of IsK are shown in Fig. 7 under Domain functions, 54-29.

Voltage-dependent activation of epithelial IsK proteins in situ 54-33-02: A proposal for activation of IsK-dependent K+ transport (efflux) in secretory epithelia due to the depolarizing effects of Na+ entry (following

111

_'---

e_n_try_5_4_

activation of Na+/sugar or amino acid co-transport systems) is outlined in Fig. 2 and associated text under Phenotypic expression, 54-14. See also Protein interactions, 54-31.

Gating of 1sK dependent on the amount of mRNA injected into Xenopus oocytes 54-33-03: Some studies (e.g. refs. 61 ,84) have shown variable activation kinetics of IsK dependent upon the amount of mRNA injected into Xenopus oocytes. Injection of larger amounts of IsK mRNA (e.g. in the 10-50ng range, versus the 100pg to 1ng range) result in slower activation kinetics (larger tau values) with a longer initial delays during activation. Similar variabilities in activation kinetics can occur with time following single injections of mRNA81 (see also Protein interactions, 54-31 and Kinetic model, 54-38). In contrast to activation, deactivation t of minK does not exhibit the same dependence upon the amount of RNA injected61 . Comparative note: Functional changes dependent upon expression of variable amounts of Kv channel cRNAs in oocytes have also been reported (for brief accounts, see the fields Current type, Inactivation and Blockers under VLC K Kv-Shak, 48-34, 48-37 and 48-43 respectively).

tSpecies differences' in activation/deactivation kinetics 54-33-04: The first 30 s of activation during depolarizations to potentials between -10 and +40mV are best described by a tri-exponential rise function for each of the human, mouse or rat IsK proteins in Xenopus oocytes59. Notably, however, the activation rates of human 18K channels have been reported as 'significantly faster' than those for either !nouse or rat IsK proteins following expression in Xenopus oocytes59 (examples of time constants required for adequate fitting of initial delay and onset: are shown in the legend to Fig 9). Deactivationt of IsK currents is also slow (requiring seconds for full relaxation t) and deactivation rates also show some 'species dependence'. Human IsK currents deactivate more rapidly than the rodent currents, although the deactivation kinetics for each of the species variants are best described by a biexponential decay function 59. Differences in deactivation rate may be attributable to acidic residues in the N-termini of the species variants82.

Electrophysiological parameters for 18K derived from isochronal currents Note: Half-points of steady-state (sic.) activation curves cannot be precisely determined since extremely long times (e.g. 200 s) are required for the current to saturate during step depolarizations42 . Using 30 s depolarizations, however, the slope factor (k) and midpoint (V 1/ 2 ) of the conductance-voltage curves have been estimated at k = 12, Vl~2 = -5 mV (for rat/mouse IsK) and k = 9, V 1/ 2 = -11 mV (for human IsK)8,21, "4.

Current-voltage relation Note: All results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40.

III

1'--_e_n_t_ry_54

(a)

_

50~

(c)

m.v_---_

1600

.-60 ..

1200

(b)

280nAL 400ms

c(

c:

'E 800 CD

t: :::J 0

400

0 -60

280nAL 4s

·40

20 0 ·20 Voltage. mV

40

60

Figure 9. Typical activation of 1sK/minK currents following heterologous expression in Xenopus oocytes*. Records were obtained following microinjection of 100ng rat 1sK cRNA, analysed 3 days post-injection. (a) Rat 1sK currents elicited by 2 second depolarizations from a holding potential -60mV to +50mV in 15mV steps. (b) Rat 1sK currents elicited by 20 second depolarizations with the same voltage protocol (note different time scale). (c) Current-voltage relations for the currents in (a) • and currents in (b) •. Comparative notes: 1. Currents could be well-fit (r = 0.998) by an exponential model of activation t containing a minimum of three independent time constants (Tactl = 0.16 s, Tact2 = 2.1 s, Tact3 = 20 s) (see text). 2. Outward tail currents are generally observed upon repolarization (see (b)), which follow a similar time course for activation, requiring many seconds to decay to baseline. 3. Native 1K ,s recorded from guinea-pig ventricular myocytes6 show similar activation and deactivation kinetics to those described here. Under the same conditions as above, guinea-pig 1K ,s can also be well-fit (r = 0.996) by the triexponential model yielding similar time constants (Tactl = 0.36 s, Tac t2 = 1.5 s, Tact3 = 20 S)6. * Note: These and other results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions (field 54-31) and Selectivity (field 54-40). (Reproduced with permission from Folander (1990) Proc Natl Acad Sci USA 87: 2975-9.) (From 54-33-01)

54-35-01: I-V relations derived for rat IsK are shown in Fig. 9c under Activation, 54-33. Current/voltage relationships of the human and rat IsK currents may 'differ significantly' (e.g. with greater depolarizations required for activation of the human channe159). For other apparent cspecies differences', e.g. in phos-

phorylation patterns, activation/deactivation kinetics, voltage dependence, and La3+ block, see Protein phosphorylation, 54-32; Activation, 54-33 and Blockers, 54-43. For the effect of the minK mutant S69A inducing shifts in the 1-V relationship, see Predicted protein topography, 54-30 and also Fig. 7.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_4------1

54-35-02: Open channel current-voltage relationships determined from macroscopic T minK currents in Xenopus oocytes show rat and human minK current has a mild inwardly rectifyingt characteristic, passing inward current 'at least 20-fold' more readily than outward current83 . Note: A similar rectification property has been reported for the slow (IK,s) component of cardiac delayed rectifier (see also Phenotypic expression, 54-14).

Inactivation 54-37-01: Following their slow, time-dependent activation, currents associated with heterologously expressed minK protein are characteristically non-inactivatingt under maintained depolarization. For deactivationt characteristics, see Activation, 54-33.

Kinetic model 54-38-01: Two kinetic schemes accounting for variable activation kinetics of IsK dependent upon the amount of mRNA injected into Xenopus oocytes have been presented84 (see also Activation, 54-33).

Selectivity Note: Results generated in the Xenopus oocyte expression system should also be interpreted in the light of findings described in paragraph 54-40-01 and those under Protein interactions, 54-31.

Distinct selectivities induced by minK peptides - direct evidence for regulatory functions 54-40-01: Short (27-34 aa) synthetic peptides derived from C- and N-termini of IsK/minK have been determined 'sufficient' to activate (respectively) slow K+ or Cl--selective currents in Xenopus oocytes68 . 'Peptide-induced' currents display biophysical and pharmacological characteristics similar to those exhibited by the native IsK protein. Together with previous (less direct) observations (Table 4 under Protein interactions, 54-31) these data were taken to provide further evidence that IsK/minK represents a regulatory subunit of pre-existing 'silent' (latent) channel complexes rather than a channel per see Figure 10 illustrates typical current traces, I-V relations and selectivity data derived for oocytes microinjected with C-27 IsK peptide (inducing a depolarization-activated K+ current) and perfused with N-34 IsK peptide (inducing a hyperpolarization-activated CI- current). The N-34 and C-27 peptide sequences are also underlined on the sequence alignment (Fig. 4) shown under Encoding, 54-19.

Comparison of IsK-induced channel selectivities with native cardiac l K ,s components 54-40-02: Expression of wild-type minK cDNA in oocytes gives rise to pores exhibiting a permeability sequence typical of 'virtually all' known K+channels: K+ > Rb+ > NHt> Cs+» Na+, Li+ 54,85,86 with tailt current reversal potentialst shifting approx. 58 mV per decade change in external

l_e_n_t_ry_5_4

----'_

K+ 53 (but see notable absence of K+ channel Isignature sequences' and effects of endogenous Cl- current activation under Protein interactions, 54-31). Some variability in permeability ratios to permeant cations have been reported between heterologously expressed IsK and native IK,s (e.g. compare the PK/Pcs of 16.4 determined for native guinea-pig IK,s 87 to 6.24 determined for rat IsK in oocytes; these may be associated with noted sequence differences between the species). Note: Hypo-osmotically induced K+ transport pathways in vestibular dark cells from the gerbil inner ear have ion selectivity and multiple blocker insensitivity consistent with involvement of minK (for details, see ref.88; see also Phenotypic expression, 54-14, especially Table 2).

Apparent MinK selectivity functions investigated by site-directed mutagenesis 54-40-03: Site-directed mutagenesis studies in oocytes with a synthetic minK sequence54 show that K+ selectivity can be altered by specific mutations at Phe55 (for location, see the [PDTM), Fig. 6 and Fig. 7 under Domain functions, 54-29). For example, the mutant F55T yields minK channels that are rv3.5-fold more permeable to Cs+ and NHt (reducing PK/Pcs to 4.6 - see previous paragraph and Domain functions, 54_29)53,54. Note: Despite the indication that minK subunits contribute 'directly' to selectivity properties54, it is still unclear whether these effects are due to formation of homomultimeric minK ion channels alone or necessarily require interaction with one or more endogenous ion channels (see Protein interactions, 54-31). In a later study89 also in oocytes, mutations in a specific minK region (spanning approx. £44 to 148) altering sensitivity influencing external blockade by tetraethylammonium and methanethiosulfonate ethylsulfonate were used to support the view that minK is directly involved in forming a K+ -selective ion conduction pathway89.

Single-channel data 54-41-01: Unitary conductances associated with minK/lsK proteins in oocytes (see Note under Activation, 54-33) are relatively low and have sometimes been described as 'below the limits of detection' (i.e. <1 pS)53. In comparison, native IK,s components (for similarities with IsK, see Phenotypic expression, 54-14) have been estimated to be 'about 3 pS' in guinea-pig atrial cells (in 150mM [K+]o)9o.

Voltage sensitivity 54-42-01: Wild-type rat kidney minK-associated conductances in oocytes (see Note under Activation, 54-33) increases e-fold per 13 mV depolarization. Notably, F55T mutations (see Selectivity, 54-40) do not change this voltage dependence. Differences in the voltage dependence of activation have been noted between IsK expressed in oocytes (half-activated at approx. -10mV); this is considerably more negative than that reported for the native IK,s component in guinea-pig cardiac myocytes (half-activated at approx. +13mV)91.

II

II (b)

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2

N-34 LPFLASLWQETDEPGGNMSADLARRSQLRDDSKL ++

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-20

01

20

40

60

Voltage (mV)

+ -- +

-0.5 20

B C-27 - ++

+ + + -

Control peptides ISSPLFKTLLSAVGSALSSSGGQE

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LLGGGGLKLALLKLGGGGGG +

P-3

+

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a 10 sec

-40

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0. -60

tij

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100

10 1

[K+]o (mM)

RGGSASVHVLPLKLSRRS +

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~-100

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D 0

~ -20

RSKKLEHSHDPFNVYIESDAWQEKGKA

P-1

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++

102

103

~

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Voltage (mV) Figure 10. Induction of Islow' currents with distinct selec- (c) 2 -250 -200 -150 -100 -50 o tivities in Xenopus oocytes following application of short I <'stvv~v~Yr" peptides derived from N- and C-termini of Is K/minK. For -2 full experimental conditions, see ref.68 (a) Definition of -4 :;( « peptides; peptides N-34, C-37 and N-13 were active in ~ -6 ::' eliciting current (see below); N-34 had no activity when c -8 ~ injected; conversely C-27 had no activity when perfused; 2.5 sec :; peptide N-20 and the control peptides P-l, P-2 and P-3 were -10 0 inactive (200 J-LM). (b) Induction of slow K+ -selective -12 currents following microinjection of C-27 peptide. (A) -14 Currents evoked by 20 s depolarizing pulses from a holding 40 D potential of -60 m V to +60 m V in 20 m V increments at 45 s >' 30 B intervals and 1 min after microinjection of C-27 (100 J-LM). I E ~ 20 (B) Tail currents of C-27-activated currents from a holding ~ 10 potential of -80 m V (C) I-V relationships for oocytes (J) (5 0 injected with water (~), lOng full-length minK/1sK mRNA « c. (ij -10 (0) and 100 J-LM C-27 peptide (e). (D) Comparison of en CD -20 selectivity predicted by the Nernst equation for a perfectly 2.5 sec > selective K+ current (straight line) to selectivity data ~ -30 derived for C-27-induced currents measured by tail current -40 104 10 1 102 103 10° reversal potentials (open symbols). (c) Induction of slow [CI-]o (mM) Cl- -selective currents following perfusion of N-34 peptide (A) Currents evoked by 5 s hyperpolarizing pulses from a holding potential of -10mV between -90mV to -170mV in 20mV decrements at 60s intervals and 1 min after superfusion of C-34 (50J-LM). (B) Tail currents of C-34-activated currents from a holding potential of -10mV (C) I-V relationships for oocytes injected with water (~), lOng full-length minK/1sK mRNA (0) and 50J-LM C-34 peptide (e). (D) Comparison of selectivity predicted by the Nernst equation for a perfectly selective Cl- current with [Cl- h == 65 mM (straight line) to selectivity data derived for C-34-induced currents measured by tail current reversal potentials (open symbols). (Reproduced with permission from Ben-Efraim et al. (1996) J BioI Chern 271: 8766-71.) (From 54-40-01)

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_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_ 4

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PHARMACOLOGY

Blockers Sensitivities of 1sK channels to tclassical' K+ channel blockers 54-43-01: MinK channels/currents expressed in Xenopus oocytes are weakly but reversibly inhibited by some 'classical' K+ channel blockers, as summarized in Table 6. Similarities in patterns of block shown by heterologously expressed IsK/minK currents and the native slow-activating cardiac K+ current component IK,s have been invoked in describing the two currents as 'equivalent' (but see Phenotypic expression, 54-14 and Protein interactions, 54-31).

Halothane inhibition of wild-type and minK activities

~N/~C-terminal

deletant

54-43-12: Current through human and rat minK channels is reversibly reduced to 68% of control values by 0.5% (0.34mM) halothane 97. Note: Doubledeletion mutants (where 30 amino acids of the extracellular N-terminus and 37 amino acids of the intracellular C-terminus are deleted) respond to low halothane concentrations similarly to the wild-type channel. This result is consistent with a model in which halothane interacts with the minK protein from within the lipid bilayer97.

Channel modulation tDiIect' and tindirect' modulation of minK protein activities 54-44-01: There are a large number of studies demonstrating both 'positive' and 'negative' regulation of IsK protein activity by intracellular second messengers; some of these effects may relate to IsK regulation under physiological or pathophysiologicalt conditions. In the summary shown in Table 7, certain 'modulatory' effects which may be related to various protein kinase activities in specified tissues are cross-referenced to Protein phosphorylation, 54-32. As noted in Table 7, physical factors such as temperature can markedly influence observed 'modulatory' properties, and should therefore be taken into account in comparative studies. MinK proteins might also be 'indirectly modulated' as a consequence of cytoskeletal interactions or interactions with other (unknown) proteins which are susceptible to independent modulation (see also Protein interactions, 54-31).

tpositive regulation' by stabilization of open 1sK channels by chloride channel blockers 54-44-24: The fenamate compounds niflumic acid, mefenamic acid, flufenamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) are commonly used in Xenopus oocytes to suppress endogenous Ca2+activated CI- channels (see ILG Cl Ca, entry 25). Following heterologous t expression of human IsK subunits in Xenopus oocytes, all compounds (10 IlM) increase IsK amplitudes and decrease rates of IsK deactivationt 108 . At higher concentrations (100 IlM) these compounds further decrease the rate

Table 6. Reported sensitivities of minK/1sK-like currents to non-selective K+ channel blockers (From 54-43-01) Blocker/inhibitor

Characteristics and references (see note 1)

Tetraethylammonium (TEA+) ions (open channel blocker at high millimolar concentrations; voltage independent)

54-43-02: External TEA+ ions block I sK 53 at Ki >40mM6,B. Comparative note: 4-Aminopyridine has no effect on IsK channel currents (for other K+ channel blockers with no effect on heterologously expressed minK currents, see note 3).

Ba2 + ions (open channel blocker at millimolar concentrations; voltage independent)

54-43-03: External Ba2+ ions block IsK currents with a Ki of approx. 1 mM6,13. Ba2+ alters channel gating favouring the closed state, and is relieved by Ktxt 53,54 (see also note 3).

Cs+ ions (open channel blocker at high millimolar concentrations; voltage dependent)

54-43-04: High external concentrations of Cs+ (>40mM) can inhibit IsK current; open-channel blockade by Cs+ is voltage dependent (see also note 3).

Clofilium (non-specific with similar inhibitory effects on other K+ channels)

54-43-05: Channels formed via heterologous expression of minK mRNA from neonatal mouse or rat heart are blocked by the class III antiarrhythmic clofiliumB. Clofilium block shows a Ki of 100JlM (neonatal mouse heart minK) or ",70% inhibition at 100JlM for neonatal rat heart minK. Clofilium block exhibits a positive shift in channel activation threshold by approx. 20 mV. Application of 100 JlM clofilium to newborn mouse ventricular cells induces increases in action potential duration, decreases in beat rate and increases in contractile forceB. A native delayed outward rectifier (similar shape but with faster activation than minK) is 'completely blocked' by 100 JlM clofiliumB.

Effects on mRNA induction following T cell activation

II

Effects of clofilium on 1L-2 mRNA induction following T cell activation 54-43-06: Oocyte current supported by cRNA encoding the human IsK protein derived from Jurkat T cell cDNA21 (see also Cell-type expression index, 54-08) is blocked by the antiarrhythmic clofilium (IC 5o 80 JlM). Clofilium has been shown to potently inhibit IL-2 mRNA induction upon mitogen-induced T cell activation, while charybdotoxin has no effect. Comparative note: The Jurkat cDNA-derived HIsK channel has been described as 'totally insensitive' to charybdotoxin21 (see also note 2).

(b

='

t""'t-

~ CJ1 ~

II

Table 6. Continued Blocker/inhibitor

Characteristics and references (see note 1)

Novel class III antiarrhythmic t compounds, e.g. NE-I0064 (azimilide) NE-I0133

Inhibition of IsK channels by novel cardiac antiarrhythmics

54-43-07: The novel class III antiarrhythmic t compound NE-I0064 (azimilide, 1-[[[5-(4chlorophenyl)-2-furanyl]methylene]-amino]-3-[4-(4-methy1-I-piperazinyl)-butyl]-2,4imidazolidinedione dihydrochloride) displays 'potent inhibitory effects' on the human IsK channel when expressed in oocytes (EC 50 of 5.4 JlM) as well as native guinea-pig I K ,s92,93. NE-I0064 block (and the reversal of block following washout) is characteristically use dependent t. NE-I0064 block also appears to be voltage dependent, being more pronounced at depolarized potentials 92. In perfusion models, NE-I0064 block induces prolongation of action potential durations without affecting upstroke t. These characteristics have been suggested to contribute to the antiarrhythmic action of NE-I00064 92.

pH-dependence of azimilide inhibition 54-43-08: Reduction of ionic strength or pH changes from pH 6.5 to 8.5 alone do not alter IsK amplitude, but inhibition of Isk by azimilide (see above) is decreased by reduced pH (but not by reduced ionic strength)94. Apparent affinity of azimilide is increased more than tenfold following an increase in pH from 6.5 to 8.5. These results suggest neutral azimilide (a weak base) may inhibit IsK via a lipophilic protein-drug interaction94. Note: These results may have some significance for interpreting variable potency of azimilide in acidosis vs. alkalosis and/or in different tissues (heart vs. kidney). 54-43-09: The class III antiarrhythmic NE-I0133 has also been shown to inhibit IsK channels expressed in Xenopus oocytes and IK,s in guinea-pig cardiac myocytes 93 . Comparative note: This study reported that NE-I0064 or NE-I0133 (at equivalent concentrations) do not induce block of (i) several cloned delayed rectifier K+ channels from the Kv family or (ii) the I K1 inward rectifier current in guinea-pig cardiac myocytes 93 (see also note 2).

B ~ CJl ~

La3 + ions (minK species differences)

Calmodulin antagonists e.g W-7, trifluoperazine, chlorpromazine (possible blockers)

'Species differences' in sensitivity to La 3+ ions 54-43-10: Low concentrations of extracellular La3 + ions (10-50 JlM, Kn approx. 30 JlM) (i) rapidly and reversibly reduce the magnitude of the mouse and rat IsK currents in oocytes during test depolarizations and (ii) increase the deactivation rates of tail currents59. In contrast, the magnitude and deactivation rates of the human IsK currents are unaffected by oJlM La3+ (Kn approx. 300 JlM). For further 1sK 'species differences', see Activation, 54-33 and Blockers, 54-43. Note: Effects of lanthanum in blocking components of cardiac 1K and screening membrane surface charge are described in ref. 91 . 54-43-11: Inhibitory effects of calmodulin antagonists (e.g trifluoperazine, chlorpromazine and W-7) on IsK protein activities (see Table 5 under Protein phosphorylation, 54-32) may occur by direct channel blockade: Half-maximal inhibitory concentrations of these compounds can not 'sufficiently' inhibit Ca2 +-mediated positive regulation of IsK (see ref.79 and Ca 2+ ions in Table 7 under Channel modulation, 54-44).

Notes: 1. For blockers shown to be ineffective on minK currents, see note 2. All results generated in the Xenopus oocyte expression system should also be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40. 2. MinK channel currents are not sensitive to amiodarone (to 300 JlM), apamin (to 100 OM), ,B-bungarotoxin (to 10 OM), bretylium tosylate (to 300 JlM), charybdotoxin (to 50 OM), dendrotoxin I (to 10 OM), mast cell-degranulating peptide (MCDP, to 100 OM), quinidine (to 300 OM; but see note 4), quinine (to 300 JlM), or tedisamil (to 300 JlM6,8). Class III antiarrhythmic drugs such as the methanesulphonanilide, sotalol and its structural analogue E-4031 (umelated to the chlorophenylfuranyls NE-10064 and NE-10133, this table) have no direct effect on native 1K,s, but inhibit the rapidly activating component 1K,r (probably encoded by HERC in humans, see VLC K eag/elk/erg, entry 46). See also Receptor/transducer interactions, 54-48. 3. Phe55 mutants (see the {PDTM}, Fig. 6) are several-fold more sensitive to open channel block by TEA and CS+ 54. 4. One group95,96 have reported quinidine suppressing IsK current in oocytes from the intracellular side of the membrane. Block of IsK by quinidine was enhanced by membrane hyperpolarization but was reduced by membrane depolarization (in contrast to effects on Kv1.4 and Kv1.2, see Blockers under VLC K Kv1-Shak, 49-43). These results were used to suggest that quinidine preferentially binds to IsK channels in the closed state, and prevents them from opening95,96.

II

(1)

=:s

~

~ CJ'l ~

_

Table 7. Reported modulatory patterns of minK/lsK-lil<e currents (From 54-44-01) Modulator class

Description/tissue- and species-specificities/references (see note 1)

Ca2+ ions (accelerating minK activation by cellular Ca2+ influx or release from internal stores; mechanism unclear (possibly indirect); markedly temperature dependent

54-44-02: Increases in [Ca2 +1 by application of the calcium ionophore A23187 (calcimycin)

or by intracellular injection of inositol l,4,5-trisphosphate (InsP3I see ILG Ca InsP3I entry 19) are associated with accelerated IsK activation and increases in IsK current amplitude47. Ca2+ -elevation of minK current amplitude may be due to indirect activation of a calcium-activated protein kinase21,47. Ca2+ -enhanced currents can be decreased by injection of BAPTA (l,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid. For related effects mediated by protein kinase C and co-expressed 5-HT2 receptor agonism, see Protein phosphorylation and Receptor/transducer interactions, 54-32 and 54-49, respectively. 54-44-03: Note: Although several calmodulin antagonists can inhibit human IsK activity in oocytes (see Protein phosphorylation, 54-32), these effects appear independent of Ca2+ concentration 79 (Le. they can occur at both physiological and enhanced [Ca2+]i, ibid.).

Interactions of temperature- and Ca 2+ -dependent effects on rat kidney minK in oocvtes 54-44-04: Increases in the batht temperature (from 22 to 32°C) markedly accelerate

minK channel activation98 . The notably high QI0 of minK channel activation is voltage-dependent, being higher at more negative potentials - compare the QI0 (-20mV): 7.02 to QI0 (+20mV): 4.0. While activation of minK channels is 'highly voltage dependent' at 22°C, voltage has little effect on minK channel activation at 32°C. Increases in [Ca2+]i, shown to increase the maximal conductance (Gma:xJ at room temperature, do not affect (Gmax ) at 32°C 98 . However, increase of [Ca2+]i causes acceleration of minK channel activation at both temperatures (as above). Similar interactions of [Ca2+]i and temperature on G max and activation rate have been observed for native IK,s in guinea-pig cardiac myocytes.

g

54-44-05: Note: Human IsK activity in oocytes can be inhibited by A23187 (compare this

~

table, above) or hypotonic t extracellular fluid (compare this table, below). These effects can not be abolished by several calmodulin inhibitors 79 (see Protein phosphorylation, 54-32).

CJ1 ~

Chromanols (inhibition of CI- secretion in colon by inhibition of cAMP-activated K+ conductance)

54-44-06: Chromanol 293B specifically inhibits channels induced by oocyte expression of IsK/minK (IC 5o approx. 7 JlM) but has little effect on Kvl.l (entry 48) or Kir2.1 (entry 33) channels expressed in the same system99 • Other chromanols display the same rank order of potency for IsK-induced channel inhibition in oocytes as demonstrated for inhibition in a native rat colon crypt cAMP-mediated Cl- secretion model. Note: Significantly, azimilide (see Blockers, 54-43) has been shown to inhibit the forskolin-induced Clsecretion in the rat colon crypt model, suggesting a common target for chromanol 293B, its analogues, and azimilide 99. Note: Observations of chromanol 293B inhibition have been extended to IK,s in guinea-pig cardiac myocytes and IsK-associated channels 100.

cAMP and analogues, e.g. 8-Br-cAMP (mechanism unclear)

54-44-07: Elevations of intracellular cAMP levels (e.g. using forskolin or 8-Br-cAMP) increase the amplitude of rat minK current in oocytes 76, treatments which are also associated with small increases in the rate of current activation61 (see also Protein kinase A in Table 5 under Protein phosphorylation, 54-32). These effects have not been observed with the mouse IsK protein8 . Effects of elevated cAMP are not associated with alterations in the surface expression of the minK protein, as determined by epitope tagging t experiments61 (see also Protein interactions, 54-31).

H+ ions (voltage-independent extracellular site modulation; mechanism unclear in native cells)

Concentration-dependent H+ modulation of IsK current in oocytes 54-44-08: External acidification reversibly decreases the rat kidney IsK current amplitude following expression in Xenopus oocytes 101 . External acidification reduces the maximal conductance (Gmax ) with a pH KD ~ 5.5, without affecting the activation kinetics. Hill coefficient data (n = 1) indicates that H+ ions bind to the channel with a one-to-one stoichiometry. The H+ modulation effect is voltage independent, indicating that the H+-binding site is located outside of the membrane electric field 101 .

pH sensitivity of transepithelial K+ transport

III

54-44-09: In vestibular dark cell epithelium, transepithelial K+ transport is sensitive to pHi and pHo (for background, see Phenotypic expression, 54-14). Determinations of cytosolic pH, transepithelial voltage (Vt ) and equivalent short-circuit current (Isc , a measure for transepithelial K+ secretion) show that acidification of the extracellular pH (pHo ) causes an increase of intracellular pH; (ii) increased dark cell V t and (iii) increased

g M"

~ CJl ~

II

Table 7. Continued Modulator class

Description/tissue- and species-specificities/references (see note 1) I sc with a decrease in transepithelial resistance Rt 102. These effects are significantly larger

when the extracellular acidification is applied to the dark cell basolateral face (as opposed the apical side of the epithelium). In these studies, cytosolic acidification appeared to activate the 18K (minK) component in the apical membrane while cytosolic alkalinization had an inhibitory effect. Note: These studies acknowledged a difficulty in determining whether the modulatory effects of pHi on 18K /minK were direct or indirect102•

Hg2 + ions (chelation/cross-linking at extracellular N -terminal site close to the membrane)

Oestrogens (and anti-oestrogens)

Organic protein cross-linking (chelating) agents (membrane-impermeable), e.g. DTSSP stabilizing voltage-dependent subunit aggregation following gating into an open conformation

Persistent (use-dependent) activation of 1sK currents 54-44-10: The heavy metal mercury induces an increase in IsK current amplitudes, mainly as a consequence of a 'markedly decreased' rate of deactivation t. Compare to similar effects with DTSSP (this table, below). Site-directed mutagenesis studies indicate that the effects of Hg2+ can be attributed to Hg2 + binding in the extracellar N-terminal domain (deletions of residues 10-25 are susceptible to Hg2+, while deletions of residues 10-39 are not (for the predicted site of 1sK protein-protein chelation, see the [PDTM], Fig. 6). 54-44-11: 'Direct' inhibitory effects of oestrogen on the IsK protein have been reported28 (see also Developmental regulation, 54-11).

Persistent (use-dependent) inhibition of 1sK currents by extracellular aggregation 54-44-12: Application of the membrane-impermeable organic cross-linker DTSSP (dithiobis-sulphosuccinimidyl propionate) to oocytes expressing minK (i) decreases time-dependent current; (ii) increases rate of activation and (iii) induces 'persistently activated' inward and outward potassium currents. These effects require membrane depolarization (Le. activation) and suggest that the activation process involves an approximation (aggregation) of the extracellular protein domains 8o (see also Protein interactions, 54-31 and Activation, 54-33). 54-44-13: Persistently activated channels retain potassium selectivity and sensitivity to block by clofilium and barium8o .

~

~ CJI ~

Osmolarity (e.g. hypotonic extracellular solutions)

54-44-14: Hypotonic t solutions increase IsK-induced K+ currents following expression in in Xenopus oocytes69 . See also information relating to the osmosensitive 'lsK-like' current of apical membranes of inner ear vestibular dark cells (this entry).

(D

::s

t"'1"

~ en ~

Nitric oxide donors, e.g. SNOC (NO-mediated mechanism unclear, but reversible, cGMP- and cGMP-kinase-independent, Cysl06/Cysl07 oxidation site-independent, PKC-dependent)

Indirect NO-donor regulation via a cGMP independent, staurosporine-sensitive pathway

54-44-15: Current amplitude-enhancing effects of the nitroso donor t S-nitroso-cysteine (SNOC, a neutral amino acid) on IsK in Xenopus oocytes are 'moderate' and occur over a slow time course (e.g. 14 ± 4.9 % for rIsK; 15 ± 6.3 % for hlsK, requiring approx. 15 min for maximal effect} 73. SNOC regulation appears to mediate a stabilization of IsK open state, as deactivation t occurs more slowly (i.e. Tdeact is increased) but there is evidence that NO acts indirectly, possibly via phosphorylation of an unidentified kinase or othe IsKregulatory protein (see below). All effects require freshly prepared SNOC i one-week-old SNOC ('inactivated SNOC') does not give responses, consistent with redox modifications of SNOC itself underlying a slow time course of NO release (half-lifetime of SNOC is approx. 20min, comparable to the IsK-enhancement time course).

54-44-16: Effects of SNOC are greatly enhanced when the amino acid exchanger rBAT is co-expressed (e.g. to 93.2±9.6% enhancement for hlsK current). The effects of rBAT require a holding potential of -50 mY, indicating an electrogenic t mode of SNOC transport into the oocyte 73 as described for transport of other neutral amino acids 103 (for significance, see Fig. 2 under Phenotypic expression, 54-14). In the presence of rBAT, extracellular application of L-cysteine (L-Cys, 1 mM) induces an outward current similar to SNOC at -50mV. L-CyS application exerts no effect on IsK currents alone. 54-44-17: SNOC can not be prevented by the guanylate cyclase t inhibitor LY-83583 (6-anilino-5,8-quinoline-dione, see Resource C) and the cGMP kinase inhibitor H-8 (N-[2-methylamino]ethyl-5-isoquinolinesulphonamide, ibid.) but is abolished in the presence of staurosporine (a relatively non-specific inhibitor of protein kinase C)73.

II

54-44-18: SNOC also increases currents induced by expression of minK protein mutants lacking intracellular sites likely to be involved in IsK regulation by oxidation and phosphorylation (e.g. rIsK CI07S, and the double deletant protein ~10-39/~94-130).

II

Table 7. Continued Modulator class

Description/tissue- and species-specificities/references (see note 1) 54-44-19: Comparative notes: 1. In other experiments, SNOC did not alter voltage-activated K+ currents through rKvl.l channels co-expressed with the renal Na+-coupled Pi transporter NaPi-2 (for Kv1.1, see VLG K Kv1-Shak, entry 48).2. For examples of NO-dependent regulation associated with apparently 'direct' oxidation of ion channel proteins see Channel modulation under ELG CAT GLU NMDA, 08-44 and Openers under ILG K Ca, 27-48. For examples of cGMP-dependent effects of nitric oxide (involving cGMP-dependent protein kinases t and cGMP-regulated cyclic nucleotide phosphodiesterases t) see ILG CAT cGMp, entry 22.

Isosorbidinitrate (ISDN)

55-44-20: ISDN inhibits an endogenous K+ conductance in Xenopus oocytes with a similar potency to that shown by expressed IsK/minK channels 104 . These inhibitory effects do not depend upon a nitric oxide messenger

Oxidative agents, e.g. peroxides (inc!. H 2 0 2 ) and DTNP (intracellular C-terminal cysteine 'oxidizable'site-dependent modulation via disulphide bond formation)

54-44-21: Native IK,s in guinea-pig cardiomyocytes is inhibited by peroxides105. Since peroxides are generated during ischaemia t and have been associated with generation of reperfusion-induced arrhythmias t 106 this type of modulation may be of pathophysiological significance. 54-44-22: Inhibition of IsK current amplitudes by peroxides and membrane-permeable oxidizing agents has been observed107. Site-directed mutagenesis has been used to show this effect is dependent upon oxidation at the Cysl06 residue in the C-terminus of the human IsK protein (Cysl07 in rat) (for the predicted site of IsK S-S bond formations, see the {PDTM}, Fig. 6). 54-44-23: Note: The predicted formation of disulphide bonds t might occur (i) between minK subunits themselves; (ti) between minK subunits and cytoskeletal proteins; (iii) between

minK proteins and other (endogenous) channel proteins or (iv) a combination of these.

Note: 1. All results generated in the Xenopus oocyte expression system should be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40.

g ~ CJ1 ~

l_e_n_t_ry_5_4

_

of IsK deactivation, resulting in 'persistent activation' of IsK (in a manner similar to the action of organic cross-linkers on IsK (see Table 7 under Channel modulation, 54-44). Notably, however, niflumic acid and flufenamic acid (at the higher concentration of 100 J.!M) decrease time-dependent outward current, whereas 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and mefenamic acid caused an additional increase108 . Complete substitution of chloride ions with gluconate results in IsK channels with altered activation properties, while niflumic acid produces similar 'positive regulatory' effects on IsK (shifting the voltage needed to evoke half-maximal IsK activation (V1/ 2 ) by approximately -20mV)108. In summary, the relative 'opening' effects of mefenamic, niflumic and flufenamic acids at 10 J.!M are +200/0, +16% and +20% while at 100J.!M concentrations these change to +1400/0, -45 % and -740/0 respectivelyl08. This study concluded that these 'positive regulatory' effects of Cl- channel blockers occurs via stabilization of open IsK channels.

Comparative note: D1DS increases K+ secretion through 1sK in dark cell epithelia 54-44-25: Inner ear vestibular dark cell epithelium secretes K+ via an 'lsKlike' pathway in the apical membrane (for background, see Phenotypic expression, 54-14). Disulphonic stilbenes like DillS (see previous paragraph) increase dark cell K+ flux (e.g. by a factor of 1.96 ± 0.71 for DillS) and cause net solute efflux109. Different effects are observed when apical membranes are partitioned (i.e. with a macropatch pipette) and DIDS is applied either inside or outside the pipette. If the DillS is applied inside, current increases across the patch and the IsK component of the membrane current shifts the reversal voltage toward the equilibrium potential for K+. When DillS is applied outside the pipette, the patch current decreases and and membrane conductance is lowered, consistent with 'shunting' of current away from the membrane patch109. Note: These authors acknowledged the difficulty of discriminating between direct actions of DillS on IsK/minK or another unknown but 'tightly co-localized' membrane component.

Receptor/transducer interactions Relative significance of IK,s and 1K,r components at high heart rates 54-49-01: Protein kinase A and Ca2 +-mediated 'up-regulation' of IsK (see Channel modulation, 54-44) may be of si~ificance under 'stress' conditions. In heart, high ,a-adrenergic drive (tonus t ) and increase of [Ca2 +Ji might be predicted to shorten action potentials with possible proarrhythmic effects. Under such conditions native IK,s becomes a dominant component for the induction of action potential repolarization, which has led to an interest in its specific pharmacological modulation (for comparative reviews, see refs.ll0-112). Notably, inhibitors of IK,r (see Current designation, 54-04) show reduced efficacy at high heart rates and/or under ,B-adrenergic tonus. Such conditions may also induce an increase in IK,s or the activation of a cAMP- and time-dependent CI- current (for discussion and associated references, see ref. 93 ).

_ L.....---

entry 54 _

MinK can be potentially regulated by a wide variety of PLC-linked receptor/transducer systems 54-49-02: The general significance for minK regulation of receptor/transducer systems that couple to activation of phospholipase C, e.g. for endothelin, ATP (e.g. P2U, see Protein phosphorylation, 54-32) or serotonin (5-HT, see below45,47) or certain oxytocin receptors (in uterine tissue, as described under Developmental regulation, 54-11). Receptor/transducer systems may couple to both functional regulation of IsK gene products as well as 'expression control' at the level of gene transcription (ibid., see also Protein phosphorylation, 54-32). In oocytes co-expressing 5-hydroxytryptamine receptors (subtype 5-HT2) receptors and IsK protein, 5-HT application increases K+ currents47 (see Channel modulation, 54-44). In oocytes pretreated with caffeine (effectively blocking release of intracellular calcium - see ILG Ca Ca RyR-Caf, entry 17) 5-HT decrease~ the current; this decrease can be prevented by staurosporine (see Protein phosphorylation, 54-32). These findings are consistent with minK current enhancement by increases in [Ca2+h (see Channel modulation, 54-44) with 'negative regulation' occurring following activation of protein kinase C (see Protein phosphorylation, 54-32). Stimulation of 5-HT2 receptors can exhibit both of these effects, with the former being 'predominant,47.

Parathyroid hormone (PTH) receptor regulation of 1sK in oocytes 54-49-03: Co-expression of minK with the parathyroid hormone (PTH) receptor in Xenopus oocytes induces (i) a shift of the IsK conductancevoltage relationship (to more negative potentials); (ii) a decrease in the rate of IsK activation and (iii) a decrease in the rate of IsK deactivation l13 . This form of PTH regulation can be mimicked by activators of protein kinase A (PKA) and significantly reduced by the PKA inhibitors staurosporine and H89. Notes: 1. Parathyroid hormone (peptide 1-34) has no apparent effect on IsK when the latter is expressed alone. 2. PTH regulation appears independent of [Ca2+]o and can be demonstrated in mutant IsK lacking the PKC consensus site l13 . 3. These results may be of significance in native kidney proximal tubule cells, where PTH receptor and IsK protein are coexpressed. Note: All results generated in the Xenopus oocyte expression

system should be interpreted in the light of findings described under Protein interactions, 54-31 and Selectivity, 54-40.

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 54-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction etJ layout of entries, entry 02);

database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in the Introduction

l_e_n_t_ry_5_4

_

eiJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouring t analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed below. Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers) author Ireference or nomenclature. Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly-reported and related sequences. Species

DNA source

Accession

Guinea-pig IsK

Genomic DNA clone ORF: 129aa Cardiac cDNA clone: Cavia cobaya adult ORF: 129aa Slow voltage-gated K channel protein ORF: 129aa Polymorphisms in the human IsK gene Jurkat T cell cDNA, retrieved by RT-PCRt ORF: 129aa

Zhang, Proc Natl Acad Sci gb: not found USA (1994) 91: 1766-70. gb: L20462 Zhang, Proc Natl Acad Sci USA (1994) 91: 1766-70.

Guinea-pig IsK HumanlsK HumanlsK Human IsK (HIsK)

Human, genomic IsK Mouse IsK Mouse IsK

Mouse IsK Rat IsK (see Developmental regulation, 54-11)

Rat IsK

Rat IsK

IsK protein clone phKI2; 436 bp ds-DNA (genomic) ORF: 129aa AT-l cardiac atrial tumour cell line eDNA Characterization of multiple cDNAs and gene organization cDNA from neonatal mouse heart cDNA library ORF: 129 aa Ovariectomized, oestrogen-induced rat uterine cDNA (cloneUtl) ORF: 130aa Ovariectomized, diethylstilbestrol-primed uterine cDNA ORF: 130aa Original expression cloning from rat kidney cRNA pools (clone pK127) ORF: 130aa Neonatal heart cDNA ORF: 130aa

Sequence/discussion

gb: L28168 Folander et al. (unpublished, 1994). gb: L33815 Lai, Gene (1994) 151: 33940. gb: not Attali, JBiol Chem (1992) found 267: 8650-7. (same sequence as human genomic clone in Murai et al.). gb: M26685 Murai, Biochem Biophys pir: A32447 Res Commun (1989) 161: 176-81. gb: not Felipe, Am JPhysiol (1994) found 267: ClOO-5. gb: X60457 Honore, EMBO J (1991) 10: 2805-11. Lesage, FEBS Lett (1992) 301: 168-72. gb: S57779 Honore, EMBO J(1991) PIR: 10: 2805-11. S17307 CIKO$RAT Pragnell, Neuron (1990) 4: (Swissprot 807-12. database) gb: M36461 Folander, Proc Natl Acad Sci USA (1990) 87: 29759. gb: M22412 Takumi, Science (1988) 242: 1042-5. gb: not found

Folander, Proc N atl Acad Sci USA (1990) 87: 29759.

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Species

DNA source

Accession

Synthetic IsK

gb: not Constructed from overlapping found oligonucleotides (based on rat kidney sequence), introducing 40 silent, unique restriction enzyme recognition sequences ORF: 130aa

Sequence/discussion Hausdorff, Biochemistry (1991) 30: 3341-46. For design of silent restriction sites, see: Oprian, Proc Natl Acad Sci USA (1987) 84: 8874-7 (see also Resource D Diagnostic tests).

Gene mapping locus designation 54-54-01: The human IsK homologue has been assigned the HGMWt name KCNE1 56,114 (see also refs under Chromosomal location, 54-18). OMIMt presently lists KCNE1 as accession number 176261: potassium voltage-gated channel, IsK-related subfamily, member 1.

Miscellaneous information Other single-transmembrane domain proteins mediating transmembrane ion flux 54-55-01: MinK is not unique in its ability to 'induce' ionic currents when heterologously expressed in Xenopus oocytes. As briefly described in the following paragraphs, other molecules possessing a single-transmembrane domain which share this property are (i) phospholemman (PLM); (ii) Mat-8 (a novel phospholemman-like protein); (iii) CHIF (aldosterone-inducible channel-inducing factor). Various similarities between the biology of minK and these proteins has been taken by some authors to lend support to the hypothesis that minK-like proteins can act as transmembrane regulators capable of activating endogenous oocyte transport proteins (see Protein interactions, 54-31). The demonstration of unitary anion currents through recombinant phospholemman channels in a cell-free system (see next paragraph) shows that single-transmembrane domain proteins can function as 'independent' voltage-gated ion channels. In other studies l15, however, phospholemman, IsK/minK (see Selectivity, 54-40) and other proteins have been suggested to act as activators of an endogenous CI- conductance in oocytes (e.g. the synthetic protein SYN-'C and the NB protein of influenza B virus l15 ).

Phospholemman induces hyperpolarization-activated currents in Xenopus oocytes 54-55-02: Phospholemman (PLM), a 7~! amino acid cardiac sarcolemmal protein with a single transmembrane domain induces a hyperpolarizationactivated chloride-selective current when expressed in Xenopus oocytes l16,117. Like minK, PLM-induced currents activate Ivery slowly' with a pronounced sigmoidal delay. PLM-induced CI- currents do not inactivate, and increase in amplitude with trains of pulses, depolarized holding potentials, and low extracellular pH. Immunoaffinity-purified recombinant PLM molecules alone

l_e_n_t_ry_S4

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can form ion channels when reconstituted into planar phospholipid bilayers and this approach has enabled measurement of unitary PLM-associated anion current 72 . Excised patches of oocytes expressing PLM show similar currents to those seen in bilayer models, and the presence of PLM can increase flux of taurine, suggesting that PLM plays a role in cell volume regulation. Moreove~ PLM-induced channels in bilayers may have anion, cation and zwitterion l selectivity (allowing fluxes of both cations and anions) and are capable of making 'instantaneous and voltage-dependent transitions' between selectivity states l18 . Point mutations within the single transmembrane region of PLM abolish the sigmoidal delay of expressed currents l17. Notes: 1. Phospholemman appears to be the smallest plasma membrane channel protein known. 2. The chloride-selective current induced by IsK expression in Xenopus oocytes is 'very similar' to the chloride current produced by phospholemman expressions (see Protein interactions, 54-31). 3. PLM is a major plasmalemmal substrate for cAMP-dependent protein kinase. 4. During hyperpolarizing pulses, defolliculated Xenopus oocytes also display endogenous time- and voltage-dependent inward chloride currents. Despite some 'striking similarities' to PLM-induced currents (summarized in ref. 119j see also VLC Gl, entry 43), these endogenous currents are generally smaller, insensitive to pH, and have a distinct pharmacology.

Mat-8 (related to but distinct from phospholemman) induces Cltransport in oocytes 54-55-03: Mat-8, an 8 kDa transmembrane protein is expressed both in primary human breast tumours and in human breast tumour cell lines. Although the extracellular and transmembrane domains of Mat-8 are homologous to those of phospholemman, its cytoplasmic domain is unrelated. The Mat-8 and PLM proteins are encoded by distinct genes and exhibit different tissue-specific patterns of expression. Expression of Mat-8 cRNA in Xenopus oocytes induces hyperpolarization-activated chloride currents similar to those induced by PLM expression120.

A corticosteroid-induced tlsK-like' K+ channel activity in Xenopus

oocytes 54-55-04: Studies of aldosterone-inducible genes expressed within a rat colon cDNA library have identified a cDNA clone designated eHIF (channelinducing factor)121. cRNA runofft and oocyte injection supports the expression of a channel activity with biophysical, pharmacological and regulatory characteristics 'very similar' to those reported for minK. These characteristics include (i) slow activation by membrane depolarization (7 >20s) with a threshold potential above -SOmVj (ii) blockade by clofilium; (iii) inhibition by phorbol ester and (iv) activation by 8-bromoadenosine 3 /,S/-cyclic monophosphate and high cytoplasmic Ca2+121 . The CHIF mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCI diet. A similar mRNA is detectable in kidney papilla but not in brain, heart or skeletal muscle. The amino acid sequence of CHIF shows no homology to IsK, but does exhibit >SO% similarity to two other short bitop!Cf membrane proteins, phospholemman (this field, above) and the Na+/K+ ATPase , subunit. CHIF mRNA is selectively expressed in the medullary

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e_n_t_ry_54_

collecting duct of the kidney and in the epithelium of the distal colon; eHIF mRNA expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion and metabolic acidosis128.

Prokaryotic single transmembrane proteins forming tslow-activating' ion channels 54-55-05: A specific comparison of minKt has been made to the colicins (bacterial peptide toxins with a single transmembrane domain) which also form 'slowly activating' ion channels 14. Despite there being minimal conserved sequence homology between the two groups, their similar topography might suggest a distant evolutionary link between them. Notably, the 97 aa influenzavirus M2 protein has an associated ion channel activity selective for monovalent ions following expression in Xenopus oocytes122. The antiinfluenzavirus drug amantadine hydrochloride significantly attenuates the inward current induced by hyperpolarization of oocyte membranes, and mutations in the membrane-spanning domain can confer amantadine resistance. The wild-type M2 channel is regulated by pH (activated at pH 5-6, as opposed to pH 7.4), which has been proposed to playa critical role in uncoating of virus particles internalized into endosomes (allowing ions to enter the virion particle)122.

Related sources and reviews 54-56-01: Major sources used for this entry, minK-type channel overviews (including discussion of structure, regulation, kinetics and possible physiosee also discussion in refs124~125. Review126, including logical roles)15~41~42~123; discussion on minK kinetic properties, mechanisms of channel formation, comparison with native currents, phenotypic roles and significance of protein structure determination. Molecular determinants of ion conduction and inactivation in K+ channels in general (comparative discussion including minK)127.

Book reference: Oiki, S., Takumi, T., Okada, Y. and Nakanishi, S. (1993) In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida) Ann NY Acad Sci, vol. 707, pp.402-6. New York Academy of Sciences, New York.

Feedback Error-corrections, enhancements and extensions 54-57-01: Please notify specific errors, omissions, updates and comments on this entry by contributing to its e-mail feedback file (for details, see Resource T- Search criteria). For this entry, send e-mail messagesTo:[email protected]. indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message (e.g. Subject: 54-40-01). Please feedback on only on~ specified paragraph or figure per

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message, normally by sending a corrected replacement according to the guidelines in Feedback eiJ CSN Access. Enhancements and extensions can also be suggested by this route (ibid.). Notified changes will be indexed from within the CSN website (www.le.ac.uk/csn/).

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. . Voltage-gated sodium channels William

J. Brammar

Entry 55

NOMENCLATURES

Abstract/general description 55-01-01: Voltage-dependent Na+ channels are responsible for the rapid

membrane depolarization that occurs during the initial 'upstroke' phase of the action potentialt in nerve and muscle. The channels are closed at resting membrane potentials, but are activated by depolarization which causes a voltage-dependent conformational change that increases Na+ permeability. Sodium permeability decreases rapidly ("-Jl ms) to the baseline level as the Na+ channels inactivate during a prolonged depolarization. Channels that have been inactivated by prolonged depolarization are refractory to activation until the cell is repolarized, which allows them to return to the resting state. 55-01-02: In skeletal muscle, the Na+ channels of the sarcolemma are essential for conducting electrical impulses generated at the neuromuscular junction along the muscle fibre and for triggering events that culminate in contraction. 55-01-03: Voltage-gated sodium channels usually underlie regenerative action potentials which carry signals in nerve and muscle. In axons, sodium channels have been shown to have free lateral mobility on the neuronal cell body but are immobile at the pre-synaptic terminal and at focal points along the axon. Brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeletont, thus controlling channel mobility and intracellular distribution (see Channel density, 55-09-02). 55-01-04: Na+ -dependent action potentials t, which are absent from the

earliest stages of neuronal differentiation, exert a major influence on synapse development. The application. of tetrodoxin, a blocker of brain voltage-gated Na+ channels, can eliminate formation of synapses and their pattern of connections (see Developmental regulation, 55-11-01). 55-01-05: Mutations in genes encoding subunits of voltage-gated Na+

channels are the cause of a number of genetic disorders ('ion channelopathies'). Missense t mutations in the human gene (SCN4A) specifying the a subunit of the adult skeletal muscle voltage-gated Na+ channel have been found in patients with two hereditary disorders of sarcolemmal excitation, hyperkalaemic periodic paralysis (HYPP or hyperPP) and paramyotonia congenita (PC). Mutations in the seN5A gene affect the function of the NaHI channel in the heart and are one of three causes of long QT syndrome, an inherited cardiac arrhythmia that can cause abrupt loss of consciousness, seizures and sudden death from ventricular tachyarrhythmia. Mutations of the med locus on mouse chromosome 15 produce a recessive t neurological disorder, 'motor endplate disease', with symptoms ranging from mild ataxia t to juvenile lethality. Molecular cloning t of the affected gene, Sen8a, has shown that it encodes an a subunit of a voltage-gated Na+ channel expressed in the brain and spinal cord (see Phenotypic expression, 55-14-01 to 55-14-14).

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55-01-06: Expression of the type II Na+ channel gene is governed by a powerful negative control element, called a {neural-restrictive transcriptional silencert '. This regulatory DNA sequence in the 5' upstream region of the gene acts as the binding site for a neural-restrictive silencer binding factor (NR8BF), which is present in non-neuronal cells and absent from neuronal cells (see Gene organization, 55-20-02). 55-01-07: The mammalian genes encoding Na+ channel Q and (3 subunits contain variable numbers of exonst (there are 27 in the mouse Scnl0a gene) and opportunities for multiple splicet variants exist. The type II and IIA isoforms of the rat brain Na+ channel are produced by differential splicing of two mutually exclusive exons from the same primary transcript (see Gene organization, 55-20-04 to 55-20-08). 55-01-08: All voltage-gated Na+ channels contain a post-translationally processed a subunit of approximately 260 kDa, consisting of four homologous domains, each having six putative transmembrane Q helices (81-86). The fourth transmembrane domain (84) of each repeat unit is amphipathict, with positively charged amino acids at every third position, and acts as the voltage sensort. Voltage-dependent movement of the 84 domains relative to the rest of the channel protein is responsible for the structural changes involved in channel gating. The largely hydrophobic region between 85 and 86 is believed to constitute most of the pore of the channel. The linker regions connecting the four homologous domains are all located on the cytoplasmic side of the cell membrane. Part of the intracellular ill-IV linker region acts as the inactivation gate of sodium channels, acting to block open channel pores and giving rise to the characteristically rapid inactivation. The four homologous domains I-IV of voltage-gated Na+ channels have sequence similarity (""32-37% identity) with the four corresponding domains of voltage-gated Ca2+ channels, reflecting the common evolutionary ancestry of these two channel types (see VLG Key facts, entry 41 and [PDTMj, Fig. 2). 55-01-09: The Na+ channneis from some tissues, including brain and muscle, also contain accessory f3 subunits which are not essential for function, as shown by the production of functional channels following the injection of cRNA encoding only Q subunits into Xenopus oocytes. The purified Na+ channel from rat brain contains a non-covalently associated f31 subunit of 36 kDa and a f32 subunit of 33 kDa, with the latter being linked to the Q subunit by disulphide bonds. The Na+ channels from skeletal muscle have only Q and (31 subunits, and those from eel electroplax contain only a single Q subunit. 55-01-10: The Q subunits of the voltage-gated Na+ channels contain multiple consensus sites for phosphorylation by PKA and PKC. Many of the channels are phosphorylatable by both enzymes in vivo and in vitro, and phosphorylation generally suppresses Na+ channel activity. 55-01-11: Voltage-gated Na+ channels are highly selective for Na+ amongst the major physiological cations. They show a high selectivity for Na+ versus K+ (permeability ratio PNa/PK ",,30) and are virtually impermeable to Ca2 +

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under physiological conditions. The 85-S6 regions of the a subunits constitute the pore t regions of the channels (see Domain functions (predicted), 55-29-04) and mutations in the S5-S6 regions affect selectivity. Molecular modelling of the pore region predicts a structure resembling a funnel, terminating in a narrowed region that serves as the selectivity filter of the channel. This region contains two carboxylt groups that are hypothesized to displace molecules of water from the hydrated Na+ ion. (Note that the voltage-gated Ca2+ channels contain four conserved glutamate residues that are involved in Ca2+ ion binding and selectivity). A positively charged lysine residue in domain ill is crucial in preventing permeation of Ca2+ ions (see Selectivity, 55-40-02 and-03). 55-01-12: Na+ channels are selectively and reversibly blocked by nanomolar external concentrations of the guanidinium-containing compounds tetrodotoxin (TTx), isolated from Fugu ('puffer fish') organs, and saxitoxin (STx). These toxins bind strongly (Ko == 1-5nM) and compete with each other for the same receptor site. The cardiac isoform (NaHl) and an isoform expressed in sensory neurones of rat dorsal root ganglia (ScnlOa) are relatively insensitive to TTx. Local anaesthetics, such as lidocaine and procaine, generally lipid-soluble tertiaryt amine compounds, block Na+ channels and inhibit propagated action potentials. A structurally diverse group of polycyclict cations, including N-methylstrychnine, pancuronium and thiazin dyes, block Na+ channels through binding within the open pore. Although these compounds are not clinically useful, lubeluzole, a benzothiazole derivative that blocks Na+ channels, has been successfully used in phase II clinical trials as a neuroprotective agent in ischaemic stroke patients. Several anticonvulsant agents, including phenytoin, carbamazepine and lamotrigine, block Na+ channels at therapeutic concentrations. Note that blockers of voltage-gated Na+ channels can have cardiac effects, including prolongation of the QTc interval of the ECG and the risk of initiating arrhythmia, which may limit dosing or limit their clinical use (see Blockers, 55-43-01 to 55-43-04). 55-01-13: The voltage-gated Na+ channels are subject to modulation by a wide range of toxins produced by animals for use in predation or defence, or by plants as protecton against herbivores. The groups of toxins have different effects on channel function, from total block to prolongation of activation. Peptide toxins from sea anemones and a scorpion toxins slow inactivation and stabilize the open state of Na+ channels, causing hyperexcitability and repetitive firing in motor neurones, leading to convulsions and ultimately respiratory failure. The pyrethroid insecticides and DDT slow both activation and inactivation of Na+ channels, prolong channel open-time and promote membrane depolarization, resulting in massive release of neurotransmitter at sensory nerve terminals, causing lethal paralysis in insects (see Channel modulation, 55-44-04).

•

Category (sortcode) 55-02-01: VLG Na.

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Channel designation 55-03-01: Na, Nav. The nomenclature in current use is generally nonsystematic, with channels being named on the basis of the tissue from which their cDNAs were first cloned (e.g. NaBI, NaBll, etc. for channels from brain, NaHI (heart), NaSkl (skeletal muscle)). A broadly agreed systematic nomenclature would clearly be of value in promoting understanding and cross-referencing Na+ channel literature. In sympathy with the system of nomenclature that is finding acceptance for voltagegated K+ channels, a system based on Nav , followed by two numbers representing the subfamily and the subfamily member is recommended. Thus Nav l.l would replace NaBI and Nav 2.1 would represent the NaH2 channel in subfamily 2. Note that the nomenclature for genes encoding voltagegated Na+ channel subunits has been specified. Scn1a is the gene encoding the Q subunit of the Nav l (NaBI) channel (SCN1A is the corresponding human gene). Scn1b encodes the ,81 subunit of voltage-gated Na+ channels (see VLC key facts, entry 41 and Channel designation under VLC K Kv-Shak, 48-03).

Current designation 55-04-01: INa, INa (subtype suffix).

Gene family A large family of genes encoding voltage-gated Na+ channels 55-05-01: Conservation of gene and amino acid sequences during evolution has allowed mammalian homologs of the eel electroplax Na+ channell to be isolated from brain, heart and skeletal muscle. At least 10 different mammalian genes encoding Q subunits of voltage-gated Na+ channels have been recognized by molecular cloning of cDNAs and genomic DNAs (see Database listings, 55-53-01).

Subfamilies of Na+ channel genes 55-05-02: On the basis of sequence comparisons, the genes encoding voltagegated Na+ channel Q subunits have been subdivided into two subfamilies2,3. Most of the Na+ channel cDNA sequences cloned from mammalian brain, heart and skeletal muscle encode proteins with >600/0 sequence identity and have been placed in the Nav l subfamily. The sequences of the hNav 2.1 2 , the rat glial Na+ channel4 and the mouse Nav 2.3 channel3 are > 70% identical to each other but <500/0 identical to the Nav l sequences, so they have been deemed to represent a second gene subfamily, Nav 23 .

Subtype classifications Subtypes based on toxin sensitivity 55-06-01: Prior to the structural groupings based on protein sequences (see above), voltage-gated Na+ channel subtypes were demonstrated functionally by the differential actions of various toxins (reviewed in ref. s).

II

_ entry 55 -

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Trivial names 55-07-01: The voltage-gated sodium channel; the depolarizing sodium channel.

EXPRESSION

Cell-type expression index Na+ channels are ubiquitous in excitable cells 55-08-01: Voltage-gated Na+ channels are essential components of electrically excitable cells in the central and peripheral nervous system, heart and skeletal muscle, where they are responsible for the depolarization during the initial upstroke of the action potential. Na+ channels which are not expressed in the differentiated nervous system include those in astrocytes 6 and tunicate eggs 7. Voltage-gated Na+ channels are !lQ! generally found in epithelial cells. Electronic analogues of INa and IK (see VLC K DR, entry 45) have been used to generate impulses in lsilicon neurones ' where analogues of I A (see VLC K A-'J: entry 44) and IAHP (see ILC K Ca, entry 27) are used to control the rate of impulse production. These analogue integrated circuits are able to emulate types of complex electrical behaviour seen in biological systems 8 . For details of the sites of expression of the various Na+ channel genes, see mRNA distribution, 55-13-02.

Differential expression of Na+ channel subtypes correlates with duration of action potential 55-08-02: Sensory neurones of frog dorsal root ganglia (DRG) express at least two subtypes of voltage-gated Na+ channel: a rapidly inactivating Na+ channel blocked by nanomolar tetrodotoxin (TTx) and an Na+ channel that inactivates 2-6 times more slowly and is resistant to blockage by up to 100 JlM TTx. In addition, the same neurones also express at least two types of voltage-gated K+ channel, differing in activation kinetics. Both the Na+ and K+ channel subtypes are differentially distributed by cell size, and duration of the action potential correlates with the channel subtypes expressed9 . At least three distinct voltage-gated Na+ channels, differing in kinetics and sensitivity to tetrodotoxin, are detectable in rat dorsal root ganglia10,11. A cDNA encoding a tetrodotoxin-resistant, voltage-gated Na+ channel a subunit has been cloned from rat dorsal root ganglion sensory neurones 12,13.

Elevated expression of type II Na+ channels in hypomyelinated axons in 'shiverer' mice 55-08-03: Type I and type ill Na+ channels are largely confined to neuronal cell bodies in mouse brain, whereas type II channels are preferentially localized in unmyelinated fibre tracts. In 'shiverer' mutant mice, which lack compact myelin due to a defect in the gene encoding myelin basic protein, immunocytochemistryt revealed elevated levels of type II Na+ channels

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were observed in the hypomyelinated axons of large-calibre fibre tracts such as the corpus callosum, internal capsule, fimbria, fornix, corpus medullare of the cerebellum, and nigrostriatal pathway. No difference was observed between wild-type and shiverer mice using antibodies specific for type I and type III Na+ channels. These findings 14 suggest that type II Na+ channels are preferentially localized in axons of brain neurones and suggest that their density and localization are influenced by myelination. The selective increase in the density of type II channels in hypomyelinated fibre tracts may contribute to the hyperexcitable phenotype of the Ishiverer' mouse.

Channel density Na+ channel densities vary and show clustering 55-09-01: A wide range of values can be measured and the parameter is not only tissue dependent but channel clustering can account for high local densities within a cell type. Direct recording of Na+ currents from patches of sarcolemma in frog sartorius muscle showed current densities that varied at least threefold over short distances, and calculations of lateral diffusion coefficients suggested that the channels are anchored in the membrane15. Typical values for channel densities are: 100-400 channels per Jlm2 (typical t giant axon); 2-20 channels per Jlm2 (mouse neuroblastoma cells)16,17; 3-10 channels per Jlm2 (cell bodies of rat central neurones)18; 3000 channels per Jlm2 (frog node of Ranvier)19; highly concentrated in eel electroplax1. Voltagesensitive Na+ channels are associated with cytoskeletal elements20,21. In rat phaeochromocytoma (PC12) cells treated with nerve growth factor there is a selective increase of sodium channel type II mRNA and sodium channel densitr2.

Axonal Na+ channels are linked to the cytoskeleton 55-09-02: The regenerative depolarization characteristic of action potential propagation in axons implies a minimum density requirement for sodium . channel molecules co-expressed and localized with axonal potassium channels. Sodium channels have been shown to have free lateral mobility on the neuronal cell body but are immobile at the axon hillock, pre-synaptic terminal and at focal points along the axon. These studies21 have also indicated that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeletont, a factor which can directly control channel mobility, intracellular distribution and/or local molecular density.

Cloning resource Expression cloning from eel electroplax leads to hybridization probes for other species 55-10-01: Sequences encoding the a subunit of a voltage-gated Na+ channel were first cloned as cDNAs derived from the mRNA of the electroplaxt of the electric eel, Electrophorus electricus 1 . Hybridization at reduced stringency with eel a subunit eDNA probes allowed the isolation of a subunit cDNAs from rat23- 25 and human26,27 brain eDNA libraries. The brain type II cDNAs were the

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probes used in the isolation of Na+ channel Q subunits expressed in skeletal muscle and heart by low-stringencyt hybridization2~8-32.

Developmental regulation Na+ -dependent action potentials are important in neuronal development 55-11-01: In developing neuronal cells, sodium currents increase in density and undergo small changes in kinetics and activate calcium currents (reviewed in ref. 33). Proton-activated sodium currents appear prior to Ca2+ and Na+ currents in some systems. Na+ -dependent action potentialstare absent from the earliest stages of neuronal differentiation and TTx or local anaesthetic blockade have no effects on many aspects of late development (reviewed in ref. 33). Sodium-dependent action potentials exert a major influence on synapse development (lTx application can eliminate synapse formation34 ) and their pattern of connections35 ). Connectivity patterns are altered by specific blockade of post-synaptic NMDA receptors36 .

NGF selectively induces expression of type II Na+ channels 55-11-02: In developing populations of cells from several regions of embryonic rat spinal cord, functional sodium channels appear prior to GABA(A) receptors, which in tum emerge prior to kainate-activated glutamate receptors. This stereotypical pattern of sequential channel development occurs individually on most cells in each region37. Nerve growth factor (NGF) has been shown to induce brain type II sodium channels selectively in rat phaeochromocytoma (PCI2) cells22 .

Parallel development of Na+ and K+ currents in Xenopus myocytes 55-11-03: In cultured developing Xenopus myocytes, the Na+ inward current and K+ -selective delayed rectifier outward current appear at around 32 h after fertilization (stage 27) and gradually increase up to more than 44h after fertilization (stage 33/34)38. The developmental time course of these two types of currents is similar, but is not co-ordinated with heterogeneous current sizes being observed.

Differential expression of Na+ channel

Q

subunit genes in rat eNS

55-11-04: The expression patterns of the genes encoding the a subunits of the voltage-gated Na+ channels in rat brain, assessed by blot hybridization with specific RNA probes39, are summarized in Table 1. The temporal expression patterns of the rNaBl, rNaB2 and rNaB3 genes in total rat brain and spinal cord are shown in Fig. 1. Note that the post-natal increase in rNaBI mRNA levels in brain coincides with a corresponding decrease in rNaB3 mRNA levels and the attainment of maximal levels of the rNaB2 mRNA. The timing of expression of the rNaB2 gene is different between brain and spinal cord: in the latter case, the rNaB2 mRNA levels peak sharply in the first post-natal week and decline to much lower adult levels. Overall, the rNaBI gene is expressed predominantly at late post-natal stages, rNaB2 throughout development with regional variation and rNaB3 is expressed mainly at foetal and early post-natal stages of development39.

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Table 1. Developmental expression of the three genes encoding Na+ channel Q subunits of the rat eNS (From 55-11-04) Q

subunit

Sites and timing of expression

rNaBI

55-11-05: The rNaBI mRNA is hardly detected at EI0 in total brain, but its level increases 'slowly and continuously' until birth in spinal cord and medulla pons and until r-..Jp7 in all other regions of the brain. It increases more rapidly during the first and second post-natal weeks in the spinal cord and medulla pons and during the second and third weeks in other regions of the CNS, remaining roughly constant thereafter. The NaBI mRNA is most abundant in caudal regions of the adult brain (midbrain, superior and inferior colliculus, cerebellum and medulla pons) and spinal cord, with low levels detectable in retina, olfactory bulb, cerebral cortex, hippocampus and corpus striatum.

rNaB2

55-11-06: The NaB2 mRNA is detectable by EI0 in total brain and spinal cord, increasing steadily until the first post-natal week. High levels of mRNA are maintained in cerebral cortex, hippocampus and corpus striatum throughout development. In midbrain, coliculi, medulla pons and spinal cord, there is a decline to the lower levels of the adult during the second post-natal week. Expression in the cerebellum shows a lag of 2-3 weeks, with the low initial level increasing rapidly during the second post-natal week to the high level of the adult. The NaB2 mRNA is generally the predominant type of Na+ channel mRNA in the rat CNS throughout development, though there are exceptions to this generalization.

rNaB3

55-11-07: Expression of the gene encoding the NaB3 a subunit

starts during embryogenesis, and significant levels of the corresponding mRNA can be detected in total brain and spinal cord by EI0. The mRNA levels are maximal around birth and decline during the first and second post-natal weeks to the very low but regionally variable adult levels. The latter are relatively high in cerebral cortex, hippocampus, corpus striatum, midbrain, colliculi and medulla pons, but very low in retina, cerebellum and spinal cord. After PIS the levels decline further in colliculi and medulla pons and become undetectable in the cerebellum.

Post-natal switching from RII to RIIA by control of alternative splicing in rat brain 55-11-08: The rat brain Na+ channel Q subunits RII and RIIA are produced by alternative splicing t of two exons t , N(RII) and A(RIIA) (see paragraph 55-2104). Assay of the two mRNA species by RNAase-protectiont and RT-PCRt shows that the RII species is abundant in rat brain at birth, but disappears

Figure 1. Temporal expression patterns of mRNAs encoding Q subunits of Na+ channels I, II and III in total rat brain (a) and spinal cord (b). Diamonds, triangles and circles represent the relative abundance of type I, II and III mRNAs respectively, assuming similar hybridization efficiencies for the three specific probes. The data represent average values for 2-5 independent RNA samples at each time point, with deviation from the means being ±10O/o. (Reproduced with permission from Beckh et al. (1989) EMBO 18: 3611-16.) (From 55-11-04)

within the first post-natal week. In contrast, the RITA mRNA increases in abundance during the first 4 weeks after birth40.

The adult skeletal muscle Na+ channel mRNA predominates at all developmental stages 55-11-09: Two genes encoding Na+ channels, homologous to human genes SCN4A and SCNSA, are expressed in mammalian skeletal muscle. The SCN4A gene encodes the adult skeletal muscle Q subunit, while SCN5A is expressed in cardiac muscle, foetal skeletal muscle and denervated adult skeletal muscle28 . The mRNA species encoding the 'adult isoform' predominates at all developmental stages in both mouse and human, representing rv75% of the total Na+ channel mRNA in human foetal muscle at 18-20 weeks gestation41 . The expression of the adult Na+ channel gene

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increases with age in the developing mouse skeletal muscle and diaphragm until 30 days after birth, while the mRNA encoding the foetal/cardiac isoform gradually declines to insignificance over this period41 . Similar observations have been made with skeletal muscle of the rat42 .

Mouse Na y 2.3 expression is developmentally regulated in heart, brain and uterus 55-11-10: The mRNA encoding the mouse Nav 2.3 Na+ channel 0: subunit increased rv2.5-fold in embryonic heart just before birth and decreased at least 20-fold by day 7 after birth. The level then increased gradually to the adult level, about half that immediately before birth3 . The developmental pattern of expression in mouse brain closely paralleled that in heart. In skeletal muscle, the Nay 2.3 mRNA levels increased gradually throughout development, reached a peak at 21 days after birth and declined about sixfold by adulthood. During pregnancy, the level of Nay 2.3 mRNA in the utems is constant until day IS, when it begins to rise. The levels increased threefold to a peak at delivery and then diminished IS-fold within 2 days post-delivery. The increase in mNay 2.3 mRNA immediately prior to delivery and steep decline immediately after suggest that the channel protein has a role in uterine contraction3 .

Reversion to the embryonic mode of Na+ channel gene expression in axotomized DRG neurones 55-II-II: The mRNA encoding the brain type III Na+ channel 0: subunit is abundant in embryonic (£17) rat dorsal root ganglion (DRG) neurones, but undetectable (by in situ hybridization) in adult rat DRG. By 7-9 days after axotomyt, type ill subunit mRNA is detectable at 'moderate-to high' levels in adult DRG, suggesting that axotomy induces a return to the embryonic pattern of type ill gene expression in DRG neurones 43 •

Expression of genes encoding Na+ channel (3 subunits during neural development 55-11-12: The mRNA encoding the Na+ channel f32 subunit is detectable by Northern blotting by embryonic day 9 in rat brain44, whereas the f31 mRNA is not detectable by this procedure until after birth45 • The levels of f32 mRNA do not vary significantly until birth, after which they increase rapidly to reach adult levels by post-natal day 1444 . The Na+ channel 0: subunit mRNA, present in the rat brain at embryonic day 10, increases rapidly in concentration after birth39,46. This pattern of developmental expression is consistent with a role for the 0: and f32 subunits in neural development. Neurogenesis in the rat central nervous system begins at embryonic day 9-10, while axonal growth and synaptogenesis increase rapidly after birth and continue actively for 2-3 weeks44 .

Parallel expression of (31 and SkM1 skeletal muscle

Q

subunits in developing rat

55-11-13: The mRNA encoding the rat Na+ channel f31 subunit is undetectable by Northern blotting in neonatal skeletal muscle, but increases post-natally from about day 5 to about day 9047. The time course for accumulation of f31

fI

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mRNA precisely coincided with that for production of SkMI a subunit mRNA and was reciprocal to that for the a subunit of the SkM2 isoform. The close link between expression of the mRNAs encoding the ,81 and SkMl a subunits also pertains after denervation of adult skeletal muscle and in primary muscle culture during the development of contracting myotubes 47.

Isolation probe Purified Na+ channels from eel electroplax potentiate cDNA cloning 55-12-01: The high-affinity binding of neurotoxins (see Blockers, 55-43) allowed the purification of Na+ channels from vertebrate brain, heart muscle, skeletal muscle and electroplax of electric eel1,5,48-54. Full-length cDNAs encoding the Na+ channel from electric eel electroplax were isolated using antibodies directed against the purified Na+ channel protein to detect expressed antigens plus degeneratet synthetic oligonucleotide probes based on amino acid sequence1 . The eel electroplax Na+ channel eDNA was used as a hybridization probe to isolate cDNAs encoding three highly homologous rat brain Na+ channel proteins (types I, IT and Ill)23,24. The cDNAs encoding the alternatively spliced type ITA Na+ channel a subunit were independently isolated by screening expression librariest with antibodies against the rat brain Na+ channel a subunit25,55. The type IT cDNAs were the probes used in the isolation of Na+ channel a subunits expressed in skeletal muscle and heart by low-stringencyt hybridization28-3o .

mRNA distribution Tissue distribution of Na+ channel a subunit mRNAs 55-13-01: The Na+ channel mRNAs encoding the four different a subunits types I, IT, ITa and III are differentially expressed within the nervous system23,56,57. Type I channel mRNA is present at 5-10% abundance of type IT56,57. Type IT channel mRNAs are most highly expressed in adult brain. Type III channel mRNAs are found at moderately high levels in embryonic brain, but are of very low abundance in the adult39,56 (see Table 1 and Fig. 1 in 55-11-04). The observations on the detection of mRNAs transcribed from 10 genes encoding mammalian Na+ channel a subunits are summarized in Table 2. Na+ channels in cerebellar Purkin;e cells 55-13-19: Mammalian cerebellar Purkinje cells contain at least two different voltage-gated Na+ conductances: a rapidly inactivating one responsible for the rising phase of the action potential and a non-inactivating conductance generating the prolonged plateau phase. Cloning of sequences encoding Na+ channel a subunits by RT-PCRt, starting with RNA from single guinea-pig Purkinje cells, showed the presence of mRNAs encoding the brain type I (RBI ortholog) and the Scn8a (rat Nach6) a subunits63 . Because mutations in the ScnBa subunit have been associated with motor endplate disease in the mouse, in which the transient Na+ currents in the cerebellar Purkinje cells are spared, it is argued that the RBI sequence encodes the a subunit of the

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Table 2. Reported tissue distributions of Na+ channel mRNAs (From 55-13-01) Scnla

55-13-02: rRBI: Northerns: An RNA probe derived from the 3' non-coding sequence of rRBI detects mRNA in caudal regions of the adult rat brain (midbrain, superior and inferior calliculus, cerebellum and medulla pons) and in spinal cord. Low levels of rRBI mRNA are also detectable in retina, olfactory bulb, cerebral cortex, hippocampus and corpus striatum. The general pattern of expression reprsents a 'caudal-to-rostral gradient'39. The levels of rRBI mRNA are low at birth, but increase gradually over the first 30 post-natal days39 (see Fig. 1). Expression of the rat 'brain' NaBI channels is not confined to the brain and peripheral nervous system: the rNaBI mRNA has also been detected in myocardium281'58. 55-13-03: hNaBI: RT-PCR: Highest in caudal regions of the human brain and spinal cord26 .

Scn2a

The rat brain RBII and RBIIA Na+ channel a subunits are produced by alternative splicing t of the same pre-mRNA40 (see paragraph 55-21-04). 55-13-04: rRBTI: Exont -specific probes showed that the RBTI a subunit mRNA is produced mostly in the midbrain and the olfactory bulb in newborn rats, but is also detectable in cerebellum and hindbrain. The levels of RTI mRNA were markedly decreased in all areas of the brain by 2 weeks after birth40. 55-1-05: rRBTIA: The RBTIA mRNA species was evident in cerebellum and olfactory bulb at birth, and increased levels were readily detectable in cortex, cerebellum, midbrain, hindbrain and olfactory bulb by 2 weeks after birth40 . 55-13-06: hNaB2: RT-PCR: Shows an approximate rostral-tocordal gradient of distribution in human brain, with highest levels in the cerebellum26 .

Scn3a

15-13-07: rRBill: Northerns: 9 kb mRNA in brain, heart and faintly in skeletal muscle of adult rat: not in small intestine or live~6.

Scn4a

55-13-08: rScn4a: Northerns: Abundant 8.5 kb mRNA in innervated adult skeletal muscle: rvtwofold higher in fast-twitch (anterior tibial) than in predominantly slow-twitch soleus muscle59. RNAase protection: mRNA found at high levels in adult skeletal muscle, low levels in neonatal skeletal muscle, but was not detected in brain or heart60. 55-13-09: hSCN4A: The mRNA transcribed from the human SCN4A gene, encoding the Na+ channel a subunit of the skeletal muscle channel, hSkMl, is found in adult skeletal muscle, but not in heart, brain or uterus31 .

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Table 2. Continued Scn5a

55-13-10: rScnSa: The Scn5a gene encodes the Q subunit of the rat heart NaHI channel, which is identical to the rNaSkM2 channel of denervated skeletal muscle. Northerns: the rNaHI mRNA is abundant in newborn and adult rat hearts, but not brain or innervated skeletal muscle, and appears upon denervation of skeletal muscle29,s9. 'Chemical denervation' with Clostridium botulinum type A toxin, a selective inhibitor of quantal acetylcholine release, produces a seven-fold increase in rSkM2 mRNA by 7 days. The levels of rSkM2 mRNA decline as functional re-innervation proceedss9. RNAase protection: rScnSa (rNaHl) mRNA could not be detected in adult skeletal muscle, but was present in neonatal skeletal muscle and after denervation of adult muscle28 . Note that both rNaSkMl and rNaSkM2 are present in denervated muscle, but only the rNaSkM2 mRNA increases after denervations9 ). 55-13-11: hSCNSA: Northerns: rv9 kb mRNA detected in heart, but not in brain, skeletal muscle, myometrium, liver or spleen32 .

Scn6a

55-13-12: hSCN6A: Northerns: The 7.8 kb transcript encoding the hNaH2 (hNav 2.1) Na+ channel Q subunit was readily detected in heart, uterus, adult skeltal muscle and cultured rhabdomyosarcoma (RD) cells, while faint signals were obtained with RNAs from brain, kidney and spleen2. 55-13-13: mNav2.3: Northerns: Strong signals corresponding to mNav 2.3 mRNA were detected in heart, uterus and the mouse atrial tumour cell line, AT-I; faint signals were present in RNA from brain, kidney and skeletal muscle3.

Scn7a

55-13-14: rScn7a: The eDNA encoding the 'glial-specific' rNaGI Q subunit, the product of the rScn7a gene, was first cloned via RNA from cultured rat astrocytes4. Northerns: The mRNA is highest in spinal cord, intermediate in midbrain and at lower levels in cerebrum and cerebellum: cultured Schwann cells, from both dorsal root ganglia and sciatic nerve, also 'express fairly high NaG mRNA levels'4. RNAase protection: rNaGl mRNA detected in skeletal muscle, spleen and intestine, not in liver or kidney. The rNaGl mRNA level in skeletal muscle increased following denervation4. ISH: Expression was limited to derivatives of the neural crest (neurones of DRG, trigeminal ganglion and mesencephalic nucleus of the trigeminal nerve, Schwann cells and satellite cells). Not all neural crest-derived elements express rNaGl, since superior cervical ganglion neurones, for example, lacked rNaGl mRNA, as did sensory neurones 61 .

SenSa

55-13-15: mscn8a: RNAase protection: present in brain, cerebellum and spinal cord; absent in skeletal muscle, heart, kidney and Schwann cells (sciatic nerve)62.

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Table 2. Continued Scn8a

55-13-16: rscn8a: ISH: Strongly expressed in granule and Purkinje cells, in the molecular layer and deep cerebellar nuclear cells. In the spinal cord, strongly expression in grey matter. The rscn8a mRNA was abundant in many neuronal populations of the CNS, which are fully documented in ref. 63 •

Scn9a

55-13-17: scn9a (rabbit): RT-PCR: detected in cultured Schwann cells, sciatic nerve, spinal cord, brainstem, cerebellum and cortex; not in lung, kidney or liver64 .

Scnl0a

55-13-18: rscnIOa: Northerns: rv6.5 kb transcript specific to neonatal and adult dorsal root ganglia. Signal substantially reduced following treatment of neonatal rats with capsaicin, suggesting the transcript is selectively expressed by capsaicinsensitive, nociceptive neurones 65 . ISH: the transcript was found only in small-diameter sensory neurones of dorsal root and trigeminal ganglia65 .

transient channels and that Scn8a specifies the Q subunit associated with the persistent Na+ current63 • Neither the RBI nor the Scn8a coding sequences have yet been expressed in heterologous t systems to test this hypothesis.

Rat PN3 Na+ channel is specific to small sensory neurones of the peripheral nervous system 55-13-20: Messenger RNA encoding the Q subunit of the rat peripheral nerve Na+ channel 3 (PN3 or SNS) was detected by Northern t blot analysis in RNA from dorsal root ganglia (DRG), and by RT-PCRt in nodose ganglia and (weakly) in sciatic nerve of the peripheral nervous system65,66. The corresponding mRNA was not detected in brain, spinal cord, superior cervical ganglia, heart or skeletal muscle. In situ hybridization showed PN3 mRNA to be present mainly in small neurones in the DRG65,66.

Differences in expression of Na{31 gene in human and mouse 55-13-21: The rNa,81 mRNA is detectable by hybridization at high levels in rat brain, spinal cord and skeletal muscle, at moderate levels in heart, and at low levels in uterus and kidney47,67. The human hNa,81 mRNA was detected at high levels in human brainstem and cerebellum, and lower levels in skeletal muscle and heart 68 . In contrast, high expression of the homologous mouse gene is confined to adult skeletal muscle and brain, and the mNa,81 mRNA was not detected in heart69.

Expression of the rat Na{32 gene 55-13-22: Northern blot analysis of total RNA from various rat tissues shows the 4.3 kb rNa,82 mRNA in brain and spinal cord, but not in liver, heart or skeletal muscle, even after prolonged exposure of the autoradiographs 44 • Note that this contrasts with the expression of the rNa,BI gene, whose

III

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transcript is found in skeletal muscle and heart67 (see paragraph 55-13-04). Within the brain, (32 mRNA was relatively abundant in cerebral cortex and cerebellum, moderately abundant in hippocampus and very weak in brainstem. The pattern of rNa(31 expression was different, with comparatively uniform levels of mRNA in all these regions of the rat brain44 •

Phenotypic expression Mutation of a mouse sodium channel gene causes motor endplate disease 55-14-01: Mutations of the med locus on mouse chromosome 15 produce a recessivet neurological disorder, motor endplate disease, with symptoms ranging from mild ataxia t to juvenile lethality. Neuronal defects, including lack of signal transmission at the neuromuscular junctiont, excess preterminal arborization t and degeneration of cerebellar Purkinje cells, are apparent in the disorder. Molecular cloning t of the affected gene, following non-targetedt insertion of a transgene 7ot , has shown that it encodes an a subunit of a voltage-gated Na+ channe162 . This gene, designated Scn8a, encodes a protein having 67-79% identity with other mammalian Na+ channel a subunits62.

Mutations in SCNSA can cause long QT syndrome 55-14-02: Long QT syndrome ('LQT') is an inherited cardiac arrhythmia that can cause abrupt loss of consciousness, seizures and sudden death from ventricular tachyarrhythmia. The disorder is associated with a prolonged QT interval on electrocardiograms, due to abnormal cardiac repolarization t. Three distinct genetic loci associated with the long QT syndrome have been mapped and one of these, 'LQT3' at 3p21, has been identified as the seNSA gene encoding a cardiac sodium channel a subunit. A deletion of 9 bp in the SCN5A gene, resulting in the loss of three conserved amino acids, Lys1505Pro1506-Gln1507, in the cytoplasmic linker between ID6 and lVI, was found in two unrelated LQT families 71 . Electrophysiological analysis of the mutant channels produced by expression of cRNA in Xenopus oocytes showed slightly decreased time constants for current decay, but the currents in cells expressing the mutant RNA were unusual in showing incomplete decay during a 200 ms depolarization. Single-channel recordings showed mutant channels fluctuating between normal and non-inactivating gating modes. This persistence of inward Na+ current would explain the prolongation of action potentials found in long QT syndrome72. (Note that mutations in the HERG gene, which encodes a subunit of the cardiac I Kr delayed rectifier K+ channel73, are responsible for the chromosome 7-linked form of long QT 74 .)

Mutations in the SCN4A gene cause myotonia t 55-14-03: Missense t mutations in the human gene (SCN4A) specifying the adult skeletal muscle voltage-gated Na+ channel a subunit, mapping to 17q23.1-25.3 75, have been found in patients with two hereditary disorders of sarcolemmal excitation, hyperkalaemic periodic paralysis (HYPP or hyperPP) and paramyotonia congenita (PMC)76-79 (see refs 8o,81 for recent reviews).

1L.-__ _ _ry_5_5 en t

_

Heterologous t expression of mutant cDNAs t in mammalian cell lines shows that PMC mutants affected at diverse locations in the subunit (G1306E,V or A, T1313M, L1433R, R1448H, R1448C, Al156T) all show reduced rates of channel inactivation82,83. Similar conclusions have been drawn from studies with biopsied muscle specimens from patients with myotonia83 . In contrast, a HYPP mutation (T704M) showed a normal inactivation rate, but altered voltage sensitivity of steady-state activation and inactivation82 . When amino acid changes corresponding to human HYPP mutations (T704M and M1592V) were introduced into the rat adult muscle Q subunit, ttl, transient t heterologous t expression in HEK-293t cells showed impaired inactivation of the mutant Na+ channels. The average amplitudes of the non-inactivating Na+ currents of the mutant channels were three-fold84 to five-fold 85 higher than those of the wild-type channel. Sporadic t cases of hyperkalaemic periodic paralysis have been reported, one of which has been shown to be due to a de novo mutation in the SCN4A gene that was not carried by either of the genetically confirmed parents77. The correlation between genotype and phenotype for the sodium channel Q subunit gene has been reviewed80,81. (Note that hypokalaemic periodic paralysis, hypoPP, is caused by mutations in the gene encoding the Ql subunit of the L-type Ca2 + channel of the T-tubules of skeletal muscle 86 (see VLGCa, 42-14-03).

Sodium channel defects in equine hyperkalaemic periodic paralysis

55-14-04: Hyperkalaemic periodic paralysis (HYPP or hyperPP), an autosomal t dominantt muscle disorder associated with high serum K+ concentrations, is prevalent in American quarter horses 87. The spontaneous muscular activity associated with the disorder increases muscular definition and hypertrophy, seen as desirable features in show horses, and selective breeding has favoured spread of the mutant gene in the quarter horse gene pool88. The causative alteration of the Na+ channel Q subunit is a Phe to Leu substitution at the cytoplasmic end of IVS3, and all cases can be traced to a single sire89.

Myotonia can be mimicked by application of anemone toxin (ATxII) 55-14-05: The small, persistent Na+ current that is characteristic of hyperkalaemic periodic paralysis can be produced in normal muscle by the in vitro use of micromolar anemone toxin (ATxII). The application of micromolar ATxII to preparations of rat fast-twitch skeletal muscle increased the steady-state open probability of Na+ channels from 0.0011 to 9 0.018 °, within the low end of the range of persistent currents seen in hyperPP myotubes. Under these conditions, whole-muscle preparations showed a tenfold slowing in the relaxation in twitch tension, the 'in vitro analogue of muscle stiffness'. In ATxII-treated muscle, a single constantcurrent pulse elicited a train of repetitive action potentials varying spontaneously in amplitude and firing frequency similarly to the electromyogram of a myotonic muscle 90 .

ScnSa mutations cause motor endplate disease in the mouse 55-14-06: A random integration event at the motor endplate disease (med) locus of the mouse resulted in a deletion of a novel gene encoding a

_'---

en_t_ry_5_5_

neuronal voltage-gated sodium channel, Sen8a62,70. The resulting med(tg) mice suffer from severe skeletal muscle atrophy, beginning by post-natal day 10 and resulting in death by day 2091 . Pathology consistent with denervation was evident in both hindlimb and forelimb musculature, but the post-natal maturation of the extraocular muscles was not altered. The onset of paralysis in the mutant mice probably coincides with a critical time for the involvement of the Scn8a Na+ channel in the initiation and/or propagation of action potentials in spinal motorneurones 91 . The original, spontaneous med mutation was caused by insertion of a truncated L1 repetitive DNA ('LINE') element into exon 2 of the Sen8a gene. The net result of the insertion is aberrant splicing t of the Scn8a transcript, from the normal donor site of exon 1 to a cryptic splice acceptort site in intron 2, producing a frame shift and a stop codon in exon 3 92 . A further spontaneous med mutation, responsible for the med J allele, involves a 4 bp deletion within the 5' donor site of exon 3, resulting in splicing from exon 1 to exon 4. The aberrant splicing alters the reading framet, resulting in a premature stop codon derived from exon 4 and a predicted protein product of only 101 residues 92 . A missenset mutation in Sen8a, resulting in the replacement of the highly conserved Ala10?1 by Thr in the cytoplasmic S4-S5 linker region of domain 11193, is responsible for the 'jolting' phenotype, involving a rhythmic tremor of the head and body and unsteady gait, characteristic of the homozygous Sen8a jo mutant 94 . Introduction of the analogous change (A1329T) into the rat brain IIA Na+ channel shifted the voltage dependence of activation by 14mV in the depolarizing direction, without affecting the kinetics of fast inactivation or recovery from inactivation. The Scn8ajo channels are thus more stable in the closed state, and a larger percentage of mutant channels remain closed at depolarizing potentials in the range -40 to OmV93 •

ScnSa mutations affect subthreshold Na+ currents and firing patterns in Purkinje neurones 55-14-07: Sen8a mutations, including the Scn8ajo missense mutation (see paragraph 55-14-06), impair the spontaneous firing of mouse Purkinje neurones 95,96. In isolated Purkinje cells from Scn8a-null t mice, peak transient Na+ current was rv60% of that in controls, but subthresholdt Na+ currents were more severely affected. The non-inactivating ('steady-state') Na+ currents elicited by voltage rampst were reduced by rv?O%, and 'resurgent t Na+ current', elicited by repolarization following strong depolarizations (mimicking action potential t -like waveforms) was reduced by rv80% 97. Spontaneous firing and bursts evoked by just suprathreshold stimulation were both strongly suppressed in Purkinje cells from Scn8a null mutant mice, demonstrating the importance of Scn8a-encoded channels in repetitive firing of Purkinje neurones 97.

Mutations at four loci affect Na+ channel function in Drosophila 55-14-08: Mutations affecting voltage-gated Na+ channel function in Drosophila melanogaster occur at four distinct genetic loci. The phenotypic consequences of these mutations are shown in Table 3.

Table 3. Mutations affecting Na+ channel function in Drosophila melanogaster (From 55-14-08) Locus

Map position

Phenotype of mutant

para ('paralytic')

x

The coding sequence predicts a protein Temperature-sensitive mutations result in paralysis at 30°C and recovery at 20°C of 1820 amino acids with 46-47% identity to rat brain and eel Na+ channel within seconds 98 . Mutations affect the amplitude of Na+ currents in Drosophila Q subunits 100,101. embryonic neurones and can alter gating properties 99 .

sei ('seizure')

chromosome 2 (60A-B)

Mutations result in convulsive seizures followed by paralysis at temperatures above 38°C 102 . One mutation increases the KD for saxitoxin-binding103 .

cDNAs encoding an Na+channel Q subunit hybridize to 60D-E, a region of chromosome 2 that is near to but not identical with the sei lOCUS 104.

nap ('no action potential')

chromosome 2

Nap mutations produce paralysis at

Nap is an allele of mle (maleless), a gene required for X chromosome dosage-compensation t and male viability107. The temperature-sensitive phenotype of napts probably results from the decreased number of Na+ channels causing a lowering of the temperature at which action potentials would normally cease108 .

37°C 105 and a decrease in the number of

binding sites for saxitoxin103 and tetrodotoxin 106.

tip-E ('temperatureinduced paralysis')

II

chromosome 3

Temperature-sensitive paralysis, with a non-permissive threshold of 39-40°C. The Na+ current in embryonic neurones from tip-E mutants is reduced109, as is the number of saxitoxin-binding sites in heads of adult flies 11o.

Characteristics of gene-product

Molecular information not yet available.

These observations are reviewed by Goldin (1994), see Related sources and reviews, 55-56.

(D

= rot-

~

CJ1 CJ1

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en_t_ry_s_S_

Protein distribution Immunocytochemical evidence for differential tissue distribution of Na+ channels 55-15-01: Voltage-gated Na+ channels are generally expressed in tissues generating action potentials t (see VLC key facts, entry 41). Although Na+ channel polypeptides from eel electroplax1 , rat brain23,24 and rat muscle30 display similar structures (see Domain arrangement, 55-27) with uniform biophysical properties56,111, there is much evidence for differential tissue distribution of Na+ channel subtypes (e.g. refs23,39,112,113). Immunochemical studies using antibodies raised against brain type ITa show that this subunit predominates in brain, while type la subunits predominate in spinal cordl14 . Immunochemical studies with an antibody against the SCAP-l voltagegated Na+ channel of the marine invertebrate Aplysia californica revealed a large cytoplasmic pool of Na+ channels and distinct clustering of the channels in Aplysia axons l15 .

Differential localization of type I and type II Na+ channels in rat brain 55-15-02.: Immunohistochemical studies using antipeptide antibodies specific for rat brain type I and type IT Na+ channels showed that staining of type I channels was relatively low and homogeneous along the rostral-caudal axis of sagittalt brain sections, in contrast to that of type II channels, which was heterogeneous and relatively dense in forebrain, substantia nigra, hippocampus and cerebellum. The somata of the dentate granule cells, hippocampal pyramidal cells, cerebellar Purkinje cells and spinal motor neurones were immunoreactive for type I but not type II channels. Areas rich in unmyelinated nerve fibres, including the mossy fibres of the dentate granule cells, the stratum radiatum and stratum oriens of the hippocampus and the molecular layer of the cerebellum, were positive for type IT and negative for type I stainingl13 .

Subcellular locations Axonal Na+ channels cluster at nodes of Ranvier 55-16-01: In myelinated axons, voltage-dependent sodium channels are

concentrated to the nodes of Ranvier l16, where they co-localize with ankyrin in an arrangement that is crucial for saltatory conduction along axons. The localization of Na+ channels was shown to be 'highly dependent on interactions with active Schwann cells' and continuing axon-glial interactions are necessary to maintain channel distribution during differentiation of myelinated axons l17 or remyelination of demyelinated axons l18 (see paragraph 55-16-02 for a different interpretation).

Axonal Na+ channel clustering is induced by a protein secreted from oligodendrocytes 55-16-02.: The immunoreactivity of Na+ channels in purified rat ganglion cells

cultured in serum-free medium is diffuse, but becomes typically clustered in the presence of optic nerve glia l19 . Sodium channel clustering, co-incident

1"--_e_n_t_ry_5_5

_

Table 4. Sizes of mRNAs transcribed from genes encoding Na+ channel Q subunits (From 55-17-01) Gene

Channel subunit

mRNA size (kb), tissue and reference

Scnla Scn2a

rRBl rRBTI rRBTIA rRBTII

9.0 (brain, cardiac muscle)23,56 9.5 (major), 8.6 (minor) (brain)23,56 9.0 (brain)121 rv9.0 (major), rv 7.5 (minor) (heart, skeletal muscle, small intestine, minor species also in brain)24,39 8.5 (adult skeletal muscle)59 rv9.0 (right atrium, left ventricle)32) 8.5 (denervated skeletal muscle)59 7.8 (heart)2 7.5 (major), 5 kb (minor) (astroglia)4 rvlO and 12 (brain, spinal cord)62,92 rvlO (weak) and rv12 (strong) (cerebellum)63. 9.4 and 7.0 (medullary thyroid carcinoma cell-line )350 7.0-7.5 (dorsal root ganglia)65,66 rv8.0 (electroplax)l > 12 giant fibre lobe neurones122

Scn3a

Scn4a SenSa Scn6a Scn7a Scn8a

rScn4a (rSkMl) hNaHl (hSkM2) rSkM2 hNaH2 (hNav 2.l) rNaGl mScn8a rScn8a (CerTII)

Scn9a

hScn9a (hNE-Na)

ScnlOa Nal (eel) Na2 (squid)

rScnlOa (PN3) eNal sNa2 (GFLNl)

with that of ankyrin-G, is also induced by glial-conditioned medium or soluble extract of optic nerve, an activity that is sensitive to proteolysis l19 . Oligodendrocytes, which develop concurrently with channel clustering in rat optic nerve, are able to induce in vitro channel clustering, but astrocytes are ineffective. Myelin-deficient (md) mutant rats that are deficient in oligodendrocytes have normal numbers of Na+ channels but develop few axonal clusters in ViVO l19 . The identical patterns of clustering of Na+ channels and ankyrin-G implicate cytoskeletal interactions in the induction and maintenance of the protein aggregates l19,120.

Transcript size Transcripts encoding

Q

subunits of Na+ channels

55-17-01: The sizes of the mRNAs encoding Na+ channel shown in Table 4.

Q

subunits are

Sizes of mRNAs encoding {3 subunits of Na+ channels 55-17-02: rNa~l Northern blot analysis reveals a 1400nt67 or l600nt47 messenger RNA for rNa~1 in rat brain, heart, skeletal muscle, uterus (low), kidney (low) and spinal cord. Probes specific for rNa~2 mRNA showed a strongly hybridizing band of rv4.3 kb in rat brain and spinal cord, but not in liver, heart or skeletal muscle, even after overexposure of the autoradiographs44 .

_______________________ en_t_ry_5_ 5

----J

SEQUENCE ANALYSES The symbol {PDTM} denotes an illustrated feature on the channel protein domain topology model (Fig. 2).

Chromosomal location 55-18-01: The chromosomal locations of the known genes encoding Na+ channel Q subunits are shown in Table 5.

The mouse Scnla, Scn2a,Scn3a and Scn9a genes are closely linked 55-18-02: Four genes encoding brain Na+ channels in the mouse are all clustered on the proximal segment of chromosome 2, with the probable gene order centromere-Hc-Neb-Pmv7-Scn2a/Scn3a-Scnla/Scn9a-Mpmv14124. The Scn2a (encoding type 2 Q subunit) and Scn3a (encoding type 3 Q subunit) genes are separated by a maximum physical distance of 600 kb, while the closely linked Scnla (encoding the type 1 Q subunit) and Scn2a genes are separated by a genetic distance of 0.7 eM (equivalent to rv700kb)124. The mouse Scn9a gene, encoding a Na+ channel Ql subunit expressed in Schwann cells, is very tightly linked to Scnla130.

Encoding Sizes of proteins encoded by Na+ channel genes 55-19-01: The sizes and molecular weights of the polypeptide chains encoded by the various cloned Na+ channel genes are given in Table 7 and paragraph 55-53-01.

Independent isolates of a sensory neurone-specific Na+ channel cDNA show minor differences 55-19-02: Full-length cDNA sequences encoding the Q subunit of a tetrodotoxin-resistant voltage-gated Na+ channel have been independently cloned from Sprague-Dawley rat dorsal root ganglia by two groups. The encoded channels, termed peripheral nerve sodium channel 3 ('PN3,)66 and sensory nerve sodium channel ('SNS,)65, are virtually identical in sequence, though the SNS protein is one amino acid longer (1957 vs. 1956) due to an additional Pro residue at position 521. Some variants of the PN3 cDNA also contained an additional GIn codon between the codons for Pr0583 and Ala584 66 . The properties of the channels are indistinguishable after expression in Xenopus oocytes, and PN°3 and SNS clearly represent products of allelic t variants of the same rat gene.

The immature rat {32 subunit contains an N-terminal signal peptide 55-19-01: The rat Na,82 gene encodes a,B2 subunit of 186 amino acids plus a 29 amino acid N-terminal signal peptide sequence that is cleaved from the mature ,82 protein44 • Only the protein with the signal peptide was functional following heterologous expression of cRNAs in Xenopus oocytes 44 .

!III

l_e_n_try_S_S

_

Table 5. Mammalian genes encoding channels (From 55-18-01)

Q

subunits of voltage-gated Na+

Gene

Chromosome location Association with disease Human Mouse Rat

SCNIA SCN2A SCN3A

2124 2124 2124

SCN4A

2q24123 2q23-24.1 125 2q2324.3 27,125,126 17q23.1-q2S.3 75

SCNSA

3p21-24128

9

SCN6A SCN7A SCN8A

2q21-23 12q13 62

SCN9A SCNI0A SCNIB 19q13.1 SCN2B

11 127

2129 15 62

3 3 3 Mutations in the human SCN4A gene, specifying the adult skeletal muscle voltagegated Na+ channel Q subunit, have been found in patients with two hereditary disorders of sarcolemmal excitation, hyperkalaemic periodic paralysis (HYPP or HyperPP) and paramyotonia congenita (PC)76-79 (see paragraph 55-14-03). HyperPP due to SCN4A mutation is prevalent in American quarter horses 87 (see paragraph 55-14-04). Defective in one form of long QT syndrome, LQT3 71 .

The mouse Scn8a gene is congenic with the med locus, mutations in which cause 'motor endplate disease'70.

2130

9131 769,132 9133

(Central region of mouse chromosome 9, close to the gene encoding the interleukin 10 receptor, 1110133 . This region of mouse chromosome 9 is orthologous t to human chromosome llq22-qter.)

Gene organization The SCN4A gene contains 24 coding exons 55-20-01: The detailed structure of the human SCN4A gene, encoding the adult skeletal muscle Na+ channel Q subunit, has been determined by

_L.--

e_n _t ry_5_5_

sequencing of genomic t clones. The coding region of the gene occupies 32.5 kbp of genomic DNA and is arranged in 24 exonst 134~135. Pairs of deoxyoligonucleotide t primers t consisting of intron t sequences for amplification of all 24 exons by the polymerase chain reaction t have been made available134.

A silencer sequence restricts expression of type II Na+ channel gene to neuronal cell types 55-20-02: Deletion of upstream sequences of the type IT Na+ channel gene resulted in an 80-fold increase in reporter genet activity in skeletal muscle cells, suggesting the presence of negative control elements136. Alignment of neural-restrictive transcriptional silencert elements (NRSE) from the 5' upstream t regions of the type IT Na+ channel gene and other neuronespecific genes has identified a consensust sequence responsible for binding neural-restrictive silencer binding factor (NRSBF), which is present in nonneuronal cells and absent in neuronal cells137~138 (see also Protein distribution, 55-15). Deletion mappingt and DNAase I footprintingt experiments delineated a 28 nucleotide sequence (-1023 to -996, ATTGGGlTTCAGAACCACGGACAGCACC) that was shown to inhibit ('silence') expression of reporter genes specifically in cell lines that do not express the type IT Na+ channel gene137, This 'silencer' element functions in either orientation and also works when placed downstream t of the reporter gene in a skeletal muscle cellline137.

cDNAs encoding the tnon-neuronal' silencer protein have been cloned 55-20-03: The human sequences encoding the silencer protein that interacts with the type IT Na+ channel silencer element (see paragraph 55-20-02) have been cloned from HeLa cell eDNA libraries139. (The silencer sequence has been termed 'repressor element I', or 'REl,137, and the silencer protein that interacts with this element has been called 'REl-~ilencing !ranscription factor', or 'REST,139). The protein of 1097 amino acids (121 kDa) contains nine putative zinc fingerst, a highly basic region and six reiterations of a proline-rich motif and a nuclear localization signal t . The REST protein is found at high levels in L6 skeletal muscle cells but at very low levels in neuronal (PCI2) cells that are 'permissive' for type IT Na+ channel gene expression. REST protein produced during transient t expression of a eDNA construct in PC12 cells was able to repress the expression of a co-transfectedt reporter t gene containing the REI silencer element. In developing mouse embryos, REST mRNA was abundant in all non-neural tissues and weakly detectable in the ependymal layer (proliferating neuroblasts)139.

Sodium channel genes with non-standard splice sites 55-20-04: Comparison of eDNA and genomic sequences encoding the mouse scnSa Na+ channel a subunit reveals four exons t , of lengths 276 (from the translation initiation codon), 119, 90 and 129 nucleotide pairs, at the promoter-proximal end of the gene 92. The positions of the splice junctionst are identical to those defined for the human SCN4A gene134~135 (see paragraph 55-20-01). The second introns t of the mouse senSa gene 92, the

l_e_n_t_ry_s_S

_

human SCN4A 134,135 and SCNSA genes (quoted in ref. 92 ) are members of a minor class of introns with non-standard splice sites: they lack the standard GT and AG dinucleotides at splice donor t and acceptort sites, and are similar to introns from yeast. Such non-standard introns in mammalian genes have been shown to bind a different set of snRNAst and splicing proteins from those binding to standard introns 140.

The mouse ScnlOa gene contains 27 exons 55-20-05: The mouse Scnl0a gene, specifically expressed in sensory neurones of dorsal root ganglia and encoding the Ql subunit of a tetrodotoxin-resistant Na+ channel, consists of 27 exonst spanning about 90kb of chromosome 9131 . The sizes of the exons and the positions of the intronst are highly conserved between the mouse Scn10a and the human SCN4A and SCNSA genes (see paragraph 55-20-01)131.

Expression of the Drosophila para gene involves alternative splicing 55-20-06: The Drosophila sodium channel gene para contains alternative exonst, a and i, encoding the first cytoplasmic loop of the channel protein. Alternative splicing of these exons results in four para transcripts that are present individually or in combination within single neurones 141 . Exon a was necessary but not sufficient for production of Na+ currents in cultured embryonic neurones. Similar alternative splicing of para mRNA was also evident in RNA isolated from whole Drosophila embryos141.

Alternative splicing of Na+ channel gene transcripts in the rat 55-20-07: Analysis of transcripts encoding the cytoplasmic loop between domains I and IT of the voltage-gated Na+ channel Q subunit in rat tissues by RT-PCRt revealed alternative splicingt of transcripts from the rat Na+ channel I and ill genes 142. Southern blot analysis confirmed the presence of introns in the corresponding regions of the rat NaCh ITI gene142. The spliced intron occurs at a homologous position in each of the three rNaCh genes. The transcripts detected in rat brain, cardiac muscle and skeletal muscle are summarized in the Table 6.

Table 6. Na+ channel gene transcripts detected in rat brain, cardiac and skeletal muscle (From 55-20-07) Channel

Brain

Cardiac muscle

Skeletal muscle

NaChl NaChIT NaChID

1,la*

1*, la * Not detected ID*, IDa, mb

1*, la* Not detected ID*, IITa*, mb*

llA* ill, IITa, mb

Data were taken from ref. 142: All RNA species were detected by RNAase protection t , while those indicated (*) were also detected by sequencing eDNA clones. Note that the experimental design would not have detected the JlI and SkM2 transcripts in skeletal muscle.

_

....

e_n_try_5_5_

Introns in the rat f32 gene 55-20-08: The coding region of the rat gene (rScn2B) specifying the Na+ channel {32 subunit has been cloned by PCR t -based amplification, using oligonucleotide t primers t based on the cDNA sequence44 • Two intronst of 243 and 483 nucleotide pairs are present, interrupting the f32 coding sequence between nucleotides 237-238 and 447-448 44 •

Homologous isoforms Tissue specialization of Na channels preceded the separation of rats and humans 55-21-01: Note that there is greater similarity between the Na+ channel sequences from the same tissues of rat and humans than there is between different channel isoforms from the same species. The tissue specialization of Na+ channels clearly preceded the separation of rats and humans (see Goldin, 1994 under Related sources and reviews, 55-56). The primary sequences of 17 Q subunits of voltage-gated Na+ channels have been aligned (see Goldin, 1994, pp. 78-82).

Rat NaHl and NaSk2 channel proteins are identical 55-21-02: Complementary cDNA clones encoding Na+ channels have been cloned from rat cardiac muscle and denervated skeletal muscle eDNA libraries. The cardiac muscle eDNA clone, rH1 29, and the skeletal muscle clone, SkM228, encode the same channel Q subunit, referred to as rNaHI or rNaSk2. Expression of the corresponding cRNAs in Xenopus oocytes gives tetrodotoxin-resistant and jL-conotoxin-resistant Na+ channels 143,144.

Two distinct cardiac Na+ channels 55-21-03: Two different sequences encoding human cardiac Na+ channels, hNaHI and hNaH2, have been identified by cloning from human heart eDNA librariest. The hNaHI sequenc.e, which encodes a protein of 2016 amino acids, expresses in Xenopus oocytes to give rise to Na+ currents that are relatively resistant to tetrodotoxin (IC so == 5.7 JlM) and sensitive to lidocaine32, as expected for the cardiac Na+ channel. The hNaH2 sequence encodes a protein of 1682 amino acids with only about 500/0 sequence identity to the rat cardiac Na+ channels2 , but has not yet been characterized by functional expression.

The rat brain RII and RIIA subunits are produced via alternative splicing 55-21-04: The rat brain Na+ channel Q subunits, RII and RIIA, are highly homologous, differing in only six amino acids25,145. The coding sequences of the RII and RIIA cDNAs differ at 36 positions, 20 of which are within a 90 nucleotide segment. These localized differences are due to the use of two alternative exonst, a 5' exon N (neonatal) in Rll and a 3' exon A (adult) in RIIA. The sequences of 30 amino acids encoded by these exons differ in only one position: Asn209 of RII is replaced by Asp in RITA. The alternative exons appear to be mutually exclusive, since no cDNAs containing both exons could be detected40 . (The additional six amino acid differences between the reported RII and RITA Q subunit sequences are presumably due

lL......-_ _ _ry_55

_

en t

to natural polymorphismst within the RII/RIIA genes of the rat strains used for the original cDNA cloning.)

Protein molecular weight (purified) The rat brain Na+ channel subunits 55-22-01: The Na+ channel complex solubilized with Triton X-lOOt and purified from rat brain as the saxitoxin-receptor has an estimated molecular mass of 316kDal46 . The complex consists of an a subunit of 260kDa and associated subunits of 36 kDa (131) and 33 kDa (132)147,148. The 132 subunit is covalently attached to the a subunit by a disulphide t bond, but association of the 131 subunit is non-covalent149. All three subunits are intrinsic membrane glycoproteins. A highly purified complex of a, 131 and 132 subunits was sufficient to mediate neurotoxin-activated Na+ flux in reconstituted lipid vesicles l48,150.

Rat skeletal muscle Na+ channels 55-22-02: The Na+ channel from rat skeletal muscle, solubilized in Lubrol PX, had an estimated molecular mass of 314kDa151 . The sarcolemmal and T tubule Na+ channels both consist of a major (a) subunit of 260 kDa and a minor 13 subunit of 38 kDa152-154.

Na+ channels lacking {3 subunits 55-22-03: The Na+ channel preparations purified from eel electroplax155,156 and chicken heart157 apparently contain only single polypeptides of 260 kDa, which have sequence homology to the a subunit from brain and Table 7. Molecular weights of Na+ channel subunits predicted from the coding sequences (From 55-23-01) Channel protein

Species

Tissue

Mol. wt (amino acids)

Refs

Na1

Electrophorus electricus Loligo opalescens

Electroplax

208321 (1820aa)

1

Giant axon

203000 (1784aa)

122

Brain Brain

228 758 (2009 aa) (2005 aa) 227840 (2005 aa) 221375 (1951 aa) 227159 (2016aa) 227417 (2019 aa) 193472 (1682aa)

23 27 23 24 32 29 2

225748 192144 220500 220500 22851 20902

64 3 66 159 67

Na2 (GFLN1) NaBl a subunit NaB2 a subunit NaB3 a subunit NaHl a subunit NaH2 a subunit

Rat Human Rat Rat Human Rat Human

Scn9a a subunit Rabbit Nav 2.3 a subunit Mouse Scn10a a subunit Rat Nat31 (13 subunit) Nat32 (13 subunit)

Rat Rat

Brain Heart Heart Schwann cells Heart uterus Dorsal root ganglia Brain

(1984aa) (1681 aa) (1956aa) (1957aa) (218 aa) (186aa)

44

_'--

•

e_n_try_5_5-----11

skeletal muscle. The purified 260 kDa protein from eel electroplax was able to mediate Na+ flux when reconstituted in lipid vesicles 158 .

Protein molecular weight (calc.) 55-23-01: The molecular weights of various Na+ channel subunits calculated from the constituent amino acids predicted from the sequences of open reading frames are given in Table 7.

Sequence motifs 55-24-01: Protein kinase A: The consensus target sites for phosphorylation by PKA in some Na+ channel Q subunits are shown in Table 8.

Table 8. Consensus PKA sites in Na+ channel Q subunits (From 55-24-01) Channel protein

Species

Nal (GFLNl)

Squid (Loligo S485 (RKL~), S496 (KRE~), opalescens) (giant fibre) T1323 (KKPI) Rat S248 (KKL~), S550 (RRD~), S620 (RRN~), S695 (RRS~) Human (brain) S250 (KKL~), S554 (KRF~), Rat (brain) S611 (RRD~), S574 (RRN~), S624 (RRH~), S686 (RRS~), S1052 (KKDS), S1930 (KKVS) S249 (KKL~), S553 (KRF~), S610 (RRDS), S623 (RRP~), S687 (RRS~), S1051 (KKD~) Rat S249 (KKL~), S573 (RRN~), S610 (RRD~), S638 (RRLS) Human (cardiac) T17 (RRFI), S483 (KRMS), S571 (RRT~), S593 (KKN~), Rat T977 (KRTI) and TI026 (RKEI) T17 (RRFI), S484 (KRL~), S594 (KRN~), TI029 (RKEI) Human (cardiac) S442 (KKR~), T777 (PKDI), S869 (IKQ~) and S905 (RRGS) S256 (KKL~) Human (skeletal muscle) S443 (KKR~), S906(RKS~) Mouse (cardiac) S528 (KRL~), Rabbit (Schwann cells) S48 7 (KKL~), S548 (RRS~), S585 (RRG~), S598 (RRS~) and S667(RRSSj

NaBI NaB2 (HBA)

NaB3 NaHI

NaH2 (Nav 2.1)

NaSkl (SkMl) Nav 2.3 Scn9a

II

Consensus PKA sites

Refs 122 23 27 23

24 32 29

2

160

3 64

----l_

l_e_n_try_5_5

55-24-02: Protein kinase C: The activation of protein kinase C in vivo or its use in inverted patches in vitro slows inactivation of sodium channels and reduces peak sodium currents (rv 80%) (see Protein phosphorylation, 55-23-03 to 55-23-05). Although there are numerous consensus sequences for PKCdependent phosphorylation in most Na+ channel Q subunits, phosphorylation of a single residue of the rat brain type ITA Na+ channel, Ser1506, located in the conserved intracellular loop between domains ill and ~ is required for PKC-dependent modulation in stably transfected CHO celllines161,162. 55-24-03: N-linked glycosylation sites: Consensus sequences (NXS/T) that are candidates for N-linked glycosylation within Na+ channel Q subunits are given in Table 9.

Table 9. Consensus N-linked glycosylation sites in Na+ channel (From 55-24-03)

Q

subunits

Channel

Species

Consensus N-glycosylation sites

Refs

Nal

Squid (Loligo bleekeri) Squid (Loligo opalescens)

248 (NAT), 258 (NYT), 1192 (NPS)

163

213 (NLS), 286 (NMT), 315 (NAS), 325 (NYT), 1113 (NLT), 1186 (NGT), 1197 (NRS), 1367 (NMS) 284 (NAS), 295 (NVT), 306 (NET), 338 (NSS), 1392 (NHS), 1403 (NET), 1758 (NPS) 285 (NSS), 291 (NIT), 297 (NNS), 303 (NGT), 308 (NRT), 340 (NSS), 1368 (NYT), 1382 (NYS), 1393 (NQT) 212 (NVS), 285 (NST), 297 (NNS), 308 (NRT), 340 (NSS), 1382 (NYS), 1393 (NQT), 1748 (NPS) 211 (NVS), 296 (NGT), 339 (NGS), 1317 (NTT), 1331 (NFS) 214 (NVS), 283 (NFT), 288 (NGT), 291 (NGS), 318 (NGT), 328 (NSS), 740 (NMT), 803 (NLS), 1365 (NQT), 1374 (NYT), 1380 (NKS), 1388 (NLT), 1736 (NGS) 215 (NVS), 319 (NGT), 329 (NSS), 741 (NMT), 865 (NYS), 1367 (NQT), 1376 (NYT), 1738 (NGS) 276 (NET), 281 (NRT), 309 (NRT), 1103 (NKS), 1113 (NES) 293 (NDT), 296 (NTT), 302 (NDT), 308 (NDT), 320 (NDS), 326 (NDT), 338 (NDT), 367 (NSS) 277 (NET), 282 (NRT), 288 (NYS), 310 (NRT), 1103 (NKS), 1113 (NES)

122

Na2

NaBl

Rat

NaB2

Human Rat

NaB3

Rat

NaHl

Human Rat

NaH2

Human

NaSkl

Human

Nav2.3

Mouse

23

27} 23

24 32} 29

2

160

3

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_5_5_

Southerns Southerns of mouse genomic DNA 55-25-01: Southern blots of restriction fragments of mouse genomic DNA separated by pulse-field t gel electrophoresis and probed with Scnla-, Scn2aand Scn3a-specific cDNAs are described in ref. 124. Blots of Mus spretus and C57BL/6T mouse genomic DNA, digested with BgID and HindIII and probed with Scn1a and Scn2a sequences have also been presented124. Southern blots of genomic DNAs from A/T and C57BL/6T mice digested with MspI and Bglll and probed with rabbit Nas (= Scn9a) cDNA sequences are presented in ref. 130.

Southern analysis of rat DNA probed with rNa III sequences 55-25-02: Southern analysis of rat genomic DNA digested with BamH1, EcoRI, Haem, HindIII, KpnI, PstI, RsaI and XbaI and probed with rat NaCh Ill-specific sequences have been described142. The data are consistent with a single, intron-containing rNaCh m gene.

STRUCTURE AND FUNCTIONS

Domain arrangement Some but not all Na+ channels have accessory subunits 55-27-01: Heterologous expression of Q subunits alone produce voltage-gated, Na+ -selective channels, although these have displayed altered (slower) inactivation properties25 than native channels. Notably, a number of smaller subunits are associated with Na+ channels from different tissues5 ,164 j coinjection of low molecular weight RNA from rat brain25 restores normal gating and increases expression. Na+ channel gene products isolated from eel electroplaxl , rat brain23,24 and rat muscle30 and other sources are relatively large (>1800 aa) with four internally homologous domains. Each domain contains multiple stretches of 19 or more predominantly hydrophobic residues that can potentially span the membrane (see VLC key facts, entry 41).

A single transmembrane segment in the {31 subunit 55-27-02: The rNa,81 subunit has a hydrophobic sequence spanning residues 142-163 that is predicted to be a single transmembrane domain. The nascent t f3 subunit carries an N-terminal signal peptide, suggesting that the N-terminus is extracellular and the C-terminus is cytoplasmic67.

The pore region has open loop' secondary structure t

55-27-03: The structure of the pore region of the rat JL1 skeletal muscle Na+ channel has been explored by serial cysteine-substitutiont mutagenesis. Replacement of consecutive amino acids in each of the four P segments gave Na+ -selective channels that showed increased sensitivity to extracellular Cd2+, which penetrates into the pore and binds avidly to free thiolt groups in cysteine side-chains. (The G1238C mutation was exceptional in producing an inactive channel.) In each of the four domains, at least two consecutive

I

~,

entry55

_

- - - - - - -

substitutions show significant increase in sensitivity to Cd2+ and to hydrophilic sulphydryl modifying agents. This finding is inconsistent with the local secondary structure being either Q helixt or {3 turnt, and suggests instead 'open loop' structures, ordered by interaction with neighbouring regions of the channel protein165.

Putative transmembrane regions of the a subunit are predominantlya helical 55-27-04: The secondary structures t of synthetic peptides representing putative transmembrane regions of the Q subunit of the rat brain type n voltage-gated Na+ channel have been analysed, in trifluoroethanol and in dodecylphosphocholine micelles, by physical techniques. All of these peptides, representing IS2, IS4, IVS4, IS3 plus IS4, and the putative linker region between segments ISS and IS6, were found to have predominantly 166 Q helical t structures in both solvent systems .

Domain conservation Na+ channel and Ca 2+ channel genes share a common ancestry 55-28-01: The four homologous domains I-IV of voltage-gated Na+ channels have discernible sequence similarity (rv32-37% identity) with the four corresponding domains of voltage-gated Ca2 + channels167, reflecting common evolutionary ancestry of these two channel types l68 .

Conservation of a region involved in Na+ channel inactivation 55-28-02: The sequence between the third and fourth homologous domains is highly conserved from Drosophila to vertebrates 1OO• Antibodies directed against this region reduce the rate of inactivation as measured in voltageclamp studies 169.

The Na+ channel {32 subunit has sequence similarities with cell adhesion molecules 55-28-03: The rat Na+ channel {32 subunit has sequence similarities with two separate segments of the neural cell adhesion molecule (CAM) contactin, a membrane glycoprotein belonging to the immunoglobulin superfamilyt 44. A sequence of 34 residues in the extracellular domain of {32 (residues 87-120 of the mature protein) is 45% identical to a segment in the N-terminal third of contactin (residues 288-321 of the contactin precursor). The adjacent sequence of 25 residues of {32 (residues 120-144) is 420/0 identical to a sequence near the C-terminus of contactin (residues 951-976). Structural prediction algorithms suggest that the extracellular domain of the Na+ channel {32 subunit forms a single 'immunoglobulin repeat', with a disulphide bond between Cys21 or Cys26 and Cys98. Contactin is predicted to contain six such immunoglobulin repeats, with the sequence similarity being in repeat three. The Na+ channel {31 subunit has a similar transmembrane topology to that of {3267, with a predicted immunoglobulin fold in its extracellular domain44 . The second region of sequence similarity between {32 and contactin is located immediately proximal and N-terminal to the cell membrane in both proteins. The sequence similarities between

_______________________ en_t_ry_5_5_

{32 and contactin, a protein whose binding to extracellular matrix proteins is implicated in the regulation of neurite migration, suggest that the extracellular domain of the (32 subunit may have a function in cell-cell interaction during development44•

Domain functions (predicted) Evidence that 84 segments of Q subunits act as voltage sensors 55-29-01: The S4 segments of the a subunits, with positively charged amino acids at every third position, are predicted to act as the voltage sensor of the voltage-gated Na+ channels. Neutralization of the positive charges in rNaB21S4 produced a decrease in the steepness of the potential-dependence of activation170. The mutation K226Q, neutralizing the last positive charge in segment IS4, causes a shift of +20mV in the potential-dependence of activation, while the stronger charge-modification resulting from a K226E mutation produces a +30mV shift. Charge-neutralizing mutations at more N-terminal positions in S4, such as R220Q or R217Q/R220Q, produce strong shifts in the opposite direction. (Reference170 presents data on the effects of single, double and triple mutations affecting charged residues in rNaB21S4 and rNaB2IIS4 on the properties of the channels analysed after expression of cRNAs t in Xenopus oocytes.) 84 segments show voltage-dependent movements 55-29-02: In ion channels with positively charged S4 segments, depolarization is predicted to cause outward movement of 54 leading to channel opening171,172. This hypothesis has been tested by probing the accessibility of cysteine residues in S4 of the hSkMl Na+ channel a subunit to extracellular attack by hydrophilic t cysteine-modifying reagents. A cysteine residue replacing Arg1448 in the a subunit from a paramyotonia congenita family76 (see Phenotypic expression, 55-14-03) is accessible to the modifying reagent methanethiosulfonate-ethyltrimethylammonium (MTSET; 20 JlM) only after depolarization173. Substitution of cysteines for each of the basic residues of IVS4 of the hSkMl a subunit ('cysteine scanning mutagenesis') has allowed investigation of the accessibility of the cysteines to cysteine-specific reagents during whole-cell recording of Na+ channel activity174. The findings of this study are summarized in Table 10. Calculations based on these findings support the conclusion that the translocation of charge in IV54 may be sufficient to account for the voltage-dependent gatingr. At least two residues, 1451 and 1454, traverse the hydrophobic core of the channel protein on depolarization. The fit to the Boltzmannt curve for the voltage-dependent reaction with the R1454C channel indicates the movement of 0.97 eo across the membrane field during exposure. Because of the greater charge on the wild-type Arg1454 (+1) than the mutant Cys (pKa "'-J8.5; fractional negative charge), the expected charge movement for the wild-type IVS4 should be "'-J2.5 eo. If the other three 54 segments translocate roughly similar quantities of charge on depolarization, the four segments could account for transfer of the total charge of about 12 eo 72,175 underlying channel gating. Note that, at depolarized voltages, Cys1454 is outside and Cys1457 is intracellular. The distance between these residues in the IV54

l_e_n_t_ry_55

-----'_

Table 10. Accessibilities of residues in IVS4 of hSkMl Na+ channel (From 55-29-02) V

R1448C

R1451C

R1454C

R1457C

Hyperpolarized Depolarized

buried outside

inside outside

inside outside

inside inside

R1460C

R1463C

K1466C

R1469C

inside inside

inside not outside

Hyperpolarized Depolarized

inside not outside

Notes: V represents the membrane potential at which the cells were exposed to the cysteine-modifying reagent. The 'hyperpolarized' condition was a holding potential of -140mV. The 'depolarized' state was achieved with the voltage being by 10 depolarizations of 900ms each at -40m~ returned to -140mV for 900ms between each depolarization. The MTS reagents had no effect on wild-type channels under these conditions. The K1466C mutant channel showed no detectable response to extracellular or intracellular cysteine-modifying reagent. These experiments are discussed in detail in ref. 174 . region will be between 4.5 A (if Q helical) and '" 11 A (if extended secondary structure), compared with ",40 Afor the lipid bilayer thickness. Thus the S4 charges need move only a relatively short distance « 11 A) across a hydrophobic core of the channel protein. It also implies that the field strength across the membrane at this point should be 'substantial' ("'10 8 VI m) for typical membrane potentials174 . The failure of extracellular pore blockers, tetrodotoxin and j.l-conotoxin, to prevent the interaction of MTSET with R1448C indicates that the hydrophobic 'channel' through which S4 moves is physically distinct from the ion-conducting pore174 . The observations and conclusions about S4 regions as voltage sensors in Na+ channels and other voltage-gated ion channels have recently been reviewed176 .

Interactions between a pore-blocking peptide and the voltage sensor 55-29-03: The jl-conotoxins are peptides that block the Na+ channels in adult skeletal muscle by binding to the outside of the pore region. A mutated (R13Q) derivative of p-conotoxin GIIIA gives incomplete block of the rSkMl Na+ channel, while binding to the normal target site, allowing study of channel function in the presence of the toxin. Binding of the mutant toxin to rSkMl in reconstituted lipid bilayers and during heterologous t expression in mammalian cells resulted in a reduction in single-channel current, a twofold reduction in Po and a shift in the activation curve towards more positive voltages. This shift in voltage-dependent activation is interpreted as an inhibition of S4 movement by the bound, cationic peptide, and allows the calculation that the centre of the S4 charge moves outward by between 3 and 8.5 A during gating177 (cf. the 4.5-11 A deduced from cysteineaccessibilty studies, see paragraph 55-29-02).

II

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_5_5_

The intracellular III-IV loop is involved in inactivation 55-29-04: Several experiments indicate that the inactivation gate of sodium

channels is located at the intracellular surface of the protein178 . A section of the Q subunit that connects domain ill to IV is predicted in one model179 to block open channel pores (leading to inactivation). Antibodies against the III-IV loop applied to the intracellular surface slow the inactivation process 169. The expression in Xenopus oocytes of two rNaB2 cRNAs, one encoding repeats I, II and ill and the other encoding only repeat ~ gives rise to functional Na+ channels that show a dramatically reduced rate of inactivation170. In these mutant channels, the time constant for inactivation, 7h, is largely voltage independent and is 30-fold greater than that of the wild-type channels at strongly depolarizing potentials (> +50mV). Single-channel recordings show a mean open time of 5.8 ms after depolarizing pulses to -20mV for the mutant channels, which is an order of magnitude larger than that of the wild-type channels 180. Small insertions at the ends of the rNaB2A ill-IV linker region slow inactivation181 . Proteolysis t (trypsin t, 50 Jlg/ml for 10min at 16°C) of inside-out patches of membrane from Xenopus oocytes expressing wild-type channels also strongly reduced channel inactivation17o. Interruptions between repeats II and ill do not have a significant effect on inactivation rates: those between repeats I and II give 'marginal' Na+ currents that are too low to allow detailed analysis 17o. See VLC key facts, entry 41, for information on S4 region voltage sensors. See also refs 169,182 under Protein phosphorylation, 55-32, and Inactivation, 55-37.

Mutations in the III-IV linker can affect inactivation 55-29-05: The conserved III-IV linker of the rat brain IIA Na+ channel

consists of 53 amino acids of which 15 are charged. Neutralization of the charged residues by mutational replacement resulted in channels which inactivated normally. Deletion of the first 10 amino acids of the ill-IV linker completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had little effect183 . Substitution of polar t glutamines for a conserved cluster of hydrophobic t amino acids (lle1488, Phe1489, Met1490) completely removed fast inactivation184 . Substitution of Met1490 alone slowed inactivation significantly, the I1488Q change alone both slowed inactivation and prevented its completion, and the F1489Q replacement alone almost entirely removed inactivation. It is proposed that the hydrophobic cluster of amino acids 1488-1490 acts as a 'hydrophobic latch', stabilizing the inactivated state in a 'hinged-lid' mechanism for inactivation of the Na+ channel184. The ill-IV linker region proved capable of rapidly inactivating a voltage-gated K+ channel (mouse Kv1.1) when attached to the N-terminus of a chimaeric channel protein185. High concentrations of external K+ accelerated recovery from inactivation in the chimaeric channels, suggesting that permeant K+ ions could displace the ill-IV linker 'inactivation particle'. The mutational change that eliminated fast inactivation in the rNaB2 channel also abolished it in the chimaeric channel185.

The pore region is located between the 85 and 86 segments 55-29-06: The Na+ channel blockers tetrodotoxin and saxitoxin are known to interact with the pore of the channeI186,187. Amino acid changes that make the

entry55

_

I - - - - - - -

channels less sensitive to both toxins lie in the S5-S6 regions of all four domains of rNaB2 53,188 and rNaB2A189. Two of the tetrodotoxin-resistant mutations in the 85-86 region of domain I of rNaB2, D384N and E387Q, reduce ion-permeability without affecting channel gating190. Changes in the 85-86 region can also affect the cadmium sensitivity of the Na+ channel. The C374Y replacement in 85-86 of rNaHI domain I increased sensitivity to tetrodotoxin and decreased sensitivity to Cd2+ 289, while the reciprocal change (Y401C) in rNa8k1 191 and a F385C change in the comparable region of rNaB2 192,193 decreased sensitivity to tetrodotoxin and increased sensitivity to Cd2+. Mutations in the 85-86 regions of rNaB2 domain III (KI422E) and domain IV (AI714E) affect the selectivity of the Na+ channe1193,194 (see Selectivity, 55-40-01). These observations combine to show that sequences in the 85-86 regions of the four domains comprise part of the permeation pathway for Na+ ions.

Cysteine substitution shows depth asymmetries of the pore-lining segments 55-29-07: Cysteine-substitutiont mutagenesis has been used to explore the positions of amino acid side-chains in the region of the Na+ channel pore195. Each domain of the channel a subunit contributes a loop ('P segment') to formation of the pore, but the contributions of the four domains are not symmetrical165 . The voltage-dependence of Cd2 + block of the cysteinesubstitution mutants, together with differential sensitivity to modification by sulphydryl-modifying reagents, shows that the P segment of domain II is external and that of domain IV is displaced internally with respect to the P segments of the first and third domains. Cysteine substitutions that show the steepest voltage dependence for Cd2+ block produce altered selectivity for monovalent cations. The substitutions E755C, K1237C, G1530C, W1531C and D1532C all give significantly increased permeability to K+ and NHt ions compared to the wild-type channel195 . The effects of the mutations in domain IV (1530-1532) suggests a unique role for this P segment in the selectivity of the channel. The findings from this cysteinesubstitution analysis have been used to modify an existing model (see North, 1994, under Related sources and reviews, 55-56) of the Na+ channel pore region195.

Binding site for scorpion a-toxin on the Na+ channel a subunit 55-29-08: A peptide neurotoxin, a-toxin, purified from the North African scorpion Leirus quinquestriatus, slows Na+ channel inactivation and greatly prolongs the action potential in nerve and muscle (see paragraph 55-37-03). The site of covalent attachment of photoreactive derivatives of a-toxin to the Na+ channel a subunit has been localized to the major extracellular loop in domain 1196 . A mutation to pyrethroid resistance in 1186 affects activation 55-29-09: A mutation giving rise to insect 'knock-down resistance' (kdr) to pyrethroids results in a Leu to Phe replacement in the IIS6 transmembrane region. The introduction of the corresponding mutation into the rat RBII a subunit shifts the steady-state activation curve of channels produced in

II

_'--

en_t_ry_s_S_

Xenopus oocytes by 14mV in the depolarizing direction. A similar change in the properties of the Na+ channel in kdr insects could account for the pyrethroid-resistant phenotype197.

Increased cell membrane surface area caused by {32 subunits 55-29-10: Expression of cRNA encoding the rat Na+ channel {32 subunit in Xenopus oocytes produced a concentration-dependent increase in whole-cell capacitance44 . Increases of up to four-fold in the capacitance were obtained with 170 ng/JlI (32 RNA, irrespective of the presence of RNA encoding Q and (31 subunits. Since cell capacitance is directly proportional to the surface area of the cell, these observations implied that (32 causes an increase in the surface area of the external plasma membrane of the oocyte. Electron microscopic examination of oocytes 2 days after injection of (32 cRNA showed I a striking increase in the nUlnber and size of microvilli in the plasma membrane and a noticeable reduction in the number of cortical granules' underlying the membrane44 . This trend is even more apparent after 4 days, when the number of pigment granules is also reduced and the vitelline membrane appears thinner. These morphological changes confirm the conclusion of the capacitance measurements and suggest that the effect of (32 in increasing the membrane surface area is achieved by increasing the fusion of intracellular lIlembrane vesicles with the plasma membrane44 .

Predicted protein topography Brain Na+ channels are heterotrimeric 55-30-01: Several models have been proposed for Na+ channel structure based

on amino acid primary sequences23,179,198-200. The Na+ channels from eel electroplax156,201 and chicken heart157 contain only an Q subunit, while those of rat skeletal muscle and heart include a (3 subunit202,203. The Na+ channel purified from mammalian brain is trimeric, with the pore-forming Q subunit (260 kDa), a non-covalently associated (31 subunit (36 kDa) and a disulphide-linked (32 subunit (33 kDa)200. Most models of the Q subunit predict a cytoplasmic localization of both the C- and N-termini together with linking regions between adjacent domains 5,114,169,182 (Fig. 2). The Na+ channel Q subunits consists of four internally homologous domains, each domain having an even number of membrane-crossing segments (see {PDTH}, Fig. 2, and VLC key facts, entry 41).

Protein interactions The {31 subunit affects the properties of the Na+ channel 55-31-01: Some voltage-gated Na+ channels contain only an Q subunit (see paragraph 55-30-01) and the heterologous t expression of sequences encoding Q subunits gives rise to functional channels. Purification of Na+ channels from rat brain has yielded one large Q subunit together with two smaller (3 subunits (see refs 52,164 and VLC key facts, entry 41). Large-subunit mRNA from rat brain Na+ channels II and III (and with less efficiency rat brain channel I) appears sufficient to form functional Na+ channels25,56,204. Inactivation is

l_e_n_t_ry_5_5

--'_

generally slower in these single-subunit channels; cO-Injection of low molecular weight mRNA encoding {3 subunits (see paragraph 55-22-01) restores normal inactivation25 . Co-expression t of sequences encoding the rNa{31 subunit with those specifying the rNaB2A Q subunit in Xenopus oocytes accelerated the macroscopic kinetics of inactivation of the channels, shifted V 1/ 2 of the steady-state inactivation in the negative direction and increased the amplitude of the whole-cell current obtained from a fixed quantity of a subunit cRNA by rv2.5-fold67. The same ,81 subunit also modifies the functional properties of the rNaB3 45, rNaSkI45~47~132, rNaSk2/rNaHl 205 and hNaSk1 206 Na+ channels, even though it is not associated with some of these Q subunits in vivo.

Protein phosphorylation PKA-dependent phosphorylation decreases Popen of the rat brain Na+ channels 55-32-01: The Q subunits of Na+ channels are substrates for phosphorylation by protein kinase A207-209 and protein kinase C161~162 (see also Sequence motifs, 55-24). The application of purified catalytic subunit of PKA (0.5 to 2 ~M) and ATP (1 mM) to the cytoplasmic surface of excised inside-out patches of rat brain primary neurones decreased Na+ channel activity by about 500/0, via a decrease in Pop en 210. Quantitatively similar results were obtained using macropatches t from Chinese hamster ovary (CHO) cells stably expressing the rat brain type ITA Na+ channel cDNA210.

Rat brain Na+ channels are substantially phosphorylated in the basal state 55-32-02: The phosphorylation of rat brain Na+ channels by the action of PKA in vitro 207~211 or in primary cultured rat neurones209 leads to the incorporation of 3-4 mol of phosphate per mol of Q subunit. Most of this phosphorylation occurs at the five potentially phosphorylatable sites in the intracellular loop between domains I and TI, where about 2.5 mol of phosphate per mol of Na+ channel subunit were located182~209. Antibodies prepared against the synthetic peptide corresponding to residues 676-692 have been used to show that the potential phosphorylation site at Ser686-Ser687 is phosphorylated by PKA in vitro and in situ 212 . Similarly, antibodies against peptide 561-575 completely inhibited in vitro phosphorylation of the purified Q subunit at Ser573211 . Reduction of basal phosphorylation by co-expression in stably transformend CHO cells of a dominant negative t mutant form of the regulatory subunit of PKA increased the number of surface Na+ channels by 54% and the peak Na+ currents in cell-attached patches by 3.36-fold210. The reduction in Na+ current by PKA-dependent phosphorylation in vitro was 'substantially restored' by treatment of the inverted patches with protein phosphatases 1 and 2A210. Note that the reduction in peak Na+ current obtained following activation of PKA in cardiac cells is due to a shift of the voltage dependence of inactivation to more negative potentials213~214. The consensus sites for cAMP-dependent phosphorylation are different between cardiac29 and brain23 Na+ channels, consistent with different molecular mechanisms being involved in PKA-dependent modulation of the two isoforms.

11 I

1

I

Repeat III

Repeat IV

I~ a-subunit

Repeat I

Repeat II

K1422E

N-glycosylation (N-gly*) sites '"

Regions binding scorpion a-toxins

S4 voltage sensor in~ repeat

<>--

Double mutant ~ighlr ~ permeable to calcIum Ions

A1714E

Region binding scorpion a-toxins ~

T704M (HYPP) COOH (aa2005)

A1156T (PMC)

Key

11488 F1489 M1490

Required for fast inactivation ("hydrophobic latch" domain)

~--·------_·_---

~

• Relative positions of motifs for phosphorylation by protein kinase A (PKA)

[9 . Relative positions of motifs for phosphorylation by protein kinase C (PKC) p. Pore-forming (P) domain by analogy to K+ channel monomers - see [PDTMJ under entry 49 Ext, Memb, Int • Extracellular, membrane, intracellular, regions ..-

---._--------

r

- - - - - - - - _ ...

NOTE: All relative positions of motifs, d~main shat?es and sizes are.dlagramm!Jtlc and a~e subject to re-Interpretatlon

-----------------------

N~!;

~~~:~I Illilrn~~

9 t"'t'

~ CJ1 CJ1

Figure 2. Protein domain topography model (PDTM) for a voltage-gated sodium channel. The model is based to scale on the rat brain type II Na+ channel, but the general structure is likely to be true of other Na+ channel isoforms. The four homologous domains (I to IV) in the a subunit each contains six membrane-spanning segments (Sl to S6). The pore-forming region is shown dipping into the lipid bilayer between S5 and S6. The small circles in these regions represent residues involved in tetrodotoxin-binding. Sites of demonstrated N-linked glycosylation (t), phosphorylation by PKA (P in a circle) and PKC (P in a diamond) and amino acids that form the inactivation particle (h in a circle) are shown. Black rectangles and ScTx show sites involved in binding scorpion toxins. Only the a and (31 subunits are shown: the topology of the (32 subunit is similar to that of (311 but (32 is linked to a by S-S bonds. The positions of mutations affecting channel function are shown. Note that many of these are due to naturally occurring mutations found in patients with genetic disorders affecting the skeletal muscle Na+

channel, hSkMl. (From 55-30-01.)

II

(t)

a

~

CJ'1 CJ'1

_'--

en_t_ry_5_5

-----J

Phosphorylation by PKC inhibits rat brain Na+ channel activity 55-32-03: Activation of PKC reduces peak Na+ currents induced by expression of embryonic rat brain RNA in Xenopus oocytes215 and in cultured rat brain neurones 161 . Currents obtained on heterologous t expression of sequences encoding rNaB2A Q subunit in Chinese hamster ovary cells are decreased up to 800/0 and their inactivation rate is slowed by activation of PKC. These effects, which are prevented by specific peptide inhibitors of PKC, can be mimicked by direct application of PKC to the cytoplasmic surface of insideout membrane patches 161 . cis-Fatty acids, which activate protein kinase C, attenuate Na+ currents in mouse neuroblastoma cells216 .

PKC phosphorylates rat brain Na+ channels in vitro 55-32-04: The in vitro action of PKC on purified Na+ channels from rat brain reconstituted into phospholipid vesicles lead to specific phosphorylation of the Q subunits217. The phosphorylation, which was dependent on the presence of Ca2 + (1.5mM), phosphatidylserine (60~M) and diolein (1.61~M), incorporated 2.5 mol of phosphate per mol of Q subunit. The phosphorylation was on serine residues only, and affected three distinct tryptic peptides. The phosphorylation of one of these peptides by PKC was prevented by prior phosphorylation with PKA, suggesting that the two kinases can phosphorylate the same serine in the Q subunit217. A single serine residue is crucial for PKC-dependent suppression of IIA Na+ channels 55-32-05: Phosphorylation by PKC of a single, conserved residue, Ser1506, located in the highly conserved intracellular III-IV loop of the rat brain ITA Na+ channel Q subunit, slows channel inactivation and reduces peak Na+ currents 162. The S1506A replacement in the Q subunit does not affect channel activity per se, but removes the ability of PKC activation to reduce peak currents and inactivation rates in stably transfected CHO celllines162.

ELECTROPHYSIOLOGY

Activation Measurement of gating charge for voltage-dependent activation

55-33-01: Measurements of gating-current fluctuation t shows that each Na+ channel gate moves 2.3 electronic charges across the membrane field during activation218, a value identical to that derived for K+ channels using singlechannel analysis219.

Atypical activation of Na+ channels in olfactory receptor neurones 55-33-02: For Na+ currents observed in acutely dissociated adult rat olfactory receptor neurones, activation was near - 70 mV and currents were fully activated by -lOmV (midpoint ~ -45mV). The current differs from those in amphibian and cultured neonatal rat olfactory neurones in its unusually negative voltage-dependence and slow recovery (~l second). Steady-state inactivation is complete at potentials more positive than -70 mV and

e_n_t_ry_55

1_ _

_

half-complete at -110 mY. Resting potentials in these cells are required to be more negative than -90mV in order for this current to underlie the action potential220 (see also Inactivation, 55-37).

Current-voltage relation 1-V relation for human cardiac channels 55-35-01: The Na+ channels produced by expression of the cRNAt encoding the human cardiac (hNaHl) Q subunit in Xenopus oocytes were activated at potentials positive to -60mV (holding potential -120mV) and showed maximum inward currents at -10mV32. A plot of the amplitude of singlechannel currents as a function of voltage showed inward rectificationt, with a slope conductance t of rv22pS in the range between -20 and +lOmY, where the 1-V relationship is approximately linear. This conductance is intermediate between those of rSkMl (32pS) and rSkM2 (lOpS) determined under identical conditions32 .

Neutral amino acid changes in S4 can affect voltage-sensitivity 55-35-02: The expression of cRNA encoding the rat IIA Na+ channel Q subunit in Xenopus oocytes gives rise to Na+ channels with a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction compared with that of channels expressed from rat brain poly(A)+ RNAt or rat IT cRNAt 25. In contrast, channels produced in oocytes from rat IT cRNA showed the same 1-V relationship as channels expressed from brain poly(A)+ RNA56 . The rat IT and ITA Q subunit amino acid sequences differ in only seven positions, at least one of which, L860F, is the result of an artefact produced in the cloning of RITA cDNA145. Changing each of the codons for the variant amino acids of rat ITA to a codon specifying the corresponding residue of rat IT showed that the artefactual F860L replacement in S4 of domain IT was entirely responsible for the shift in the 1-V relationship of the rat ITA channel145.

Activation of the peripheral nerve Na+ channel requires strong depolarization 55-35-03: Expression of cRNA encoding the rat peripheral nerve PN3 Na+ channel Q subunit in Xenopus oocytes generates a voltage-gated Na+ channel with a 'strikingly depolarized activation potential,66. These PN3 channels shows little activation at -lOmV and a peak inward current at +20mV. (Most Na+ channels produced by heterologous expression in oocytes begin to activate at potentials between -60 and -30mV).

Inactivation Inactivation is coupled to activation of Na+ channels 55-37-01: There are three functional states of the sodium channel, closed t , opent and inactivatedt. Inactivation is faster from the open state221 and has little or no intrinsic voltage dependence222 (reviewed in ref. 223) (see Domain functions, 55-29, for location of the inactivation gate). Inactivation is coupled to activation, which itself is steeply voltage dependent. An early model for the mechanism of inactivation, the 'ball-and-chain' model, proposed that an

_'--

e_n_try_5_5_

N-terminal'ball', tethered by a flexible peptide 'chain', acts as an inactivating particle by binding to a blocking site that is revealed on activation of the channel222. Support for this model has largely derived from studies of the inactivation of Shaker K+ channels224,225 (see VLC K Kvl-Shak, entry 48). For the voltage-gated Na+ channels, the evidence favours a segment of the intracellular Ill-IV loop as the inactivating 'particle', stabilizing the inactivated state of the channel by interaction with the pore region via a 'hinged lid' mechanism184 (see Domain functions (predicted), 55-29).

Inactivation is impaired by limited internal proteolysis 55-37-02: Limited cytoplasmic applications of pronase, trypsin or chemical

reagents such as N-bromoacetamide selectively prevent inactivation of Na+ channels: following such treatment Na+ channels are blocked by TEA in a manner similar to that of delayed rectifiers 170,226-228 (see VLC K DR, entry 45). Interruption of the intracellular loop between domains ill and IV of the Na+ channel a subunit gives rise to channels that are very slow to inactivate170 (see paragraph 55-29-02). Antibodies directed towards the region between domains ill and IV (see {PDTM} and Domain conservation, 55-28) reduce the rate of channel inactivation169.

Peptide a-toxins inhibit inactivation 55-37-03: A family of peptide toxins from scorpions and sea anemones, the

0:-

toxins, slow inactivation of Na+ channels by binding reversibly and competitively to an external receptor site with K o values in the range from 0.5 nM to 20 JlM. The binding site, 'neurotoxin receptor site 3', is distinct from that for saxitoxin and tetrodotoxin ('site I'), and a-toxin binding does not interfere with ionic flux. The effect on nerve and muscle is an exaggerated extension of the action potentials, from a few milliseconds to several seconds. The a-toxins bind preferentially to resting and open channels, making inactivation of the drug-associated channel energetically less favourable. The action of a-toxins on Na+ channels has been reviewed in ref. 229 and by Hille, 1992 (see Related sources and reviews, 55-56) (see also Channel modulation, 55-44-03).

Na+ channels in olfactory receptor neurones are inactivated at resting potentials 55-37-04: Somatic sodium channels of frog olfactory receptor neurones

(ORNs) are inactivated at rest230• Steady-state inactivation studies have shown that the mean voltage for half-inactivation (V1/ 2 ) is -82mV (range -72 to -98 mV), which indicates that the voltage-dependent Na+ channels in the cell body or soma of frog ORNs are not available for conducting currents at the resting membrane potential. Thus voltage-dependent Na+ channels may not play a significant role in sensory transduction at the soma, but are directed towards the efficient depolarization of the axons leading to the brain230 (see also Activation, 55-33.)

Paralysis due to impaired inactivation of muscle Na+ channels 55-37-05: Mutations in the SCN4A gene encoding the a subunit of skeletal

muscle Na+ channels are a cause of hyperkalaemic periodic paralysis

e_n_t_ry_5_5

1_ _

.......1_

(hyperPP), an autosomalt dominantt disorder involving recurrent attacks of paralysis (see Phenotypic expression, 55-14-03). The primary defect in Na+ channel function in hyperPP is an impairment of the rapid inactivation that is characteristic of the wild-type channel. The steady-state Popen for Na+ channels in depolarized muscle is normally about 0.001 231 : on depolarization, channels open after a brief delay and close within a millisecond to an inactive state221 . The hyperPP mutant channels containing the M1592V replacement in cultured myotubes show frequent repetitive openings and closings, resulting in a steady-state Popen of 1"V0.025232 . A persistent Na+ current results from intermittent failure of inactivation occurring in clusters of consecutive trials232 . Other hyperPP mutants have an abnormally persistent current, including the Phe to Leu substitution in rvS3 of the equine hyperPP, which shows an eight-fold increase in Popen with bursts of reopenings 233 .

Paramyotonia congenita mutations slow inactivation of muscle Na+ channels 55-37-06: The autosomal t dominantt muscle disease paramyotonia congenita (PMC) is caused by mutations in the gene encoding the Q; subunit of the adult human skeletal muscle Na+ channel. PMC mutant channels containing the amino acid replacements R1448H, R1448C, T1313M, A1156T and L1433R all inactivate more slowly and with less voltage-dependence than wild-type channels82,234. In some cases (e.g. V1589M, G1306E) the mutations are also associated with increased persistent Na+ currents235 .

Mutant SeNSA channels in long QT syndrome show incomplete inactivation 55-37-07: Mutations in the human SCN5A gene encoding the human heart voltage-gated Na+ channel Q; subunit hHI are responsible for the LQT3 version of the long QT syndrome, in which a prolonged QT interval on electrocardiograms results from abnormal cardiac repolarizationt . A deletion of 9 bp in the SCN5A gene results in the loss of three conserved amino acids, LysI505-ProI506-GlnI507, in the cytoplasmic linker between domains III and rv 71 . The mutant channels produced by expression of cRNA in Xenopus oocytes showed slightly decreased time constants for current decay, but the currents in cells expressing the mutant RNA were unusual in showing incomplete decay during a 200 ms depolarization. Single-channel recordings showed mutant channels fluctuating between normal and noninactivating gating modes. This persistence of inward Na+ current would explain the prolongation of action potentials found in long QT syndrome 72 (see also Phenotypic expression, 55-14-02, and Chromosomal location, 5518-01).

External K+ concentration can affect gating of mutant Na+ channels 55-37-08: The external K+ concentration has been shown to affect the gating of Na+ channel mutants implicated in hyperkalaemic periodic paralysis (hyperPP). For the two hyperPP-associated mutations - M1592V and the Phe to Leu substitution in rvS3 of the equine hyperPP (see Phenotypic

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_5_5_

expression, 55-14-04} - a threefold increase in the steady-state open probability of the channels was produced by the inclusion of 10mM K+ in the extracellular solution232,233. These observations are specific to muscle preparations, and the effect of external K+ could not be seen in heterologous expression systems84,234,235.

Na+ channels must deactivate to recover from inactivation 55-37-09: The Na+ channels of rat hippocampal neurones show a small but detectable current, equivalent to rvO.5% of the channels being open, during a recovery period at hyperpolarizing potentials after activation and inactivation at 0 mV. This current decayed with a time constant of rv4 ms, similar to the time course of recovery of channel activity. There is a voltage-dependent delay in the onset of recovery from inactivation, suggesting charge movements during the associated conformational t change of the channel protein. The rate of recovery increases with increasing hyperpolarization in the range -70 to -170mV, where it saturates with a rate constant of rv4ms- 1 . A model for the recovery process suggests that the same voltage sensors that are responsible for the voltage dependence of both activation and inactivation must move back to the 'off' position before removal of the inactivation particle. This coupling between deactivation and recovery from inactivation ensures that very little 'leak' Na+ currents pass during the recovery phase, and ensures a highly voltage-sensitive priming of the channels for the next impulse236 . Note that this behaviour of Na+ channels contrasts with that of Shaker K+ channels, which pass substantial current during the recovery phase due to unbinding of the inactivating particle before deactivation of the channe1237,238 (see Inactivation under VLC K Kv1-Shak, 48-37).

Cardiac Q subunits expressed in Xenopus oocytes mimic native cardiac channels 55-37-10: The Na+ channels produced by expression of cRNAt encoding the human cardiac Q subunit (hNaHl) in Xenopus oocytes shows voltagedependent inactivation that could be fitted with a Boltzmannt distribution with a midpoint of -61.9mV and a slope factort of 7.7mV32. The kinetics of inactivation during a voltage pulse exhibited a single exponential with a time constant (7h) that decreased e-fold per 53 mV with depolarization32. These kinetics closely resemble those observed in native cardiac tissue239,24o.

Rat brain Na+ channels expressed in Xenopus oocytes inactivate slowly 55-37-11: The rapid decay of Na+ currents during sustained depolarization of rat embryonic neurones in culture or acutely dissociated adult rat central neurones is mirrored by that of currents obtained by stable expression of the rNaBIIA coding sequence in transformed Chinese hamster ovary (CHO) cells. In these transformed cell preparations, the Na+ currents decayed with a 71/2 of 0.7ms at +10mV121 . Steady-state inactivation showed a steep voltage dependence, with a half-maximal steady-state inactivation at -50 mV. In contrast, the Na+ currents resulting from expression of the rIIA cRNA in Xenopus oocytes inactivated much more slowly, being incomplete after a 16 ms pulse121 . Co-expression of cRNA encoding rNa,81 subunits in Xenopus

1"---_e_n_t_ry_55

_

oocytes restores inactivation kinetics to resemble those of native neuronal channels67. Co-expression of cRNA encoding the a and {32 subunits also increased the proportion of rapidly inactivating channels, though f32 was not so influential as the f31 subunit44 . Channel inactivation was most rapid when the Q, f31 and f32 subunits were produced together in Xenopus oocytes, when the slow component in the time course was virtually eliminated44 . The rat peripheral nerve Na+ channel, PN3, shows slow inactivation after heterologous expression in Xenopus oocytes, but in this case the inactivation rate is not affected by co-expression of a Na+ channel f31 subunit, suggesting that PN3 may possess inherently slow inactivation kinetics 66 .

A small fraction of Na+ currents fail to inactivate 55-37-12: A small fraction (""1-3%) of Na+ currents in a variety of neurones, glial cells and other excitable cells fail to inactivate even after prolonged depolarization241 (reviewed in ref. 242). These 'sustained' or 'persistent Na+ currents' are usually (though not always243) blocked by tetrodotoxin (30 JlM) and by local anaesthetics. The sustained currents are regenerativet , due to membrane depolarization and consequent recruitment of additional channels, and explain the long-lasting plateau potentials that are particularly prominent when repolarizing K+ currents are blocked243,244. Single channel analysis of Na+ currents in acutely isolated rat neocortical neurones showed about 1 % of traces having prolonged bursts of channel openings lasting tens of milliseconds or more, sometimes appearing in runs lasting for several seconds241 . Sustained Na+ channel openings have also been seen in skeletal and cardiac muscle241 , in mammalian cells transfected with eDNA encoding rat brain type ITA Na+ channel Q subunits 161 and in Xenopus oocytes expressing cRNAt encoding rat brain type ill Q subunits245 . It is hypothesized that sustained Na+ currents are a consequence of occasional gating transitions of normal channels to a conformational state from which rapid inactivation is unlikelr41,245. Non-inactivating Na+ currents amplify small changes in membrane potential in photoreceptor cells and other neurones246, increase excitatory post-synaptic potentialst in hippocampal pyramidal cells247,248 and act as a major determinant of the rate of repetitive firing of action potentials248 . Sustained Na+ currents have been implicated in ischaemic damage in the CNS249 and cardiac myocytes250, and drugs that block Na+ channels, including the highly selective tetrodotoxin251 as well as the less selective local anaesthetics, are effective at preventing ischaemic damage in model systems.

Kinetic model The Hodgkin-Huxley model for Na+ conductance 55-38-01: The Hodgkin-Huxley model for sodium conductance assumed that there are two kinds of charged gating particles, three m particles controlling activation and a single h particle controlling inactivation. The probability that all four gating particles are in the permissive position is m 3h. The Hodgkin-Huxley equation for sodium conductance is gNa == gNa(max)m

3

h

III

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_5_5_

(This is analogous to the Hodgkin-Huxley expression gK = gK(max)A4 B governing the potassium conductance (gK) of rapidly inactivating A-type K+ channels - see VLCK A-'J: entry 44.) The Hodgkin-Huxley equations were developed from observations on macroscopic currents in the squid giant axon. More elaborate kinetic models have been developed that take account of parallel voltage-controlled transitions in the four 84 segments of the channel} a voltage-independent hydration step and two distinct open states252,253.

Computer simulations of cells with mutant Na+ channels predict myotonia 55-38-02: Mutations affecting the voltage-gated Na+ channel of skeletal

muscle give rise to a small} persistent Na+ current leading to myotonia or paralysis (see Phenotypic expression, 55-14-03 to 55-14-05). Computer simulation of a model muscle cell showed that when a small proportion of the Na+ conductance failed to inactivate myotonic discharges were generated254. The predicted behaviour of the muscle was normal if the fraction of non-inactivating channels was less than 0.8%. In the range 0.8-2%} myotonia was predicted} while inactivation of 3-4% of the channels produced an initial myotonia in which progressive K+ accumulation in the T tubules led to paralysis. Disruption of inactivation above the 4% level was predicted to cause paralysis without myotonia254 . These computer simulations are consistent with the phenotypic effects of known functional defects of mutant Na+ channels in myotonia and hyperkalaemic periodic paralysis254.

Selectivity Several monovalent cations permeate Na+ channels 55-40-01: The permeability ratios of the voltage-gated Na+ channels in squid

giant axons255 and myelinated sciatic nerve fibres of the frog (Rana pipiens)256 to monovalent cations are shown in Table 11. Data on the permeability of the Na+ channels of the frog myelenated sciatic nerve to organic cations} including the observation that methyl and methylene groups rendered the organic cations impermeant} lead to the conclusion that the channel has a selectivity filter of rv3 Ax 5 A in cross-section} lined by oxygen atoms that act as H-bondt acceptors during ion transport257• The transport of ions is blocked by titration with acid} with half-maximal block (= pKa ) at rvpH5.2} suggesting the involvement of carboxylic acidt residues in the selectivity process258 . These suggestions made in 1971 are consistent with more recent (1994) models of the Na+ channel pore} in which two acidic residues} Asp384 and Glu942} are brought into proximity in the narrowest region of the funnel-shaped pore259.

Mutations in 85-86 regions define the selectivity filter 55-40-02: The voltage-gated Na+ channels are highly selective for Na+ ions.

For example} the rNaB2 channel expressed in Xenopus oocytes has a permeability ratio PK../PNa of 0.03 194. The 85-86 regions of the Q subunits constitute the poreT regions of the channels (see Domain functions (predicted), 55-29-04). Mutations in the 85-86 regions of rNaB2 domain ill (K1422E) and domain IV (A1714E) affect the selectivity of the Na+

1

----J_

e_n_t_ry_55

Table 11. Permeability ratios of voltage-gated Na+ channels (From 55-40-01) Ion

Pion/PNa (squid giant axon)

Pion/PNa (frog sciatic nerve)

Na+ NHaOH+ (hydroxylamine) Li+ NH2NHj (hydrazine) TI+

1.0

1.0 0.94 0.93 0.59 0.33 0.16 0.14 0.13 0.12 0.086 0.06 <0.012 <0.013

1.1

NHt

NH2:CHNHi (formamidine) NH2:C(NH2)i (guanidine) NH2:C(NHOH)(NH2)+ (hydroxyguanidine) K+ NH2:C(NH2)(NH.NH2)+ (aminoguanidine) Rb+ Cs+

0.083 0.025 0.017

Data for squid giant axon taken from reference255 and for frog sciatic nerve from reference256 . channel194. These mutant channels have lost the high selectivity for Na+ over K+ characteristic of the wild-type channel, and are blocked by Ca2+ and other divalent cations at micromolar concentrations. The mutant channels containing the K1422E change are permeable to Ca2+ and Ba2+ at high concentrations (rv 100 mM), and the doubly mutant channel (K1422E, A1714E) is selective for Ca 2+ over Na+ at their physiological concentrations194. These findings suggest that the sites of the Na+ channel Q subunit defined by these mutations contribute to the selectivity filter t of the channel194. Molecular modelling of the pore region of the NaB2 and NaSkl Na+ channels predicts a structure resembling a funnel, terminating in a narrowed region that might serve as the selectivity filter of the channel. This region contains two carboxylt groups, from Asp384 and Glu942, that are hypothesized to displace molecules of water from the hydrated Na+ ion. (The presence of carboxylate ions in the selectivity filter was predicted from the observation that ion transport is blocked by protonation of one or more groups with an acidic dissociation constant (pKa ) of rvS.225B .) Modelling of mutations in this region that have produced Ca2+-permeable Na+ channels (see above) brought three carboxyls (Asp384, Glu942 and Glu1714) into close proximitr59. Note that the voltage-gated Ca2+ channels contain four conserved glutamate residues ('the EEEE locus') that are involved in Ca2+ ion binding and selectivity (see Selectivity under VLC Ca, 42-40-07).

The importance of the positively charged Lys in domain III in excluding Ca 2+ 55-40-03: Measurements of the ionic selectivity of mutant J.L1 (rSkM1) rat muscle Na+ channels confirmed that the Asp, Glu and Lys residues at the

_ entry 55 ~--------

selectivity filter (see paragraph 55-40-02) are crucial for discrimination of physiological cations and pointed to the importance of the positively charged Lys residue in domain ill in preventing permeation of Ca2+ ions26o. Mutations replacing the Lys by neutral or negatively charged amino acids resulted in Na+ channels that were permeable to Ca2+, whereas the Lys to Arg substitution that retains the positive charge did not affect Ca2 + permeability. The Lys to His substitution resulted in Ca2 + permeability at an external pH of 7.2, when the histidine side-chain would be uncharged, but impermeability at pH 6.0, when the side-chain would be partially protonated and positively charged. These findings suggest that the positive charge at the selectivity filter is the critical feature for prevention of Ca2+ permeation through the Na+ channel pore and have led to the proposal that the alkylammonium t ion of the Lys residue of domain ill might act as an endogenous cation within the selectivity filter of the Na+ channel, to 'tune the kinetics and affinity of inorganic cation binding within the pore', analogous to the ionic interactions that occur in multi-ion conduction26o .

Single-channel data Single-channel conductances of Na+ channels 55-41-01: The original direct measurements of single-channel Na+ conductances in cultured rat muscle cells261 and mouse neuroblastoma cells262 gave values of 12-18 pS. These conductances are higher than those obtained by indirect measurements of the unit conductance, because Na+ channels open only briefly on depolarization and macroscopic measurements of Na+ currents systematically underestimate peak single-channel contributions. Single-channel recordings with excised patches from reconstituted vesicles containing purified eel electroplax Na+ channels showed a single-channel conductance of 11 pS263, while reconstituted Na+ channels from rat brain showed single-channel conductances of 25 pS264. The open probability of the reconstituted rat brain channels was voltage dependent, Popen being 0.5 at -91 m~ and the apparent gating charge 'was 3.8 my264.

Two native Na+ channel types in skeletal muscle 55-41-02: Single-channel recording with outside-out patches from rat muscle myoblasts and myotubes revealed two classes of Na+ channels in both cell types. Single-channel currents elicited by depolarizations to -40 mY were either 1.4 or f'.Jl.0pA and were all sensitive to 15 JlM tetrodotoxin (TTx). TTx concentrations in the range 5-500nM selectively eliminated the 1.4pA currents. The slope conductances were f'.J 12 pS for the TTx-sensitive channels and f'.J9.8 pS for the TTx-resistant class. The proportion of TTxsensitive channels was higher in myotubes than in the myoblasts, consistent with the idea that these channels supplant the TTx-resistant class during development265 .

Characteristics of channels encoded by SkMl and SkM2 cRNAs 55-41-03: Single-channel recordings with outside-out patches from Xenopus oocytes expressing cRNAs encoding the rat skeletal muscle channel SkMl and SkM2 Q subunits show a linear 1-V relationship in the range -60 to

11II

l_e_n_t_ry_s_S

_

(b)

(a) V = -60 rnV

~

A

~~

!

~~~U\f-~

.J V

5 ms

-60

(d)

V = - 10 mV

,

-·10 mV

~~1~~

"''''V~·~

(c)

v=

,

-40

-20

o

Voltage (mV) -0.5

A

~J~ o

-3.0

Figure 3. Single-channel currents through the human cardiac Na+ channel, hNaH1. Data were obtained from an outside-out patch of a Xenopus oocyte expressing hNaH1 cRNA. The traces show openings elicited at -60 (a), -40 (b) and -10 (c) mV from a holding potential of -120mV. The start and end of pulses are indicated by the downward and upward arrows. Panel (d) shows the amplitudes of the single-channel currents for the same patch, superimposed on the single-channel 1- V relationship from oocytes injected with cRNA for rSkM1 and rSkM2. (Reproduced with permission from Gellens (1992), Proc Natl Acad Sci USA 89: 554-558.) (From 55-41-03)

+IOmV, with single-channel conductances of 32pS and lOpS respectively32. Under the same conditions the channel encoded by the human cardiac (hHl) cRNA showed inward rectification t , with a slope conductance t of rv22pS in the range -20 to +IOmY, where the I-V relationship is relatively linea~2 (Fig. 3).

Voltage sensitivity Voltage sensitivity depends on the structure of 84 55-42-01: The voltage dependence of activation of different voltage-gated Na+ channels is variable, depending on the structure of the relevant a subunit and particularly of the voltage-sensing 84 domains. Single amino acid changes in

_ entry 55

- - - - - - - - one S4 segment can shift the voltage-dependence of activation by as much as 30mV170 (see Domain functions (predicted), 55-29-01). The translocation of charge obtained from the voltage-dependent movement of S4 segments is sufficient to account for the gating charge172 (see VLG key facts, entry 41, Voltage-sensitivity under VLG K Shak, 48-42). Most Na+ channels produced by heterologous t expression in Xenopus oocytes begin to activate at potentials between -60 and -30m~ but the rat peripheral nerve PN3 Na+ channel 0 subunit under these conditions shows little activation at -10mV and a peak inward current at +20mV66 (see Current-voltage relation, 55-3503). Note that inactivation has little or no intrinsic voltage dependence222 (reviewed in ref. 223), but is intrinsically coupled to activation, which is steeply voltage dependent.

PHARMACOLOGY

Blockers Guanidinium-containing toxins are selective blockers of Na+ channels 55-43-01: The guanidinium-containing compounds tetrodotoxin (isolated from Fugu ('puffer fish') organs, often abbreviated to TTx), and saxitoxin (STx), act as selective, reversible blockerst of Na+ channels at nanomolar external concentrations. These toxins bind strongly (Kd = 1-5 nM) and compete with each other for the same receptor site. They have long residency times (TTx, 70 Sj STx, 37 s at 20°C) and give rise to periods of block of tens of seconds in single-channel recordings266 . The toxins are membrane impermeant and act only from the extracellular side of the membrane. The rate of block by TTx and STx at the node of Ranvier is not affected by stimulation of the axon, showing that channel opening is not essential for drug-binding (see refs 267,268 for reviews of the action of TTx and STx on voltage-sensitive Na+ channels). Channels blocked by TTx or STx continue gating, without conducting Na+ ions, since their gating currentst are unaffected by the presence. of the drugs. A molecular model of the binding pocket for TTx and STx, involving antiparallel {3 hairpinst formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel, has been developed259. The cardiac isoform of the Na+ channel is relatively insensitive to TTx: channels produced by expression of the human cardiac cRNA in Xenopus oocytes had an IC so value of 5.7 JlM for TTX32. An isoform expressed in sensory neurones of rat dorsal root ganglia is also insensitive to TTx. This isoform contains the 01 subunit encoded by the rat Scn10a gene 131 and expression of the Scnl0a cRNA in Xenopus oocytes gives rise to a Na+ channel with an ICso for TTx of 59.6 JlM 12 .

Local anaesthetics block the Na+ channel of nerve axons 55-43-02.: Local anaesthetics, such as lidocaine and procaine, are generally

lipid-soluble tertiaryt amine compounds that inhibit propagated action potentials by blocking Na+ channels. The quaternaryt amine analogue of

l_e_n_t_ry_55

_

lidocaine, QX-314, which is positively charged and not lipid soluble, blocks Na+ channels only when applied to the inside of the membrane following opening of the channels by depolarization269 . The effects of local anaesthetics in generating use-dependent block t and altering the gating kinetics of Na+ channels have been discussed by Hille, 1992, pp. 403-411 (see Related sources and reviews, 55-56).

The skeletal muscle Na+ channel is sensitive to J-l-conotoxin 55-43-03: The 22 amino acid peptide p-conotoxin (GIIIB), isolated from the fish-hunting cone snail, Conus geographus, is a selective inhibitor of Na+ currents in skeletal muscle270. The reversible blocking action of Jl-conotoxin on single Na+ channels from skeletal muscle is voltage dependent, with a KD of 100nM at OmV and 22°C 271 . The human cardiac Na+ channel expressed in Xenopus oocytes is insensitive to 100nM Jl-conotoxin32 (see also Blockers under ILG CAT cGMp, 22-43).

Polycyclic cations block Na+ channels by binding to the open pore

55-43-03: A structurally diverse group of polycyclic t cations, including Nmethylstrychnine, pancuronium and thiazin dyes, block Na+ channels through binding within the open pore. Once the drugs have bound, the channel is frozen in the open state, being able to close neither by inactivation nor by deactivation until the drug leaves the pore272 (discussed in Hille, 1992, p. 409). (Note that these compounds are experimentally useful for the study of Na+ channels, but are not of known clinical value.)

Na+ channel blockers as neuroprotective and antiarrhythmic agents 55-43-04: Lubeluzole, a benzothiazole derivative, has been successfully used in phase II trials as a neuroprotective agent in ischaemic stroke patients. The alkaloid N-desacetyllappaconitine is a selective blocker of the TTxinsensitive cardiac Na+ channel and has potent antiarrhythmic, antifibrillatory, anti-inflammatory and local anaesthetic actions. Other blockers include sevdinedione (steroid alkaloid antiarrhythmic); diacetylkorseveriline (alkaloid antiarrhythmic); nitrarine dihydrochloride (alkaloid with coronary dilator, sedative, hypotensive and spasmolytic effects); heteratisine (antiarrhythmic, antifibrillatory alkaloid); hetisine hydrochloride (antiarrhythmic, local anaesthetic); lappaconitine hydrobromide (also known as allapinine; potent antiarrhythmic).

A non-specific blocker of Na+ channels 55-43-05: The morphinan dextromethorphan blocks NMDA-induced currents and voltage-operated inward Na+ and Ca2+ currents in cultured cortical neurones and PC12 cells273 (see Blockers under VLG Ca, 42-43, and under ELG CAT GLU NMDA, 08-43).

Sodium channel blockers decreasing endogenous glutamate release 55-43-06: Several sodium channel blockers can act to decrease pre-synaptic glutamate release. These compounds possess anticonvulsive properties and include riluzole (see Receptor agonists under ELG CAT GLU NMDA,

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_5_5_

08-50), phenytoin, lamotrigene and lafarizine. Characteristics of riluzole and other antagonists of glutamate release have have been reviewed274 . Felbamate appears to be a competitive antagonist at the binding site of NMDA receptors (see Receptor antagonists under ELG CAT GLU NMDA, 08-51), but may also act on sodium channels275 . (Note that lifarizine, a diphenylpiperazine analogue, was originally introduced as a Ca2 + channel modulato~76. Subsequent studies have shown displacement of batrachotoxinin (BTx-B), a specific ligand for Na+ channel neurotoxin-binding site 2, from rat cortical membranes (IC so = 55 nM)277 and voltage-dependent inhibition of Na+ currents in mouse and human neuroblastoma cells278,279.)

Na+ channel blockage by anticonvulsants protects against anoxia 55-43-07: Several anticonvulsant agents, including phenytoin, carbamazepine and lamotrigine, block Na+ channels at therapeutic concentrations. These compounds act via use-dependent t inhibition of Na+ conductance by stabilizing the inactivation state (reviewed in ref. 280). Phenytoin (r-v20mM) has been shown to provide protection against the effects of anoxia in the rat optic nerve281 , rat hippocampal slices282, gerbil hippocampal CAl neurones283 and several models of focal cerebral ischaemia (reviewed in ref. 277). Lamotrigine and its derivatives BWI003C87 and BW19C89 are neuroprotective during ischaemic and traumatic injury in animal models (reviewed by Obrenovitch, 1997, see Related sources and reviews, 55-56).

Block by binding of a phenylacetamide to the local anaesthetic-binding site 55-43-08: PD85639, a phenylacetamide structurally related to phenytoin and local anaesthetics, binds to the local anaesthetic receptor of voltage-gated Na+ channels (Ki 0.26 mM)284,285. Whole-cell voltage-clamp recordings from Chinese hamster ovary cells transfected with a eDNA encoding the rat brain type lIA Na+ channel and from dissociated rat brain neurones showed that PD85639, applied either in the external bath or in the internal pipette solution, decreased Na+ currents. Block included both a component that occurred in the absence of stimulus pulses (ECso 30 JlM) and an additional use-dependent component first detectable at I JlM286 .

Clinical use of Na+ channel blockers as neuroprotective agents 55-43-09: Blockade of voltage-gated Na+ channel function is of potential clinical use to augment neuroprotection during ischaemic or traumatic injury. Inhibition of Na+ conductance minimizes residual energy demand during ischaemia by avoiding the energetically expensive maintenance of Na+ and K+ gradients and delays anoxic depolarization, with benefits for Ca2+ homeostasis, acid-base regulation, glutamate uptake and cell volume regulation. A number of drugs interacting with voltage-gated Na+ channels are being used in clinical trials for neuroprotection during ischaemia287. Lubeluzole, a benzothiazole derivative, has been successfully used in phase IT trials as a neuroprotective agent in ischaemic stroke patients288 . Blockers of voltage-gated Na+ channels can have cardiac effects, including prolongation of the QTc interval of the ECG and the risk of initiating arrhythmia, which may limit dosing or limit their clinical use287.

PIlI

l_e_n_t_ry_5_5

_

Cd2+ and Zn 2+ block the cardiac Na+ channel 55-43-10: Mammalian cardiac Na+ channels are particularly sensitive to block by Cd2 + and Zn2+ (Kd 100 JlM at 0 mV for both ions }191. The block is more pronounced at increasingly negative potentials and for both divalent cations the fractional electrical distance t for blockage is ",,22% 191. Single-channel recordings from guinea-pig ventricular myocytes show discrete blocking events as 'complete occlusions' of Na+ flUX 191 . The rat skeletal muscle JlI Na+ channel can also be blocked by extracellular Cd2+, but is two orders of magnitude less sensitive than the cardiac isoform. A difference in the SS2 region of domain I, Tyr401 in JlI versus Cys in the heart isoform, completely accounts for the difference in Cd2+_sensitivity191,289. The Y401C mutation in JlI channels also alters the affinity for tetrodotoxin (lTx), with the IC so changing from 16 OM for the wild-type to >50 JlM for the mutant channel191 . The reverse mutation (Cys to Tyr) in the rat heart channel decreases sensitivity to Cd2+ and increases that to lTr89. A Na+ current with kinetic and pharmacological properties similar to those of the cardiac Na+ current, including resistance to lTx (IC so 146nM) and sensitivity to Zn2+ (ICso 9.1 JlM), has been characterized290 in neurones derived from the superficial layers of the rat entorhinal cortex.

Ca 2+ is a weak extracellular bocker of Na+ channels 55-43-11: Not only are Ca2+ and other divalent cations practically impermeant through vertebrate Na+ channels, but they are also relatively weak extracellular blockers of Na+ currents through these channels. The KD of extracellular Ca2+ for blockage of Na+ currents at OmV has been estimated as ",,35 mM, with a voltage dependence equivalent to ",,20-30% of the transmembrane field291,292.

Na+ channel blockers induce expression of the rScn2a gene in cardiac muscle 55-43-12: The class I antiarrhythmic drug mexiletine, a blocker of voltagegated Na+ channels, increased rat cardiac Na+ channel number during chronic in vivo treatment293 . Chronic treatment with mexiletine (5075 mg/kg per day, subcutaneously) produced a threefold increase in the mRNA encoding the Na+ channel Q subunit in the heart, without affecting the mRNA encoding the brain Na+ channel subunits. (Similar observations have been made using the Na+ channel blocker bupivacaine to induce expression of the SkMl Q gene in skeletal muscle294,295.) The class IV antiarrhythmic verapamil, a blocker of voltage-activated Ca2+ channels, similarly produced increases of the Na+ channel Q subunit mRNA in heart without increase in brain mRNA296 . The combination of mexiletine and verapamil produced no further increase in the level of the cardiac channel mRNA. In cultured neonatal cardiac myocytes, chronic elevation of cytosolic Ca2 + by treatment with the Ca2 + ionophore, A23187, produced a fivefold decrease in Na+ channel Q subunit mRNA296 . These data are consistent with the suggestion that changes in cytosolic Ca2+ concentration regulate the expression of Na+ channel genes in heart and skeletal muscle296 . (Note that essentially similar observations have been made on

expression of the nicotinic acetylcholine receptor (nAChR). Verapamil and

-

entry 55

Table 12. Toxin-binding sites on voltage-activated Na+ channels (From 55-44-01)

Binding site

Toxin

Chemical type

Effect

1

Tetrodotoxin (TTx) Saxitoxin (STx) JL-Conotoxins

Heterocyclic, guanidinium

Ion channel block, via binding site at the extracellular opening of the pore of the channeI267,271, 298,299. Result in blockade of nerve and muscle action potentials.

Veratridine Batrachotoxin Aconitine Grayanotoxins (GTx)

Alkaloid

3

Scorpion a-toxins Sea anemone toxins

Peptides, 64 aa Peptides, 46 aa (I), 47 aa (II) and 27 aa (III)

Voltage-dependent binding causes slowing of inactivation and stabilization of the channel in the open state229,267. Binding at site 3 enhances the effects of activator toxins binding to site 2 267,302. The macroscopic effects include hyperexcitability and repetitive firing in motor units, accelerated respiration, convulsions, spastic paralysis and eventual respiratory failure.

4

Scorpion ,B-toxins

Peptides, 66 aa

Shift voltage-dependent activation in positive direction: toxin plus conditioning pre-pulse shift activation in negative direction303-30s. Hyperexcitability results, with heavy perspiration and tremor.

5

Brevetoxins Ciguatoxins

Cyclic polyethers

Inhibit inactivation and shift voltage-dependent activation to more negative potentials.

2

III

Small peptides ('" 17-22 aa)

Diterpenoid

Slow inactivation, shift activation to more negative potentials and reduce selectivity267,300,301 (note: veratridine also blocks some K+ channels). Result in hyperexcitability due to long-lasting membrane depolarization.

IL_e_n_t_ry_55

_

Table 12. Continued Binding site

Toxin

Chemical type

Effect

5

Brevetoxins Ciguatoxins

Cyclic polyethers

Binding at site 5, detected using a radiolabelled brevetoxin derivative306, enhances the binding of site 2 and site 4 ligands307,30B. Ciguatoxins inhibit brevetoxin binding to site 5309 . The macroscopic consequence is repetitive firing in neurones310,311.

6

Pyrethrins Pyrethroids

Organic esters

Slow activation and inactivation, prolong channel open-time and promote membrane depolarization, leading to lethal paralysis in insects312.

Data based on Trends in Neurosciences, Neurotoxins Supplement, 1996.

the Na+ channel blocker tetrodotoxin increased and A23187 decreased the number of nicotinic acetylcholine receptors, with corresponding effects on mRNA levels, in skeletal muscle cells297.)

Channel modulation Six distinguishable toxin-binding sites on voltage-activated Na+ channels 55-44-01: The voltage-gated Na+ channels are subject to modulation by a wide range of toxins produced by animals for use in predation or defence, or by plants as protection against herbivores. At least six different toxin-binding sites have been identified on sodium channels, as summarized in Table 12. The groups of toxins have different effects on channel function, from total block to prolongation of activation. The interactions of toxins with voltageactivated Na+ channels are comprehensively covered in the 1996 Supplement to Trends in Neurosciences. The chemical structures of various toxins acting as Na+ channel modulators are shown in Fig. 4.

Scorpion peptide a-toxins inhibit Na+ channel inactivation and prolong the action potential 55-44-02: The family of scorpion peptide a-toxins prolong the action potential duration in muscle313 and nerve314. The scorpion a-toxin neurotoxin IV (65 aa peptide isolated from Leiurus quinquestriatus) modulates Na+ channels in excitable membranes by inhibiting inactivation and eliciting a non-inactivating conducting state17,315 (see Inactivation, 55-37-04).

II

_ entry 55

""--------------'

GUANIDINIUM TOXINS OH

STX

TIX

BREVITOXIN A

ALKALOIDS

Batrachotoxin

CH~ 3 ".

CH CH 3

2

HO"

Verotrldine

OH ••• (OCH! ._., -OCOC H

··OH

.' OCOCH a ~ 20CH! OCH! Aconitine

•

5

H

9H

HO~ OH

~

OH

Gra,anotoxin I

Figure 4. Examples of modulators of voltage-gated Na+ channels. Tetrodotoxin (TTx) and saxitoxin (STx) are paralytic natural products that act as specific blockers of Na+ channels. Aconitine, batrachotoxin, grayanotoxin I and veratridine are alkaloids that cause Na+ channels to remain open. Brevetoxin A is a member of a family of related toxins that inhibit inactivation of Na+ channels. The pyretmoids, synthetic analogues of pyrethrin, bind to open channels and delay inactivation. The local anaesthetics stop propagated action potentials by blocking Na+ channels. Note that benzocaine is permanently uncharged, lidocaine and procaine are ionizable tertiary amines, but quaternary ammonium derivatives like QX-314 are membrane-impermeant cations. (From 55-44-01)

Scorpion {3-toxins modulate Na+ channel activation 55-44-02: Purified peptide 'scorpion ,a-toxins' from the scorpions Centruroides sculpturatus and C. suffus suffus modulate the activation of skeletal muscle Na+ channels at nanomolar concentrations304,305. During a test depolarization

l_e_n_try_5_5

_

LOCAL ANAESTHETICS

HzN

-0-

00- 0 -

C,Hs

Benzocaine

PYRETHROIDS TYPE II

TYPE I

o eN

~o~o'O CI Allethrin

~O-N~

Fenvalerate

o

Tetramethrin

Deltamethrin

Figure 4. Continued

from rest, f3-toxin slowed both activation and inactivation, increased the depolarization needed for activation and lowered the maximum macroscopic permeability316. A conditioning pulse before the test depolarization reversed the effect of the toxin on activation, making it faster, achievable at more negative potentials and increasing the maximum permeability. The inactivation was further slowed by f3-toxin after the conditioning pulse. The overall effect of the f3-toxin in depolarizationconditioned nerves is to produce conducting Na+ channels that close very slowly at rest and result in inward Na+ currents capable of supporting repetitive firing in response to minimal stimulation316 . The scorpion a- and f3-toxins do not bind to the same site on Na+ channels, since they can bind simultaneously to the same channeI317,318.

_'--

en_t_ry_5_5_

Sea anemone toxins act similarly to scorpion a-toxins on Na+ channels 55-44-03: A family of homologous peptide toxins isolated from sea anemones have electrophysiological effects on macroscopic Na+ currents in nerve fibres similar to those of scorpion a-toxins (see paragraphs 55-44-02 and 55-37-03), despite having no sequence homology with scorpion toxins. Chemical modification of the Anemonia sulcata toxin, ATxll, showed that the carboxylatet side-chains of Asp7,9 and the C-terminal carboxyl group (Gln47) are essential for toxicity and electrophysiological activity, though the modified derivatives remained able to bind to the Na+ channel319. Chemical modification of the c-amino groupst and the a-amino groupt (Gly1) also eliminated toxicity and reduced the affinity for the target Na+ channel319.

Voltage-activated Na+ channels are primary targets for insecticides 55-44-04: The insecticides DDT and the pyrethroids, which cause repetitive firing in the invertebrate nervous system, inhibit inactivation of voltageactivated Na+ channels and thereby allow prolonged Na+ currents. Singlechannel recording shows that channels that normally close within about 5 ms of activation by depolarization remain open for up to several seconds in the presence of pyrethroids. The onset of channel opening following a depolarizing pulse can also be delayed by the insecticide32o,321. Pyrethroids, particularly the 'type II' compounds which contain a cyanot group at the a position, also cause membrane depolarization due to the prolonged opening of Na+ channels. This effect, resulting in massive release of neurotransmitter at sensory nerve terminals, is the basis of paraesthesia t caused by type II pyrethroids322. Increasing the depolarizing afterpotential t to the threshold for initiation of repetitive discharges requires only rvO.1 % or less of the Na+ channel population to be modified, which is achieved at nanomolar concentrations of pyrethroids323 .

Non-specific modulation of Na+ channel activities by fatty acid metabolites 55-44-05: In addition to intracellular ligand-gating activities of fatty acid metabolites (e.g. see JLG Ca AA-LTC4 , entry 15), modulation of ion channels through alterations of membrane composition have also been suggested for the squid giant axon Na+ channel324 and aortic smooth muscle K+ channels325. Note: In general, lipophilict compounds may have detergent effects when at or near the critical micellar concentration, where aggregates of monomers disrupt membranes and create lipidic pores326 .

Equilibrium dissociation constant K D values for binding of alkaloid Na+ channel openers 55-45-01: The frog-derived alkaloid batrachotoxin (BTx) is a neurotoxin that opens Na+ channels in nerve and muscle and leads to failure to inactivate. The binding of a radiolabelled derivative of BTx to rat brain synaptosomes is antagonized by the alkaloids BTx (Kn 0.05 JlM), veratridine (Kn 7.0 JlM) and aconitine (Kn 1.2 JlM)327.

l_e_n_try_5_5

_

Tetrodotoxin has high affinity for brain and skeletal muscle Na+ channels 55-45-02: Tetrodotoxin (TTx), a powerful neurotoxin found in the internal organs of the Fugu puffer fish, acts as a specific blocker of voltage-gated Na+ channels. The toxin inhibits Na+ currents produced by the expression of the rat brain rIIA Q subunit coding sequence in stably transformed Chinese hamster ovary cells with a KD of ",,25 nM121, in reasonable agreement with values obtained from biochemical studies of rat brain Na+ channels328. In general, brain and skeletal muscle Na+ channels are blocked by nanomolar tetrodotoxin, but the 'TTx-resistant' cardiac channels require micromolar concentrations for effective blockage. The rat peripheral nerve channel PN3 is highly resistant to TTx (IC so >100 JlM 66 ).

Saxitoxin has high affinity for neuronal and skeletal muscle Na+ channels

55-45-03: Saxitoxin (STx), a positively charged heterocyclic t toxin from marine dynoflagellates, blocks neuronal and skeletal muscle Na+ channels at nanomolar concentrations, but is less effective on cardiac channels. The KD values for saxitoxin binding to Na+ channels from various sources have been estimated from single-channel kinetics to be as follows: dog heart, 32 nM329; calf heart, 29 nM329; rat brain, 0.8 nM266; rat skeletal muscle, I.4nM266 . The binding of labelled STx to rabbit vagus nerve preparations gave a K D of I.8nM at 3°C299.

Cardiac Na+ channels are sensitive to external Zn 2+ 55-45-04: External Zn2+ blocks cardiac Na+ channels by binding with a dissociation constant estimated as 18-24JlM (dog, -70mV) and 24-29JlM (calf, -50mV)329. Other Na+ channel subtypes are approximately IOO-fold less sensitive to blockage by Zn2+ (The binding of external Zn2 + is competitive with that of saxitoxin (STx), a paralytic guanidinium-based heterocyclic toxin from marine dynoflagellates that binds to 'binding site I' on voltage-gated Na+ channels329 - see paragraph 55-45-03.)

Hill coefficient (n) Zn 2+ binding to cardiac Na+ channels involves a single-site interaction 55-46-01: Mammalian cardiac Na+ channels are approximately IOO-fold more sensitive than other Na+ channel subtypes to block by external Zn2+. Hill plots of Zn2+ titrations yield Hill coefficients of 1.17 for dog and 1.09 for calf cardiac Na+ channels, suggesting that the binding of Zn2+ to block the cardiac Na+ channel involves a single-site interaction329.

Ligands Radiolabelled toxins as ligands 55-47-01: Available radioligands for voltage-gated Na+ channels include [3]H-batrachotoxin, [3H]-saxitoxin, [3H]-tetrodotoxin.

_ L....----

entry 55 _

Openers Alkaloid neurotoxins open Na+ channels at resting potentials 55-48-01: A group of lipid-soluble alkaloid neurotoxins, including batrachotoxin (BTx), aconitine, veratridine and grayanotoxin I, depolarize resting nerve and muscle cells and increase the Na+ permeability of excitable cells by opening Na+ channels (reviewed in refs 267,301,330). Batrachotoxin, isolated from the skin of the Columbian frog Phyllobates aurotaenia, binds rapidly to open channels and produces modified behaviour including activation at potentials 40-50 mV more negative than unmodified channels, failure to inactivate, reduced ion selectivity310 and decreased sensitivity to local anaesthetics331 . Veratridine, isolated from the lily genus Veratrum, binds to activated Na+ channels to prevent inactivation and shift activation to more negative potentials, with the consequence that the modified channels remain open at the former resting potentiaI332,333. The voltagedependence of the opening of channels obtained by expression of rat brain rIIA eDNA in Chinese hamster ovary cells was displaced by rv -lOOmV by treatment with 100 JlM veratridine121 . The plant steroid alkaloid veratroylzygadenine (an analogue of veratridine) is a potent Na+ channel opener, neurotoxin and activator of nerve and gland cells.

Aconitine as Na+ channel opener and arrhythmiagenic agent 55-48-02: The plant alkaloid aconitine, purified from Aconitum napellus (monk's hood), is a potent neurotoxin and generates cardiac arrhythmias by acting as an opener of the TTx-sensitive Na+ channel. Aconitine-modified Na+ channels in nerve and skeletal muscle cells show shifts in the voltage dependence of activation of rv _SOmV334,335, though those in neuroblastoma cells are shifted by only rv -20mV336 .

Grayanotoxin affects selectivity, inactivation and tetrodotoxin sensitivity of Na+ channels 55-48-03: Grayanotoxins (GTx), a series of lipid-soluble toxins isolated from rhododendrons and other ericaceous plants, produce persistent activation of Na+ channels via interaction at binding site 2 (see Channel modulation, 5544-01). Sodium channels modified by GTx are less Na+ selective than unmodified channels, fail to inactivate337-339 and are less sensitive to block by terodotoxin339.

Pyrethrins and other insecticides cause repetitive firing in nerve axons 55-48-04: The pyrethrins, natural insecticides isolated from the flowers of the genus Chrysanthemum, and their synthetic analogues, the pyrethroids, prolong Na+ channel activation and inhibit inactivation in nerve axons, causing repetitive firing and depolarizing after-potentials312. The synthetic insecticide DDT (dichlorodiphenyltrichloroethane) acts in a similar fashion (see Channel modulation, 55-44-04).

Brevetoxins behave like alkaloids in modifying Na+ channels

55-48-05: The brevetoxins (BvTx) are a series of lipophilic t neurotoxins containing fused cyclic ethers t produced by the marine dinoflagellate

l_e_n_t_ry_55

_

Ptychodiscus brevis, the organism responsible for 'red tides'. Like the alkaloids (see paragraph 55-48-01), the brevetoxins shift the voltage dependence of the axon Na+ channel by rv - 35 mV, inhibit inactivation and modify ion-selectivity34o. The depolarization of crayfish giant axons by BvTx-E is inhibited by the local anaesthetic, procaine340.

Receptor/transducer interactions Muscarinic receptor agonists suppress Na+ currents in rat hippocampal neurones 55-49-01: Activation of muscarinic receptors in acutely isolated pyramidal neurones from the rat hippocampus by carbachol (20 JlM) reduces peak Na+ current and slows macroscopic inactivation341 . These effects, which are prevented by the muscarinic receptor antagonist, atropine (10 JlM), require the activation of PKC, being eliminated by the specific inhibitor PKCII9-36 (2 JlM) and mimicked by the membrane-permeant PKC activator l-oleoyl-2acetyl-sn-glycerol (OAG, 20 JlM). Hippocampal pyramidal neurones express the ml receptor subtype predominantly, with lower densities of the m2, m3, m4 and ms subtypes342. The subtype of the receptor responsible for the modulation of the Na+ channel via PKC activation has not yet been determined.

Modulation of cardiac Na+ channels by cAMP receptors 55-49-02: In addition to the PKA-dependent suppression of cardiac sodium channel activity by elevation of intracellular cAMPt (see paragraph 55-3201), extracellular cAMP can also modulate the Na+ channels in myocytes. Extracellular cAMP (100JlM) decreased the Na+ current by about 550/0 in rat, guinea pig and frog ventricular or atrial myocytes343 . Suppression was rapid « 50ms), reversible and dose dependent and occurred in the presence of a PKA inhibitor (H-7, 10 J,1M) in the internal dialysate. The decrease in the sodium current was inhibited by guanosine-5 ' -O-(2-thiodiphosphate) (GDP,BS, 1 mM) or pertussis toxin (0.5 J,1g/ml), suggesting that the suppression of Na+ channel activity by extracellular cAMP involves a cell surface receptor and the activation of a pertussis toxin-sensitive G protein343 .

Nerve growth factor stimulates transcription of the rScn2a gene 55-49-03: The treatment of cultured cells (e.g. rat phaeochromocytoma PC12 cells) with nerve growth factor (NGF) results in increased whole-cell Na+ currents. This effect of NGF is due to a stimulation in gene expression, since there is a selective increase of type IT sodium channel mRNA and sodium channel densitr2.

INFORMATION RETRIEVAL

Database listings/primary sequence discussion 55-53-01: The relevant database is indicated by the lower case prefix (e.g. gb:) which should not be typed (see Introduction &, layout of entries, entry 02);

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _en_t_ry_5_5_

database locus names and accession numbers immediately follow the colon. Note that a comprehensive listing of all available accession numbers is superfluous for location of relevant sequences in GenBank® resources, which are now available with powerful in-built neighbouringt analysis routines (for description, see the Database listings field in the Introduction eiJ layout of entries, entry 02). For example, sequences of cross-species variants or related gene familyt members can be readily accessed by one or two rounds of neighbouringt analysis (which are based on pre-computed alignments performed using the BLASTt algorithm by the NCBIt). This feature is most useful for retrieval of sequence entries deposited in databases later than those listed belo"~ Thus, representative members of known sequence homology groupings are listed to permit initial direct retrievals by accession number, unique sequence identifiers (Seq ID: numbers) author Ireference or nomenclature. Following direct accession, however, neighbouringt analysis is strongly recommended to identify newly-reported and related sequences. Nomenclature

Species, DNA source

Original isolate

Accession

dNa1

Drosophila melanogaster genomic DNA library Drosophila melanogaster genomic DNA library

DSC 1 (partial)

gb: M32078-80 Salkoff, Science (1987) 237: 74448.

para (1820 aa)

gb: X14394-8

eDNA, eel (Electrophorus electricus) electroplax eDNA, guineapig cerebellar RNA

Eel Na channel (1820aa)

gb: X01119

Guinea-pig Cerli (partial)

gb: AF003373

eDNA, human brain eDNA, human brain

HBSCI (partial)

gb: X65362

hNaHl

eDNA, human heart

hHl (2016 aa)

gb:M77235

hNaH2

eDNA, human heart

hNav 2.1 (1682aa)

gb:M91556

dNa2

eNal

gpScn8a

hNaBl hNaB2

• • II

HBSCII (2005 aa) gb: X65361 gb:M94055 HBA

Sequence/ discussion

Loughney, Cell (1989) 58: 114354. Ramaswami, Proc Natl Acad Sci USA (1989) 86: 2079-82. Noda, Nature (1984) 312: 1217. Vega-Saenz de Miera, Proc Natl Acad Sci USA (1997) 94: 705964. Lu, FEBS Lett (1992) 303: 53-8. Lu, FEBS Lett (1992) 303: 53-8. Ahmed, Proc Natl Acad Sci USA (1992) 89: 8220-4. Gellens, Proc Natl Acad Sci USA (1992) 89: 554-8. George, Proc Natl Acad Sci USA (1992) 89: 4893-7.

I

entry 55

-

Nomenclature

Species, DNA source

Original isolate Accession

Sequence/ discussion

hNaSkl

cDNA,human skeletal muscle

SkMl (1836aa)

gb:M81758

hScn9a (hNE-Na)

cDNA,human medullary carcinoma cellline eDNA, jellyfish Cyanea eapillata neuronal eDNA, mouse AT-1 atrial tumour cell-line

hNE-Na (1977aa)

em: X82835

George, Ann Neurol (1992) 31: 131-7. Wang, Bioehem Biophys Res Commun (1992) 182: 794-801. Klugbauer, EMBO J(1995) 14: 1084-90.

CYNA1 (1739aa)

gb:L15445

mNav2.3 (1681 aa)

gb:L36179

jNal

mNav2.3

mScn8A

eDNA, mouse brain

Scn8A (l,732aa) gb: U26707

mScnl0a

genomic DNA, mouse

Scn10a

rScnla (rNaBl)

eDNA, rat brain Rat I (2009 aa)

gb: X03638

rScn2a (rNaB2)

eDNA, rat brain Rat II (2005 aa)

gb: X03639

rNaB2A

eDNA, rat brain Rat IIA (2005 aa) gb: X61149

rNaB2 5' flanking region rScn3a (rNaB3)

Rat genomic DNA

gb:M31433

eDNA, rat brain Rat ill (1951 aa)

gb: Y00766

rScn8a (rNaCh6)

eDNA, rat brain Rat NaCh6

gb:L39018

rabbitScn9a

eDNA, rabbit Schwann cells

gb: U35238

rScnl0a

eDNA, rat dorsal SNS (1957 aa) root ganglia PN3 (1956aa)

em: X92184 gb: U53833

rNaGI

eDNA, rat astrocyte

gb:M96578

Rabbit Nas (1984aa)

Na-G (partial)

em: Y09108

Anderson, Proe Natl Aead Sci USA (1993) 90: 7419-423. Felipe, J Bioi Chem (1994) 26229:3012531. Burgess, Nature Genet (1995) 10: 461-5. Souslova, Genomies (1997) 41: 201-09. Noda, Nature (1986) 320: 18892. Noda, Nature (1986) 320: 188192. Auld, Neuron (1988) 1: 449-61. Maue, Neuron (1990) 4: 223-31. Kayano, FEBS Lett (1988) 228: 187-94. Schaller, J Neurosei (1995) 15: 3231-42. Belcher, Proe Natl Aead Sci USA (1995) 92: 11034-8. Akopian, Nature (1996) 379: 25762. Sangameswaran, J Bioi Chem (1996) 271: 5953-6. Gautron, Proe Natl Aead Sci USA (1992) 89: 7272-6.

II

-

entry 55

Nomenclature

Species, DNA source

Original isolate

Accession

Sequence/ discussion

rNaHl

cDNA, rat cardiac muscle cDNA, denervated skeletal muscle

RH1 (2018 aa) SkM2 (2018aa)

gb:M27902

rNaSkl

cDNA, rat skeletal muscle

1840aa

gb:M26643

sNal

cDNA, squid Loligo bleekeri axon

1522aa

gb: 014525

sNa2

cDNA, squid GFLN1 (1784aa) gb: L19979 Loligo opalescens giant fibre cDNA,human Human,81, gb:L10338 brain SCN1B (223 aa)

Rogart, Proc Natl Acad Sci USA (1989) 86: 8170-4. Kallen, Neuron (1990) 4: 233-42. Trimmer, Neuron (1989) 3: 33-49. Sato, Biochem Biophys Res Commun (1992) 186: 61-8. Rosenthal, Proc Natl Acad Sci USA (1993) 90: 10026-30. McClatchey, Hum Mol Genet (1993) 2: 745-9. Isom, Science (1992) 256: 83942. Isom, Cell (1995) 83: 433-42. Isom, Cell (1995) 83: 433-42.

hNa,81 rNa,81

cDNA, rat brain Rat ,81 (199 + 19aa)

rNa,82

cDNA, rat brain Rat ,82 gb: U37026 rat genomic gb: U37147 (186 + 29aa) DNA PCRproduct withrat genomic DNA template

gb:M91808

Related sources and reviews 55-56-01: Sodium channel defects in genetic disease8o,344-346j structure and function of voltage-sensitive Na+ channels l64,347,348j Na+ channels as targets for insecticides322j Diversity of sodium channels in the nervous system349j voltage sensors and pores 176 .

Book references: Goldin, A.L. (1994) Molecular analysis of sodium channel inactivation. In Handbook of Receptors and Channels (ed. R.A. North) pp. 73-111. CRC Press, Cleveland, Ohio. Guy, H.R. and Durell, S.R. (1995) Structural models of Na+, Ca2 + and K+ channels. In Ion Channels and Genetic Diseases (eds C. Dawson and R.A. Frizzell) pp. 1-16. Rockefeller University Press, New York. Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA. North, R.A. (ed.) (1994) Handbook of Receptors and Channels. CRC Press, Cleveland, OH. Obrenovitch, T.O. (1997) Sodium and potassium channel modulators: their role in neuroprotection. In Neuroprotective Agents and Cerebral Ischaemia, pp. 109-135. Academic Press, London.

1__e_n_t_ry_5_5

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REFERENCES 1

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Entry Number Rubric Entry 13 resume The rubrics are intended to form a 'quick-reference' method to locate information by means of a channel type/field combination. Entry (channel type) numbers form the first two numbers xx of the six-figure index number (xx-yy-zz) according to the rubric below. yy is the field i.d. number (see the accompanying field number rubric) and zz is the datatype i.d. number within a field (in the current entries zz simply indicates 'paragraph running order' but this number will eventually define and index specific types of data 'falling under' each fieldname). 'Coverage' under each page header/sortcode as listed below is indicated under Cumulative contents. entry 02. Entry

Page header/sortcode See also accompanying FIELD NUMBER RUBRIC

Entry 01-yy-zz Entry 02-yy-zz Entry 03-yy-zz Entry 04-yy-zz Entry 05-yy-zz Entry 06-yy-zz Entry 07-yy-zz Entry 08-yy-zz Entry 09-yy-zz Entry 10-yy-zz Entry ll-yy-zz Entry 12-yy-zz Entry 13-yy-zz Entry 14-yy-zz Entry 15-yy-zz Entry 16-yy-zz Entry 17-yy-zz Entry 18-yy-zz Entry 19-yy-zz Entry 20-yy-zz Entry 21-yy-zz Entry 22-yy-zz Entry 23-yy-zz Entry 24-yy-zz Entry 25-yy-zz Entry 26-yy-zz Entry 27-yy-zz Entry 28-yy-zz Entry 29-yy-zz Entry 30-yy-zz Entry 31-yy-zz Entry 32-yy-zz Entry 33-yy-zz Entry 34-yy-zz

Cumulative tables of contents Introduction &.. layout of entries Abbreviations ELG [key facts] ELG CAT 5-HT3 ELG CAT ATP ELG CAT GLU AMPA/KAIN ELG CAT GLU NMDA ELG CAT nAChR ELG CI GABAA ELG CI GLY Feedback &.. CSN access Rubrics ILG [key facts] ILG Ca AA-LTC4 [native] ILG Ca Ca InsP4 S [native] ILG Ca Ca RyR-Caf ILG Ca CSRC [native] ILG Ca InsP3 ILG CAT Ca [native] ILG CAT cAMP ILG CATcGMP ILG CI ABC-CF ILG Cl ABC-MDR/PG ILG CI Ca [native] ILG K AA [native] ILG K Ca ILG K Na [native] INR K [key facts] INR K ATP-i [native] INR K G/ACh [native] INR K [native] INR K [subunits] INR K/Na Ifhq [native]

Entry e-mail feedback file [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

II

_L..Entry

e_n_try __ 13_re_s_u_m_e_1

Page header/sortcode See also accompanying FIELD NUMBER RUBRIC

Entry 35-yy-zz Entry 36-yy-zz Entry 37-yy-zz Entry 38-yy-zz Entry 39-yy-zz Entry 40-yy-zz Entry 41-yy-zz Entry 42-yy-zz Entry 43-yy-zz Entry 44-yy-zz Entry 45-yy-zz Entry 46-yy-zz Entry 47-yy-zz Entry 48-yy-zz Entry 49-yy-zz Entry 50-yy-zz Entry 51-yy-zz Entry 52-yy-zz Entry 53-yy-zz Entry 54-yy-zz Entry 55-yy-zz Resource A Resource B Resource C Resource D Resource E Resource F Resource G Resource H Resource I Resource J Resource K

JUN [connexins] MEC [mechanosensitive] MIT [mitochondrial, native] NUC [nuclear, native] OSM [aquaporins] SYN [vesicular] VLG [key facts] VLGCa VLGCI VLG K A-T [native] VLG K DR [native] VLG K eag/elk/erg VLG K Kv-beta VLG K Kvl-Shak VLG K Kv2-Shab VLG K Kv3-Shaw VLG K Kv4-Shal VLG K Kvx [unassigned] VLG K M-i [native] VLG (K) minK VLGNa G Protein-Linked Receptors Electrical Effectors Compounds &.. Proteins Diagnostic Tests Book References Subject Reviews Consensus Sites &, Motifs Cell-Types Cell-Sig. Mol. Types Search Criteria &, CSN Dev. Multidisciplinary Glossary Framework

Entry e-mail feedback file [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN-5 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

Note: Entry 'running order' is only of significance in book-form publications; computer updates will use the xx-yy-zz numbers as hypertext pointers. Resource documents will be supported on-line following completion of the (printed) entry series.

Field Number Rubric Entry 13 resume Field numbers form the third and fourth numbers yy of the six-figure index number (xx-yy-zz) according to the rubric below. zz is the datatype Ld. number within a field (in the current entries zz simply indicates 'paragraph running order' but this number will eventually define and index specific types of data 'falling under' each fieldname). Omission of a field number within the main entries implies information was 'not applicable' or was 'not found' during compilation. NOMENCLATURES SECTION Abstract - general description Category (sortcode) Channel designation Current designation Gene family Subtype classifications Trivial names

xx-0l-zz xx-02-zz xx-03-zz xx-04-zz xx-05-zz xx-06-zz xx-07-zz

EXPRESSION SECTION xx-08-zz Cell-type expression index xx-09-zz Channel density xx-l0-zz Cloning resource xx-ll-zz Developmental regulation xx-12-zz Isolation probe xx-13-zz mRNA distribution xx-14-zz Phenotypic expression xx-15-zz Protein distribution xx-16-zz Subcellular locations xx-17-zz Transcript size SEQUENCE ANALYSES SECTION Chromosomal location Encoding Gene organization Homologous isoforms Protein MW (purified) Protein MW (calc) Sequence motifs Southerns

xx-18-zz xx-19-zz xx-20-zz xx-21-zz xx-22-zz xx-23-zz xx-24-zz xx-25-zz

STRUCTURE &, FUNCTIONS SECTION xx-26-zz Amino acid composition xx-27-zz Domain arrangement xx-28-zz Domain conservation xx-29-zz Domain functions (predicted) xx-30-zz Predicted protein topography xx-31-zz Protein interactions xx-32-zz Protein phosphorylation

ELECTROPHYSIOLOGY SECTION Activation Current type Current-voltage relation Dose-response Inactivation Kinetic model Rundown Selectivity Single-channel data Voltage sensitivity

xx-33-zz xx-34-zz xx-35-zz xx-36-zz xx-37-zz xx-38-zz xx-39-zz xx-40-zz xx-41-zz xx-42-zz

PHARMACOLOGY SECTION Blockers Channel modulation Equilib. dissoc. constant Hill coefficient (n) Ligands Openers Receptor/transducer ints Receptor agonists Receptor antagonists Receptor inverse agonists

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INFORMATION RETRIEVAL SECTION xx-53-zz Database listings xx-54-zz Gene map. locus desig. xx-55-zz Miscellaneous information xx-56-zz Related sources &, reviews xx-57-zz Feedback In-press updates REFERENCES SECTION

II

Index Page numbers in italics represent entries in figures and tables A23187, 192, 754, 819, 821 Absence (petit mal) epilepsy mouse model, 12,23,49-50 rat model, 51 Abstract/general description field VLG Ca, 22-4 VLG CI, 154-6 VLG K A-T [native], 196-8 VLG K DR [native], 226-8 VLG K eag/elk/erg, 275-9 VLG K Kv-beta, 327-9 VLG K Kvl-Shak, 374-8 VLG K Kv2-Shab, 524-7 VLG K Kv3-Shaw, 559-64 VLG K Kv4-Shal, 617-19 VLG K Kvx [unassigned], 647-8 VLG K M-i [native], 657-9 VLG (K) minK, 703-6 VLG Na, 768-70 Accessory subunits Kv channels, 210, 226, 327, 332 VLG Ca, 30-5, 368 VLGNa, 368, 769, 796,802-3 Acetazolamide, 49 Acetylcholine, 51, 112, 220, 505, 682, 698 Acetylcholine receptors, 118, 157,219 see also Muscarinic receptors Aconitine, 820, 822, 824, 826 ACPD (l-aminocyclopentane-1s,3R-dicarboxylic acid),694 Actin, 535 Activation field VLG Ca, 85-8 VLG CI, 177-8 VLG K A-T [native], 209 VLG K DR [native], 249-50 VLG K eag/elk/erg, 306-7, 308-9 VLG K Kv-beta, 359-60 VLG K Kv1-Shak, 460-2, 461-2 VLG K Kv2-Shab, 545 VLG K Kv3-Shaw, 599-601, 602 VLG K Kv4-Shal, 634, 635 VLG K M-i [native], 669-70 VLG (K) minK, 743-4, 745 VLG Na, 806-7 A-current, 196-7, 198, 199, 200 see also A-type K+ channels Adaptive radiation, 281 Adenosine, 112, 220, 698-9 Adenosine receptors AI, 117, 118, 119 A2b , 117, 120 Adhesion, integrin-mediated, 285 ADP,94 Adrenal chromaffin cells, 41, 86, 94, 117, 125-8 Adrenal cortical cells, 117 Adrenal gland, 37, 47, 160, 202 Adrenal zona fasciculata cells, 208, 213 Adrenal zona glomerulosa cells, 232, 264 p-Adrenoceptor agonists, 78, 111-14, 264-5, 690, 695

p-Adrenoceptor blockers (P-blockers), 276, 320-1, 658 ~-Adrenoceptors, 264-5 p-Adrenoceptors, 264-5, 505 Adrenocorticotrophic hormone, 396 adr mutant mice, 154-5, 162, 172 After-hyperpolarization, 246, 405, 470, 688 w- Agatoxin mA, 108 w-Agatoxin IVA P-type channel sensitivity, 23, 37, 40, 108, 123, 131 subunit sensitivity, 26, 30 Agitoxins, 486 Airway chemoreceptors, 563-4, 608 A-kinase anchoring protein (AKAP), 79-80 AKTl, 298 AKv5.1 clone, 647-56, 657, 662 Alanine transport, 407 Alcohol see Ethanol Aldo-keto reductase superfamily, 329, 330, 341, 346-8,353,369-71 Aldosterone-inducible genes, 763-4 Alkaline phosphatase, 457, 668 n-Alkanols, 609 4-(Alkylamino)-1,4-dihydroquinolines, 491 ~-toxins, peptide, 770, 801, 808, 820, 821 Alveoli, 232 Alzheimer's disease, 244 Amantadine hydrochloride, 764 Amidation, protein, 328, 351-2 Amiloride, 105, 110 Amino acid composition field VLG K eag/elk/erg, 298 VLG K Kv-beta, 352 VLG K Kvl-Shak, 436-7 Amino acid transport, 407 4-Aminopyridine (4-AP), 679 Kv~ subunits, 491-2, 498-9, 551, 563, 607-8, 618,638-9 native Kv channels, 212, 214, 253, 255, 256 4-Aminopyridine-sensitive, Ca2+-insensitive A-type currents, 200 Amiodarone, 105 Amlodipine, 130 Ammonium (N~+) ions, 96, 312 Anandamide, 492 Anchoring protein, 662, 665 Anemone toxin (ATxll), 783, 824 Angiotensin AT2 receptors, 264 Angiotensin IT Ca2+ channel regulation, 112, 117, 126 Kv channel regulation, 221, 264 ~M-channel regulation, 682, 694 Anipamil, 130 Ankyrin, 74, 633, 768, 773, 786, 787 Annexins, 139 Anorexia (anx) mutation, 420 9-Anthracene-carboxylic acid (9-AC), 156, 164, 186, 187 Antiarrhythmics, 817 class ill, 253, 278, 313, 752

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Anticipation, genetic, 423 Anticonvulsants, 131,770,817-18 Antioxidants, 467 Antipsychotics (neuroleptics), 108, 110 Aorta, 41, 46 Apamin, 255 Aplysia aKv5.1 clone, 647-56 bag cell neurones, 240-1, 525, 534-5 Apoptosis, 506 Arachidonic acid (AA) Ca2 + channel regulation, 83, 118-19 KvtX subunits and, 378, 500, 618, 639, 640-1 M-channel modulation, 683, 684-5, 688 native Kv channel regulation, 214, 215, 259, 265 Arginine vasopressin, 267 Arrhythmias, cardiac, 244, 276, 288, 320 drug-induced, 315, 770, 818 suppression, 309, 313, 320-1 Arylaminoalkyl benzoates, 187-8 Ascidian embryos, 192 Astemizole, 317 Astrocytes, 202, 205, 232, 238, 410, 413 AT-1 atrial tumour myocytes, 284, 305, 529, 710, 739, 780 Ataxia, episodic hKv1.1 gene mutations, 12, 375, 404, 420 type-2 (CACNL1A4 mutations), 49 ATP Ca2+ channels and, 94, 125-8, 126 I K regulation, 247, 266 M-channel regulation, 661, 666-8, 672-3, 682, 686, 688, 690, 691-2 ATP-y-S, 672, 688 ATP-inhibited K+ channels, 661 Atropine, 689, 827 Atrotoxin, 129 AtT20 pituitary cells, 569 A-type K+ channels (VLG K A-T [native]), 196-225,227,374,383,405 Kv3 Shaw-related channels, 562, 603 Kv4 Shal-related channels, 617, 620, 624-6, 627,637-8 Kvp subunits and, 210, 339-40 Auditory pathways, 246, 560, 578-80 Autoimmune disorders, 579 Autonomic neurones, 216 Auxiliary subunits see Accessory subunits Awareness, 599 Axons, 580, 581, 627, 768, 772-3, 786-7 myelination, 237, 414 Azidopine, 71, 122 Azimilide, 316-17, 752

Baclofen, 81, 121 Bacteriorhodopsin, 390 Ball-and-chain model, 465, 807-8 Ball domain peptides, synthetic, 468, 562-3, 603, 604-5 Ball receptor domain, 353-4, 468-9 Barium (Ba2 +), 254, 314, 319, 553 Ca2+ channel effects, 85, 89, 95, 98

M-current effects, 659, 664, 674, 679-80, 682, 689 minK effects, 735, 751 Basketcell~ 413,414 Batrachotoxin (BTx), 818, 820, 822, 824, 826 Bay K 8644, 35,103,104,105,115,128-9 Becker's disease (recessive generalized myotonia), 12, ISS, 163, 172-3 Behaviour, 197, 204 Benign familial neonatal convulsions, 423, 538 Benzoate, 180, 186-7 Benzocaine, 823 Benzodiazepine derivatives, 318 Benzothiazepines, 22, 36, 101, 104, 105, 122 binding site, 130 structure, 103 Bepridil, 105 Berberine, 317 Beta-blockers (p-adrenoceptor blockers), 276, 320-1, 658 P-toxins, scorpion, 820, 822-3 Blockers, 20 Blockers field VLG Ca, 101-10 VLG CI, 186-8, 187 VLG K A-T [native], 212-14, 214 VLG K DR [native], 252-3,254-8 VLG K eag/elk/erg, 313, 314-18 VLG K Kv-beta, 363-4 VLG K Kv1-Shak, 484-95, 496, 498-9 VLG K Kv2-Shab, 550-3 VLG K Kv3-Shaw, 607, 607-8 VLG K Kv4-Shal, 638-9 VLG K M-i [native], 679-82 VLG (K) minK, 750, 751-3 VLG Na, 816-21 Blood cells, 232 B lymphocytes, 232 BMY20064, 106 Bradykinin, 657, 682, 692-3 Brain VLG Ca, 37, 46-8, 52-3 VLG CI, 160 VLG K eag/elk/erg, 276, 286 VLG K Kv-beta, 328, 335, 336-8, 339, 343, 344 VLG K Kvl-Shak, 391, 400-3, 411 VLG K Kv2-Shab, 525, 530, 532-3, 535 VLG K Kv3-Shaw, 559, 568, 571-3, 581-2 VLG K Kv4-Shal, 623, 624, 628 VLG K Kvx [unassigned], 651, 652 VLG Na, 773-6, 777, 778, 779-81, 781-2, 786, 791 Brainstem, 246, 560, 571, 575-7, 578, 579-80 Braking current, 657, 665 Breast cancer cells, 240 Brevetoxins, 820-1, 822, 826-7 BRL-32872, 318 Bromide (Br-), 181, 182 N-Bromoacetamide, 211, 564, 609, 808 8-Bromoadenosine-cyclic AMP (8-Br-cAMP), 278, 305, 313, 755 8-Bromoadenosine-cyclic GMP (8-Br-cGMP), 278, 313

-----~---8-Bromoadenosine-cyclic monophosphorothioate (8-Br-cAMPS), 455 Brown fat cells, 244 p-Bungarotoxin, 363-4, 366, 490, 543 Bupivacaine, 819 Bursting behaviour, 211, 252, 311 2,3-Butanedione monoxime (BDM), 108-9,213, 218 tert-Butyl hydroperoxide, 504 BW1003C87, 818 BW19C89, 818 CACNL1A3 gene, 23, 30, 56 CACNL1A4 gene, 26-8, 42, 58, 69 mutations, 23, 49 CACNL2A gene, 56 Cadmium (Cd2+), 187, 819 Ca2+ channel sensitivity, 26, 30, 42, 102, 104-5, 130 Kv channel sensitivity, 259, 314 Caenorhabditis elegans, 570 Caesium (Cs+) channel inhibition, 254, 314, 319, 679, 682, 751 channel permeability, 96, 675 Caffeine, 257, 687 Calcineurin (protein phosphatase 2B), 84, 94, 658, 665, 666, 672 Calciseptine, 109, 129-31 Calcitonin gene-related peptide (CGRP), 126 Calcium (Ca 2+) channel modulation, 20, 254,378 channel selectivity, 95, 97, 110, 813-14 -dependent inactivation, 71, 91-2, 93 domains, Helix neurones, 74 external, 89-90, 216, 735 extracellular, 259-60, 500, 688, 819 influx, 74-6, 227 intracellular, 22, 139, 457, 561, 583 channel blockade, 91, 314, 500 M-currents and, 659, 682, 685, 686-8 minK/lsK and, 735, 754 oscillations, 200, 278, 314 -responsive genes, 43-4, 52 shielding effects, 89 Calcium (Ca 2+)-activated K+ channels (Kca ), 8, 74, 368, 665, 681-2 Calcium (Ca 2+)-activated protein kinase, 741, 754 Calcium (Ca 2+)-binding motif, 71, 92 Calcium (Ca2+ )-calmodulin-dependent protein kinase (CaM kinase), 43-4, 690 Calcium (Ca 2+)-calmodulin-dependent protein kinase IT (CaMKll), 84, 287, 306, 458, 459, 741-2 Calcium (Ca 2+) channel for inositide-mediated Ca2+ entry, 9 Calcium (Ca2+) channels, voltage-gated (VLG Cal, 4, 22-153, 770, 797 accessory subunits, 30-5, 368 IXI subunits, 6, 26-8, 46-8, 62-3, 73-4, 81 domain I-IT linker, 71-2, 82 G protein interactions, 116-17 SS2 repeat ill, 72

III

subunit, 30-2, 67, 73-4, 124 gene, 56, 57, 59, 61 mRNA, 48, 63 Psubunits, 32-5, 42 functions, 81-2, 92-3, 100, 115 genes, 59, 61 molecular weights, 63-4, 68 mRNA, 48, 63-4 blockers, 24, 101-10, 129-31 l' subunit, 35, 48, 59, 64, 68 subunits, 25, 26-35 Calcium (Ca 2+)-release channels, 74 Calmodulin (antagonists), 119, 688, 742, 753, 754 Calpastatin, 94 Canaliculi, 43 7, 476 Candidate Ca2+ channel subunit 1 (CCCS1), 66 Cannabinoids, 112, 118,492 Capsaicin, 222, 257, 492 Carbachol, 125, 663 Carbamazepine, 770, 818 Carboxypeptidase A, 93 Cardiac ischaemia, 206 Cardiac muscle Kv channels, 202, 233, 623, 624 VLG Ca, 22, 36, 41, 46 Cardiac pacemaking, 207-8, 219, 316 Cardiac Purkinje cells, 221 Casein kinase IT (CKll), 359, 600-1, 634 Catecholamines, 86, 125-8 Category (sortcode) field VLG Ca, 24 VLG CI, 156 VLG K A-T [native], 198 VLG K DR [native], 228 VLG K eag/elk/erg, 279 VLG K Kv-beta, 329 VLG K Kv1-Shak, 378-9 VLG K Kv2-Shab, 527 VLG K Kv3-Shaw, 564 VLG K Kv4-Shal, 619 VLG K Kvx [unassigned], 648-9 VLG K M-i [native], 659-60 VLG (K) minK, 706 VLG Na, 770 Cation-selective voltage/second-messenger-gated channel gene superfamily, 5, 6-10 CChb1 gene, 50 Cdc2 (p34), 277, 285 CDRK (cdrk), 524, 528, 535, 536 Cell adhesion molecules, 797-8 Cell division, 192 Cell-type expression index field VLG Ca, 39-40 VLG K A-T [native], 201, 202-3 VLG K DR [native], 231, 232-6 VLG K eag/elk/erg, 284 VLG K Kv-beta, 332 VLG K Kv1-Shak, 387-9 VLG K Kv2-Shab, 529 VLG K Kv3-Shaw, 568 VLG K Kv4-Shal, 621 VLG K M-i [native], 661-2 VLG (K) minK, 708, 709 VLG Na, 772-3 1X2/~

--------Centruroides limpidus limpidus toxins, 214 Cerebellar ataxia, autosomal dominant, 12,23, 49 Cerebellar granule cells, 216, 238, 261, 625-6, 628 Cerebellar Purkinje cells VLGCa, 23, 37,46, 107, 108 VLG K Kv3-Shaw, 570, 581 VLG Na, 778-81, 784 Cerebellum Kva subunits, 412, 571, 572, 577, 628 VLG K eag/elk/erg, 276, 286 VLG Na, 775, 779, 780,782 Cerebral cortex, 775, 782 Cerebral cortical neurones, 114 Ceruloplasmin, 259 CGP 28392, 128 CGS 21680, 120 Channel density field VLG Ca, 41-2 VLG CI, 157 VLG K A-T [native], 201-4 VLG K DR [native], 237 VLG K Kv-beta, 333 VLG K Kv1-Shak, 389-91 VLG K Kv2-Shab, 529 VLG K Kv4-Shal, 621 VLG (K) minK, 708 VLG Na, 773 Channel designation field VLG Ca, 24 VLG CI, 156 VLG K A-T [native], 198 VLG K DR [native], 228 VLG K eag/elk/erg, 279 VLG K Kv-beta, 330 VLG K Kv l-Shak, 379-81 VLG K Kv2-Shab, 527 VLG K Kv3-Shaw, 564-5 VLG K Kv4-Shal, 619 VLG K M-i [native], 660 VLG (K) minK, 706-7 VLG Na, 771 Channel-inducing factor (CHIF), 762, 763-4 Channel modulation field VLG Ca, 110-21 VLG CI, 188 VLG K A-T [native], 214, 215-19 VLG K DR [native], 253, 259-63 VLG K eag/elk/erg, 313-20 VLG K Kv-beta, 364-6 VLG K Kv1-Shak, 495, 500-4 VLG K Kv2-Shab, 553 VLG K Kv3-Shaw, 608-9 VLG K Kv4-Shal, 639-42, 640-1 VLG K M-i [native], 682-5, 686-9 VLG (K) minK, 750-9, 754-8 VLG Na, 821-4, 822-3 Chapsyn-110, 376, 389 Charybdotoxin (CTx), 254, 551-2 VLG K Kvl-Shak sensitivity, 394-5, 407, 486-7,490,496,498-9 CHIF (channel-inducing factor), 762, 763-4 Chloramine-T, 473,504,609

Chlorate (CI0 4 -), 187 Chloride (CI-) cAMP-mediated secretion, 717 electrochemical gradient, 184 external concentration, 156, 179-80, 184-5 phospholemman/minK-induced currents, 191, 735,762-3 Chloride (CI-) channels blockers, 156, 186-8, 750, 759 Ca2+ -activated, 311-12 voltage-gated (VLG CI), 14-15, 154-95 2-Chloroadenosine, 81, 119 Chlorpromazine, 742 CHO cells, 599, 605, 738, 739 Cholecystokinin (CCK), 697 Chondrocytes, 232, 245 Chromaffin cells, 41, 86, 94, 117, 125-8 Chromanols, 755 Chromosomal location field VLG Ca, 56, 57 VLG CI, 166-7 VLG K eag/elk/erg, 291-6, 292-3 VLG K Kv-beta, 346 VLG K Kvl-Shak, 417-25, 420-3 VLG K Kv2-Shab, 537, 538 VLG K Kv3-Shaw, 582-3, 584-5 VLG K Kv4-Shal, 629, 630 VLG K Kvx [unassigned], 653 VLG (K) minK, 723 VLG Na, 788, 789 Ciguatoxins, 820-1 Ciliary ganglion neurones, 205 Cinnarizine, 105 Circadian rhythms, 208-9 CIC channels, 14-15, 154-95 CIC-K channels, 154, ISS, 159, 161 Clentiazem, 130 Clofilium, 278, 313, 320, 493, 719, 735, 751 Cloning resource field VLG Ca, 42 VLG CI, 157-8 VLG K eag/elk/erg, 284 VLG K Kv-beta, 333 VLG K Kv1-Shak, 391, 393 VLG K Kv2-Shab, 529 VLG K Kv3-Shaw, 568 VLG K Kv4-Shal, 622 VLG K Kvx [unassigned], 651 VLG K M-i [native], 662 VLG (K) minK, 710 VLG Na, 773-4 Clostridium botulinum type A toxin, 780 Clotrimazole, 218, 259 Cobalt (Co 2+), 102 Cocaine, 317-18 Cochlea, 232 Cochlear hair cells, 40, 201, 202, 232, 243-6, 714 Coeliac ganglion neurones, 618, 639, 682, 692 Cognition enhancer, 698 Cold stress response, 396 Colicins, 764 Colony-stimulating factor-1 (CSF-1), 405-6 Combretastatin B1, 318

"'------------Conductance chord, 18 slope, 18 Conductance-voltage relations, 19, 82 Cone-rod dystrophy (CORD2), 585 kappa-Conotoxin PVllA, 485 jl-Conotoxin, 799, 817, 820 ro-Conotoxin, 123 GVIA (ro-CTx), 23, 36-7, 67, 73, 107-8, 114 MVllA, 108, 131 MVTIC, 26, 37, 40, 83, 123, 131 Contactin, 797-8 Convulsions, benign familial neonatal, 423, 538 Copper (Cu2+), 187 Corneal keratocytes, 232, 238 COS cells, 536, 544, 556, 738 CP-339, 818, 394, 473, 492 CRE-binding protein (CREB), 375, 397 CTLL-2 cytotoxic T lymphocyte cells, 405, 406 Current designation field VLG Ca, 24 VLG CI, 156 VLG K A-T [native], 198-9 VLG K DR [native], 229 VLG K eag/elk/erg, 279-80 VLG K Kv1-Shak, 381 VLG K Kv2-Shab, 527 VLG K Kv3-Shaw, 565 VLG K Kv4-Shal, 619-20 VLG K M-i [native], 660 VLG (K) minK, 707 VLG Na, 771 Currents generation of families, 16 native vs cloned, 5-11 Current type field VLG Ca, 88 VLG K A-T [native], 209-10 VLG K DR [native], 250 VLG K eag/elk/erg, 307 VLG K Kv-beta, 360 VLG K Kv1-Shak, 462-3 VLG K Kv2-Shab, 545, 546 VLG K Kv3-Shaw, 601, 603 VLG K Kv4-Shal, 634, 636 Current-voltage (I-V) relations, 17 Current-voltage relation field VLG Ca, 88-9, 90 VLG CI, 178-9, 179 VLG K A-T [native], 210 VLG K DR [native], 250 VLG K eag/elk/erg, 307 VLG K Kv-beta, 360 VLG K Kvl-Shak, 464 VLG K Kv2-Shab, 545-7 VLG K Kv3-Shaw, 602-3 VLG K Kv4-Shal, 634 VLG K M-i [native], 670 VLG (K) minK, 744-6 VLG Na, 807 Cyclic AMP VLG Ca, 78, 80 VLG K eag/elk/erg, 278, 287, 305, 313-19 VLG K Kv1-Shak, 375, 396-7

VLG K Kv3-Shaw, 560, 583-5 Cyclic AMP-dependent protein kinase see Protein kinase A Cyclic AMP-response element (CRE), 375, 396-7, 585 Cyclic GMP (analogues), 83,304 Cyclic GMP-dependent protein kinase (PKG), 83, 84,634 Cyclic nucleotide-binding domains, 275, 281,303, 319 Cyclic nucleotide-gated (CNG) channels,S, 9, 277,298 Cyclin B, 277, 285 8-Cyclopentyl-1,3-dimethylxanthine (CPT), 120 Cyclosporin A, 672 I-Cysteine, 757 Cysteine string proteins (CSP), 66 Cysteine-substitution mutagenesis, 476, 481-2, 798-9, 801 Cytochalasin-B, 551 Cytochrome P450 inhibitors, 218, 259, 364-6 Cytokinesis, 240 Cytomegalovirus (CMV), 239 Cytoskeleton, 358, 633, 750, 768, 773

D600, 20, 103, 110,213 D800, 104 DADL, 695 DAMGO, 695 Darodipine, 130 Database listings/primary sequence discussion field VLG Ca, 131-9 VLG CI, 189-91 VLG K eag/elk/erg, 321-2 VLG K Kv-beta, 366-8 VLG K Kv1-Shak, 507-10 VLG K Kv2-Shab, 554, 555 VLG K Kv3-Shaw, 610-13 VLG K Kv4-Shal, 642-3 VLG K Kvx [unassigned], 655 VLG (K) minK, 760-2 VLG Na, 827-30 DI)T, 15, 770, 824, 826 Deafness, 704, 712, 713 deafwaddler (dfw) mutation, 422-3 De-differentiation, epithelial, 206, 240 Dehydration, 159 Delayed-rectifier K+ channels (VLG K DR [native]), 226-74, 374, 383 cardiac, 226, 229, 244, 253,307 Kv2 Shab-related channels and, 524, 526, 530, 534 Kv3 Shaw-related channels and, 562, 599, 603 ultrarapid, 229, 265 Denatonium, 265, 554 Dendrites, 52, 53-5, 88, 414, 627, 628 Dendrotoxin (DTx), 254-5, 266, 363-4, 543, 607 acceptor complex, 327, 333, 349, 358, 376, 411 ex (ex-DTx), 212, 349, 355, 363-4, 366, 487 sensitive K+ channel complex, 327, 333, 349, 351 ~ (~-DTx), 363, 366 VLG K Kv1-Shak sensitivity, 490, 498-9

_ _ _ _ _IIIIL....--Dentatorubral and pallidoluysian atrophy (DRPLA), 423 Dent's disease, 12, 155, 164-5, 166 Depolarization, 24, 85, 91, 98, 209, 664-5 secondary, 288 Dequalinium, 681 N-Desacetyllappaconitine, 817 Desmethylastemizole, 317 Devapamil, 108, 122, 130 Development, 13, 197, 227, 375 Developmental regulation field VLG Ca, 43-4 VLG CI, 158-9 VLG K A-T [native], 204, 205-6 VLG K DR [native], 237-41, 238-41 VLG K eag/elk/erg, 285 VLG K Kv-beta, 333 VLG K Kv1-Shak, 391-8 VLG K Kv2-Shab, 530-3 VLG K Kv3-Shaw, 568-70 VLG K Kv4-Shal, 622 VLG K M-i [native], 662-3 VLG (K) minK, 710-11 VLG Na, 774-8, 775, 776 Dexamethasone, 396 Dextromethorphan, 109-10, 817 Diabetes mellitus, streptozotocin-induced, 201-4,218 Diacetylkorseveriline, 81 7 Diacylglycerol (DAG), 493, 501, 598, 668, 695 3,4-Diaminopyridine, 679 Diazepam, 394 Diazipine, 71 Dibutyryl-cAMP, 249 Dibutyryl-cGMP,83 Diclofurime, 130 DillS see 4,4'-Diisothiocyano-2,2'-stilbenedisulphonate Diethylpyrocarbonate, 552 Diethylstilboestrol, 710 l,4-Dihydropyridines (DHP), 22, 36, 101-4, 105, 130, 131

agonists, 98, 128-9 binding site, 27, 70-1 dissociation constants, 122 K+ channel actions, 213 Dihydrotestosterone (DHT), 711 4,4'-Diisothiocyano-2,2'-stilbenedisulphonate (DillS) CI- channel blockade, 156, 183-4, 186, 187, 192 minK blockade, 735, 750, 759 Diltiazem, 103, 104, 110, 122, 130, 493 N6-[2-(3,5-Dimethyloxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), 120 2-Dioctanoyl-sn-glycerol (DOG), 493, 501, 598, 668 Diphenylalkylamines, 105 Diphenylamine-2-carboxylate (DPC), 156, 187, 187 Diphenylbutylpiperidines, 108, 110, 122 Diphosphoglyceric acid (DPG), 668 Discopyge ommata, 60-1 Disulphide bonds, 67, 437-8, 443

_

2'-Dithiobis-5-nitropyridine (DTBNP), 504 Dithiobis-sulphosuccinimidyl propionate (DTSSP), 756 Dithiothreitol, 262, 467, 504, 563, 608 Diurnal modulation, 259 DMB protein, 431, 488 DM-nitrophen, 91 Docosahexaenoic acid (DHA), 215, 493, 503 n-Dodecyl-guanidine, 503 Doe, 60-1, 62, 63, 93 Dofetilide, 278, 313, 315-16, 320 Domain arrangement field VLG Ca, 69 VLG K eag/elk/erg, 298 VLG K Kv-beta, 352 VLG K Kv1-Shak, 437-8 VLG K Kv2-Shab, 541 VLG K Kv3-Shaw, 591-3 VLG K Kv4-Shal, 632 VLG (K) minK, 728 VLG Na, 796-7 Domain conservation field VLG Ca, 70 VLG K eag/elk/erg, 298-303 VLG K Kv-beta, 352-3 VLG K Kv1-Shak, 438-41, 442-3 VLG K Kv2-Shab, 541, 542 VLG K Kv3-Shaw, 593-5 VLG K Kvx [unassigned], 654 VLG (K) minK, 728, 729 VLG Na, 797-8 Domain functions (predicted) field VLG Ca, 70-3 VLG CI, 173-4 VLG K Kv-beta, 353-4 VLG K Kv1-Shak, 441, 444-5 VLG K Kv2-Shab, 541-2, 543 VLG K Kv4-Shal, 632 VLG (K) minK, 729-32, 730 VLG Na, 798-802 Dopamine receptors D 1,l17 D 2,610 Dorsal raphe neurones, 221 Dorsal root ganglion neurones, 107, 216 VLG K DR [native], 228, 229, 248, 252 VLG Na, 772, 777, 781, 781 Dose-response field, VLG Ca, 89-90 Double-barrelled channel model, 155, 184 Down's syndrome marker, 704, 716, 723 Doxorubicin, 257-8 DPDPE, 695 DRK1, 524, 528, 535,536, 540,547-8 associated 38 kDa protein, 543 minimal channel, 548 Drosophila cysteine string proteins (CSP), 66 ether-a-go-go (eag) mutants, 275, 284, 287 Hyperkinetic (Hk) gene, 341-2, 353 Na+ channel genes, 784, 785, 791 Shab gene/channels, 524, 525, 529, 534, 626-7 Shaker K+ channels see Shaker K+ channels trp gene, 66 Drug design, rational, 484-5

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DTNP, 758 Ductus arteriosus, 261 Duodenum, 709,712 Duplication, genetic, 376, 417-18, 439, 582-3, 587 Dynorphin, 695 E-4031, 253, 278, 313, 315, 316 E4080, 213 eag/elk/erg gene family K+ channels, 230, 275-326, 657, 662 Ear, inner, 232, 704, 709, 712, 718 Econazole, 131 Eel electroplax, 773-4, 778, 793-4, 814 Ef-hand, 71, 92 Egg-laying hormone, 534 EGL-36, 570 EJ-ras oncogene, 569 Electric organ, Torpedo, 155, 157, 159-61, 165 Electron microscopy, 73 Electrophysiology section VLG Ca, 84-100 VLG CI, 177-85 VLG K A-T [native], 206-11 VLG K DR [native], 249-52 VLG K eag/elk/erg, 306-12 VLG K Kv-beta, 359-63 VLG K Kv1-Shak, 460-84 VLG K Kv2-Shab, 545-50 VLG K Kv3-Shaw, 599-606 VLG K Kv4-Shal, 634-8 VLG K M-i [native], 669-78 VLG (K) minK, 743-7 VLG Na, 806-16 Elgodipine, 130 Elk, 275 Encoding field VLG Ca, 56 VLG CI, 167, 168 VLG K eag/elk/erg, 294-5, 296 VLG K Kv-beta, 346-8 VLG K Kv1-Shak, 425, 426-8 VLG K Kv2-Shab, 538-40, 539 VLG K Kv3-Shaw, 583, 586 VLG K Kv4-Shal, 629, 631 VLG K Kvx [unassigned], 653 VLG (K) minK, 723, 724 VLG Na, 788 Endocrine organs, 22 Endogenous brain peptide, 118 Endothelin, 44, 112, 121, 126, 215, 505, 682 Endothelium, 202 Enkephalin, 121 Epidermis, 202 Epilepsy benign familial neonatal, 423, 538 petit mal (absence) see Absence (petit mal) epilepsy see also Seizures Epithelia, secretory, 231, 709, 712-18, 717, 720-2, 743-4 Equilibrium dissociation constant field VLG Ca, 121-3 VLG Na, 824-5

erg genes, 226, 275-6 Erythromycin, 317 Escherichia coli CI-- channel, 158, 167 Kv homologue, 378 Eserine, 689 Ethanol, 119, 501, 609, 689, 698 ether-ti-go-go (eag) mutants, 275, 284, 287 Ethosuximide, 110 Evolution, 5,11-12 eag/elk/erg K+ channels, 281, 282-3 Kv channel gene family, 5, 376, 381-3, 387, 417-18,419,587 Excitable cells, 3-4, 13, 201, 227-8, 387-8, 772 Excitation-contraction (E-C) coupling, 8, 36 cardiac, 257-8 skeletal muscle, 50, 70, 74 smooth muscle, 405 Excitation-secretion coupling, 36, 37, 108, 208, 407 Excitatory-junction potentials (EJPs), 287 Excitatory post-synaptic potentials (EPSPs), 88, 657, 664, 689, 694, 811 Exocytosis, 39-40 Expression section VLG Ca, 39-55 VLG CI, 157-65 VLG K A-T [native], 201-4 VLG K DR [native], 231-46 VLG K eag/elk/erg, 284-91 VLG K Kv-beta, 332-46 VLG K Kv1-Shak, 383-417 VLG K Kv2-Shab, 528-37 VLG K Kv3-Shaw, 565-82 VLG K Kv4-Shal, 621-9 VLG K Kvx [unassigned], 651-3 VLG K M-i [native], 661-5 VLG (K) minK, 708-22 VLG Na, 772-87 Eye, 232

Facilitation, 78, 86-8 pre-pulse, 114, 116-17 rebound, 114 Fas receptor, 457, 506 Fast-inactivating voltage-gated K+ currents, 200 Fatty acid ethyl esters, 501 Fatty acid metabolites, 824 Fatty acids, 83, 118-19, 639, 640-1 Feedback field VLG Ca, 140 VLG CI, 193 VLG K A-T [native], 222-3 VLG K DR [native], 268-9 VLG K eag/elk/erg, 323 VLG K Kv-beta, 371 VLG K Kv1-Shak, 512-13 VLG K Kv2-Shab, 556 VLG K Kv3-Shaw, 614 VLG K Kv4-Shal, 644 VLG K Kvx [unassigned], 656 VLG K M-i [native], 699 VLG (K) minK, 764-5 VLG Na, 831

_ _ _ _11III'---Felbamate, 818 Felodipine, 130 Fendiline, 105 Fibroblast growth factor, basic (BFGF), 265 Fibroblast growth factor (FGF) receptors, 506 Fibronectin, 285 Flecainide, lOS, 317, 494 Flies, fast- and slow-flying, 245 Flordipine, 130 Flufenamic acid, 750, 759 Flunarizine, 105 Fluoxetine, 260 Fluspirilene, 108, 110, 122 FMl-43,39 Fodrin, 535 Forskolin, 78-9 FPL 64176, 129 Free radicals, 83, 262, 504 FTx (funnel-web spider toxin), 23, 37, 108, 130 GABA (y-aminobutyric acid), 112 GABAergic interneurones, 559, 568-9, 571 GABA (y-aminobutyric acid) receptors, type B, 118 Gabapentin, 124 Gadolinium ions (Gd2+), 102, 130, 679 Galanin, 118 Gallopamil, 130 Gastric smooth muscle cells, 661, 664, 668, 690 Gating charges, 14, 88-9, 480-1 currents, 14, 100, 460, 482-3 modal, 66, 98-100,555-8,658,661, 667, 673-8 voltage-dependent, 89, 97-8, 185, 278 GEF1 yeast mutants, 165, 168 Gene family field VLG Ca, 25 VLG CI, 156-7 VLG K A-T [native], 199-200 VLG K DR [native], 229-30 VLG K eag/elk/erg, 280, 280-1 VLG K Kv-beta, 330,331 VLG K Kv1-Shak, 381-3,384-6 VLG K Kv2-Shab, 527-8, 528 VLG K Kv3-Shaw, 565, 566-7 VLG K Kv4-Shal, 620, 620 VLG K Kvx [unassigned], 649-51, 650 VLG K M-i [native], 660-1 VLG (K) minK, 707 VLG Na, 771 Gene mapping locus designation field VLG K Kv-beta, 368 VLG K Kv1-Shak, 510 VLG K Kv2-Shab, 554 VLG K Kv3-Shaw, 613 VLG K Kv4-Shal, 643 VLG K Kvx [unassigned], 655 VLG (K) minK, 762 Gene organization field VLG Ca, 58-9 VLG CI, 167-8,169 VLG K eag/elk/erg, 296-7 VLG K Kv-beta, 348 VLG K Kv1-Shak, 425-9, 430

_

VLG K Kv2-Shab, 540 VLG K Kv3-Shaw, 583-7, 588-9, 590-1, 592-5 VLG K Kv4-Shal, 629 VLG K Kvx [unassigned], 653-4 VLG (K) minK, 724-5, 726-7 VLG Na, 789-92 General anaesthetics, 219 Gene silencer element, 375, 397, 790 Genetic Absence Epilepsy Rat (GAER), 51 Genetic disorders, 12-13, 23, 768 Ginsenoside Rf, 111 Glia, 232, 392, 401, 402, 413 Glucagon, 126 Glucocorticoid agonists, 396 Gluconate, 759 Glucose utilization, 201-4 Glutamate, 241, 817-18 Glutamate receptor-channels, 678 Glutamate receptors, metabotropic (mGluR), 81, 118 Glutamatergic agonists, 694 Glutamatergic synapses, 246 Glutamic acid decarboxylase (GAD67), 571, 610 Glutathione, 366, 467, 503-4, 563, 608 N-Glycosylation, 68, 328 Kvcx subunits, 435, 436, 541, 596, 632 VLG CI, 171-2, 172, 174-6 VLG K eag/elk/erg, 277, 297-8 VLG (K) minK, 70S, 724, 725-8 VLG Na, 795, 795 O-Glycosylation, 596 Gonioporotoxin, 129 G protein-linked receptors A-type K+ channels, 214-19 delayed-rectifier K+ channels, 263-7 VLG Ca, 24, 110-11, 112-13, 118, 120 G proteins, 192 M-channels and, 659, 671-2, 685, 695-6 pertussis-sensitive, Ill, 457, 827 VLG Ca and, 110-17, 118, 120-1, 124-5 Grayanotoxins (GTx), 820, 822, 826 GRB-PAP1 kidney medullary cell line, 406 Growth, 44 Growth factors, 525, 560, 585 GTP, 114 GTPyS, 114, 120-1, 192, 672 Guanidinium-containing toxins, 816, 822 Guanosine 5'-O-(2-thiodiphosphate) (GDPPS), 114,827 Guanylate kinases, 376, 389, 414, 434, 447, 451 Guanylyl-imidodiphosphate (GppNHp), 115 H-7 (1-(5-isoquinoline-sulphonyl)-2-methylpiperazine), 44, 83, 121, 305, 598, 669 H-8, 757 HA1004 (N(2-guanidinoethyl)-5isoquinolinesulphonamide), 83 Halothane, 553, 609, 750 Hanatoxin 1 (HaTxl), 526, 550-1 Hanatoxin2 (HaTx2), 526, 550-1 Harvey-ras gene, 292 Hdlg, 376, 389, 451 Heart, 4, 38, 160, 339 VLG Ca, 38, 43, 47

'------------------Heart (Cont.) VLG K A-T [native], 204 VLG K DR [native], 226, 229, 244, 253, 307 VLG K eag/elk/erg, 286, 287 VLG K Kv1-Shak, 388, 397, 401, 403, 409-10, 411 VLG K Kv2-Shab, 525, 532, 535 VLG K Kv4-Shal, 619-20, 623, 624, 626, 628 VLG (K) minK, 709,711 VLG Na, 772, 777, 779, 780, 791, 792, 793-4 HEK-293 cells, 125, 179, 340-1 Helix neurones, 74 Heparin, 131, 688 Hepatocytes, 232 HERG gene, 276, 279-80, 281, 287 ~1261 mutation, 277, 289, 304 genomic structure, 296-7, 297 mutations, 276,288, 289-90, 291-6 Heteratisine, 817 Hetisine hydrochloride, 81 7 High voltage activated (HVA) Ca2 + channels, 39, 85,94 Hill coefficient (n) field, VLG Na, 825 Hinged-lid inactivation mechanism, 800, 808 Hippocampal neurones VLG Ca, 40, 53-5, 83-4, 117 VLG K A-T [native], 205, 217, 220, 221 VLG K DR [native], 238, 248 VLG K Kv2-Shab, 531-2 VLG K Kv4-Shal, 622, 625-6 VLG K M-i [native], 668, 683, 689, 693, 698 VLG Na, 810, 827 Hippocampus, 266,335 VLG K eag/elk/erg, 276, 286 VLG K Kv1-Shak, 391,395,407,412 VLG K Kv2-Shab, 532-3 VLG K Kv3-Shaw, 559, 560, 568-9, 572, 574, 580 VLG K Kv4-Shal, 624, 628, 633 VLG Na, 775,782 Histamine, 126 lllT-T15 insulin-secreting cells, 47, 651-2 Hodgkin-Huxley models, 227, 249, 251, 524, 811-12 HOE-166, 106, 122, 130 Homologous isoforms field VLG Ca, 59-66 VLG CI, 169-71, 170 VLG K eag/elk/erg, 297 VLG K Kv-beta, 348 VLG K Kv1-Shak, 431 VLG K Kv3-Shaw, 587 VLG K Kv4-Shal, 630 VLG (K) minK, 725 VLG Na, 792-3 Hormonal regulation, 375 Horses, American quarter, 783, 789 Huntington's disease, 423 Hydrogen ions (H+) see pH Hydrogen peroxide, 565, 609, 758 Hydrogen transfer mechanism, 328, 352, 353, 370-1 Hydrophobic core, central, 302, 382-3, 443, 541, 591-3 Hydroxylamine, 469

Hydroxyl (OH-) ions, internal, 186-7 5-Hydroxytryptamine see Serotonin Hyperkalaemic periodic paralysis, 12, 768, 782-3, 789, 808-10, 812 Hyperkinetic (Hk) gene, 341-2, 353, 368 Hyperosmotic media, 218-19 Hyperpolarization, 177-8, 179, 209, 211 Hyperpolarizing shift, 359-60, 361, 362 Hyperthermia, malignant, 56 Hypokalaemia, 279, 320 Hypokalaemic periodic paralysis (hypoPP), 12,23, 48-9, 56 Hyposmotic (hypotonic) conditions, 155, 164, 178,192,405-6,757 Hypothalamic neurones, 119, 238 Hypoxia, 260-1,366

lA, 198-9, 201, 204, 206-11, 669-70 mMX (isobutylmethylxanthine), 257,663 I ca , 24 I Catl 229, 252 I-channels (I-type channels), 228 ICI , 156 IDRK ,229 IK8 clone, 383, 524, 647-56 I K, 226-7, 229,230-1,669-70 IK (M),660 IK,r, 229, 707, 739, 759 VLG K eag/elk/erg and, 279-80, 284, 287 IK,s, 191, 229, 707, 708, 738, 746-7 at high heart rates, 759 protein underlying, 704, 718-19 I K1rr , 229, 265, 565 1M , 660, 669-70 Imidazole antimycotics, 131 Immediate-early/early genes, viral, 239 Immediate-early genes, 43-4, 52 Immunoglobulin superfamily adhesion molecules, 797-8 INa, 771 Inactivation, 15-20 A-type, 360, 562 Ca2 +-dependent, 71, 91-2, 93 C-type, 365, 637 HERG channels, 277, 309 VLG K Kvl-Shak, 391, 457, 470-3, 473 N-type, 209-10,356,362-3,637 HERG channels, 309 VLG K Kv1-Shak, 459-60, 464, 465-70 VLG K Kv3-Shaw, 562-3, 599, 604-5 P-type, 473, 548 voltage-dependent, 73, 90, 93, 310 Inactivation field VLG Ca, 90-3 VLG CI, 179-80 VLG K A-T [native], 210-11 VLG K DR [native], 250-1 VLG K eag/elk/erg, 307-10 VLG K Kv-beta, 360-3 VLG K Kv1-Shak, 464-73, 465-73, 474-5 VLG K Kv2-Shab, 547-8 VLG K Kv3-Shaw, 603-5 VLG K Kv4-Shal, 637-8 VLG K M-i [native], 670

-----~---

Jervell-Lange-Nielson (JLN) syndrome, 293, 716 JKL1073A, 218 Jurkat T cells, 708, 719-20, 751

Ketoconazole, 317 Kidney medullary K+ transport, 406, 409 VLG CI, 159, 160-1, 169-71 VLG (K) minK, 709, 711, 712, 720, 738, 760 Kinetic model field VLG Ca, 93-4 VLG CI, 180-1 VLG K A-T [native], 211 VLG K DR [native], 251 VLG K eag/elk/erg, 310 VLG K Kv1-Shak, 473-5 VLG K Kv3-Shaw, 605 VLG K M-i [native], 670-2 VLG (K) minK, 746 VLG Na, 811-12 Kinetic slowing, 115 Kir gene family, 374 K1eak,682 K(M) channels, 660 KST1, 298 KST-5452, 698 Kv8.1 clone, 647-56 Kvrx/ p subunit complexes, 230, 383 Kvrx subunits, 7, 226, 230, 327, 374, 383 accessory subunits, 210, 226, 327, 332 Kv1 (Shaker-related), 374-523 Kv2 (Shab-related), 524-58 Kv3 (Shaw-related), 559-616 Kv4 (Shal-related), 617-46 Kvf3 subunits (VLG K Kv-beta), 210, 226,327-73, 374, 381, 633 Kv1rx interactions, 328, 351, 355, 447, 464, 466 Kv4rx interactions, 618, 633 novel Kv2-interacting, 543 Kv channels, 228, 229-30 rx/f3 subunit interaction model, 328, 345 A-type see A-type K+ channels conductances, IS, 16-19 delayed-rectifier see Delayed-rectifier K+ channels dendrotoxin (DTx)-sensitive complex, 327, 333, 349,351,355 eag/elk/erg gene family, 230, 275-326, 657, 662 evolution, 5, 376, 381-3, 387, 417-18, 419, 587 heterotetrameric assemblies, 354-5 mutations, 12 primordial, 329, 330 subunits see Kvrx subunits; Kvf3 subunits unassigned cDNA clones, 647-56 KvLQT1, 191, 226, 647, 704, 707 expression, 708, 712, 718 minK co-expression, 738, 739 Kx channels, 660, 664

K13 clone, 383,647-56 KA channels, 198 Kaliotoxin (KTx), 254, 488, 490, 496 KAT1, 298 KCa channels, 8, 74, 368, 665, 681-2 Kcn1/gen, 647 KCNA1 gene mutations, 404, 420 Km (KDR , K-DR) channels, 228 Ketamine, 553

L691,121, 278, 316 Lacidipine, 130 Lactotrophs, 693 Lafarizine, 818 Lambert-Eaton myasthenic syndrome (LEMS), 45, 55 Laminin, 285 Lamotrigine, 770, 818 Lanthanum (La3+), 101, 102, 216-17, 261, 314, 753

VLG (K) minK, 746 VLG Na, 807-11 Influenza virus M2 protein, 764 Information retrieval section VLG Ca, 131-40 VLG CI, 189-93 VLG K A-T [native], 219-23 VLG K DR [native], 268-9 VLG K eag/elk/erg, 321-3 VLG K Kv-beta, 366-71 VLG K Kv1-Shak, 507-13 VLG K Kv2-Shab, 554-6 VLG K Kv3-Shaw, 610-14 VLG K Kv4-Shal, 642-4 VLG K Kvx [unassigned], 655-6 VLG K M-i [native], 699 VLG (K) minK, 760-5 VLG Na, 827-31 Inhibitory post-synaptic potentials (IPSPs), 88 Inositol l,3,4,5-tetrakisphosphate (InsP4 ), 119 Inositol trisphosphate (InsP3), 85-6, 119, 506 Insecticides, 15, 770, 824, 826 Insulin, 139 Integrins, 285, 321 Interleukin-2 (IL-2), 243, 395, 720, 751 Interneurones, 525, 535, 559, 568-9, 571, 578 Intestine, 46, 160 Iodide (1-), 181, 182 lodopine, 122 Ion channelopathies, 12-13, 768 Iron transport, yeast, 165 I sA , 210, 619-20 Ischaemic damage, 811, 818 IsK see MinK Isoflurane, 304 Isolation probe field VLG K eag/elk/erg, 285-6 VLG K Kv-beta, 333-4 VLG K Kv1-Shak, 398-9 VLG K Kv2-Shab, 533 VLG K Kv3-Shaw, 570 VLG K Kv4-Shal, 622-3 VLG K Kvx [unassigned], 651 VLG (K) minK, 711 VLG Na, 778 Isoproterenol, 678, 690 Isosorbidinitrate (ISDN), 758 Isradipine, 128, 130 ITO (Ito), 198-9, 201-4, 619-20 Ix, 229

II

-----~---Lappaconitine hydrobromide, 817 L-cells, mouse, 340-1, 390-1 Leaner mutation, 50 Leaming, 197, 204, 306 Leg-shaking phenotype, 275, 287, 341 Leucine zipper (heptad repeat) motifs, 435, 481, 560, 589 Leukotriene C4 , 683 Leupeptin, 94 Levemopamil, 130 Lidocaine, 20, 816-17, 823 Ligands field VLG Ca, 123-4 VLG K eag/elk/erg, 320 VLG K Kv-beta, 366 VLG K Kv1-Shak, 496 VLG Na, 825 Linopirdine, 689 5-Lipoxygenase-activating protein (FLAP), 683-4 5-Lipoxygenase inhibitors, 683, 684 Lithium (Li+), 96, 212, 254 Liver, 47, 160 Lobster neurones, 525-6, 533, 534, 540, 623 Local anaesthetics, 20,770,811,816-17,823 Locus coeruleus neurones, 210, 220 Locus control region, 375, 398 Long QT (LQT) syndrome, 12, 13, 288-91, 321 acqurred, 278,288,313 earlier hypotheses, 291 extracellular K+ modulating, 319-20 gene-specific therapy, 291 genetic linkages, 292-3 LQT1 (KVLQTl), 292, 584, 718, 738 LQT2 (HERG), 276, 279, 288, 289-90, 291-6, 292-3 LQT3 (SCN5A), 288, 293, 768, 782, 789, 809 Long-term potentiation (LTP), 52, 222, 306 Louckes lymphoma cells, 579 Low voltage activated (LVA) Ca2+ channels, 23, 85,95 Ltk- mouse fibroblasts, 407-8 L-type Ca2+ channels, 22, 23, 36, 51 blockers, 101-7, 106, 108-10 dissociation constants, 121-3, 122 electrophysiology, 85, 86-9, 90-6, 97-9, 100, 678 expression, 39, 40, 41, 42, 52, 53-5 modulation, 111,112-13,114,117,118,119 mutations, 12, 23, 39, 45, 48-9 phosphorylation, 78, 79-80, 82, 83, 84, 247 purified protein, 66-7 radioligands, 123 receptor interaction/agonists, 124-5, 126-7, 128-31 structure and function, 70-3, 77 subunits, 28, 29, 31, 50, 66-7 Lubeluzole, 770, 817, 818 Lung, 46, 160 Lurcher mutation, 570 Luteinizing hormone-releasing hormone (LHRH), 81, 112, 120, 668, 669, 694, 695 Lymphocytes see B lymphocytes; Thymocytes; T lymphocytes

Machado-Joseph disease, 423 McN5691, 106 McN6186, 130 Magnesium (Mg2+) Ca2+ channel inhibition, 94, 101, 110 extracellular, 279, 320 intracellular, 210, 212-13, 545-7, 602-3, 604 Malignant hyperthermia susceptibility (MRS), 56 MAP2, 633 MAPK/ERK kinase MEK1, 51 MARCKS/80K, 663 Margatoxin (MgTx), 254, 394, 407, 488, 496 Mast cell degranulating peptide (MCDP), 222, 266,489,490,498-9,607

Mat-8, 762, 763 Maurotoxin, 489 MCD peptide, 214 M-channels (muscarinic-inhibited K+ channels), 230, 657-702 MCI176, 130 M-current, 278, 647-8, 657-8 MDCK cells, 415, 536, 560, 581 mdg mutant mice, 12,23,39,45,59, 69 MDL1233A, 106 Mechanosensitivity,218-19 Medial nucleus of trapezoid body (MNTB), 246 med mutations, 13, 768, 782, 783-4, 789 Mefenamic acid, 750, 759 Melanoma cells, 202, 232, 240 Melanotrophs, 217, 220 Membrane glycoproteins, type ill, 703, 706 Membrane potential, 3,90,407 Menthol, 119 p-Mercaptoethanol, 262 Mercury (Hg2+), 756 Merkel cells, 202 Metabotropicglutamatereceptors (mGluR), 81,118 Methanesulphonanilides, 315 Methanesulphonate, 156, 185, 188 Methanethiosulfonate-ethyltrimethylammonium (MTSET), 482, 798 Methanethiosulphonate (MTS), 471, 476 Methoctramine, 698 Methoxyflurane, 110 N-Methyl-LU 49888, 122 N-Methylstrychnine, 770, 817 Methylxanthine, 687 Mexiletine, 819 Miconazole, 131 Microcystin (MC), 79,247,668 Microglia, 405-6 Migraine, familial hemiplegic (FHM), 12, 23, 49 Minimal K protein subunits see MinK (lsK) MinK (lsK), 226, 229, 230, 277, 305,703-67 CI- current activation, 191, 735 gene knockout mice, 704, 712, 713-16 synthetic peptides, 734, 746, 748-9 Miscellaneous information field VLG Ca, 139 VLG CI, 191-2 VLG K Kv-beta, 368-71 VLG K Kv1-Shak, 511 VLG K Kv2-Shab, 554-6 VLG (K) minK, 762-4

'----------'---------

Mitogen-activated protein kinases (MAPKs), 51 Mitosis-promoting factor, 277, 285 MK-499, 278, 313, 315 MK-886, 684 Mole-fraction dependence, anomalous, 95, 252, 477,674 Monobutyryl cAMP, 267 Monocyte-macrophage differentiation, 239 Morphine, 375, 398 Motor endplate disease, 13, 768, 778, 782, 783-4, 789 Motor nerve terminals, 243 Mouse erythroleukaemia cells (MELC), 58, 65, 69 mRNA distribution field VLG Ca, 45, 46-8 VLG CI, 159, 160-1 VLG K eag/elk/erg, 286 VLG K Kv-beta, 334-9, 336-8 VLG K Kv1-Shak, 399-404, 400-3 VLG K Kv2-Shab, 532-3, 533 VLG K Kv3-Shaw, 570-3, 571-3, 574-7 VLG K Kv4-Shal, 623, 624 VLG K Kvx [unassigned], 651-2 VLG (K) minK, 711-12 VLG Na, 778-84, 779-81 mRNA stability, 429 Muscarine, 661, 669, 671, 684, 696, 698 Muscarinic agonists, 220,657,696,698, 827 Muscarinic-inhibited K+ channels (VLG K M-i [native]), 230, 657-702 Muscarinic receptors M I , 456, 505, 691, 695 M 2 , 125 M 3 , 505, 691 Mt,124 Muscle, 3, 4 VLG K DR [native], 227, 231, 232-4 see also Cardiac muscle; Excitationcontraction coupling; Skeletal muscle; Smooth muscle Muscular dysgenesis mice, 12, 23, 39, 45, 59, 69 Mutations, gene, 12-13 Myasthenic syndrome, Lambert-Eaton (LEMS), 45,55 Myocardial infarction, 622 MyoD, 159 Myogenin, 159 Myokymia, 12, 404, 420 Myosin light chain kinase (MLCK), 658, 665, 666-8 Myotonia, 154-5, 162-4, 187, 188 adr mutant mice, 154-5, 162, 172 mutant Na+ channels, 782-3, 812 recessive (CI- channel mutation), IS, 163-4 recessive generalized (RGM, Becker's disease), 1~ 15~ 163, 172-3 Myotonia congenita, dominant (Thomsen's disease), 12, ISS, 162-3 Myristic acid, 119 NAB domains, 277, 304, 328, 351, 447, 448 NADPH binding motif, 328, 352, 353, 370-1 NAD(P)H-dependent aldo-keto reductase superfamily, 329, 330, 341, 346-8, 353, 369-71

NADPH oxidase, 608-9 Na (Nav ) channels see Sodium (Na+) channels, voltage-gated nap mutations, 785 Narke ;aponica, 123 NE-10064 (azimilide), 316-17, 752 NE-10133, 752 Neostriatal neurones, 220 Nephrolithiasis, hypercalciuric see Dent's disease Nerve growth factor (NGF), 44, 525, 531, 535, 774,827 Nerve terminals, 414, 581, 627 Nervous system, 3-4 VLG Ca, 37, 46-8,52-3 VLG K Kv-beta, 328, 335, 336-8 VLG K Kv l-Shak, 391, 400-3 VLG K Kv3-Shaw, 559 VLG K Kv4-Shal, 623, 624, 625, 627 VLG Na, 772, 774-5 Neural crest-derived cells, 392, 780 Neural-restrictive silencer binding factor (NRSBF), 769, 790 Neural-restrictive transcriptional silencer, 769, 790 Neurite extension, 285, 321, 393, 535 Neuroblastoma cells, 259, 276, 285, 321, 456, 773 see also NG108-15 mouse neuroblastoma x rat glioma hybrid cells Neuroepithelial bodies (NEB), pulmonary, 608-9 Neurogenesis, 375, 392, 777 Neuroleptic drugs (antipsychotics), 108, 110 Neurological phenotype mutants, 408, 422-3 Neuromuscular junction, 55 Neurones oxidative stress, 327, 340 VLG Ca, 36, 43, 52, 53-5, 91, 124 VLG K A-T [native], 197,202-3,204,205,207, 209 VLG K DR [native], 227, 231, 234-5, 238, 243, 248 VLG K eag/elk/erg, 275, 276-7 VLG K Kv-beta, 328, 335, 339, 341 VLG K Kv1-Shak, 392-3, 404-5,411-14 VLG K Kv2-Shab, 524-5, 529, 530-1, 533, 534-7 VLG K Kv3-Shaw, 560, 568-9, 570-3, 580-1, 605 VLG K Kv4-Shal, 617, 623, 624-6, 627 VLG K M-i [native], 658, 661, 663-4, 665, 671 VLG Na, 772-3,774 Neurontin, 124 Neuropeptide secretion, 534-5 Neuropeptide Y (NPY), 112, 114-15, 126, 221, 534 Neuroprotection, 20, 131, 770, 818 Neurotoxin IV, 821 Neurotransmitters modulation, 20, 112-13 Kv channels, 198, 220-2, 243, 618, 625 release, 689 Kv channels, 197, 204, 227, 243, 287, 405 VLG Ca, 37, 39-40, 51-2, 77-8, 80, 83

1....----

NG108-15 mouse neuroblastoma x rat glioma hybrid cells, 203 Ca2+ channels, 114-15, 118 M channels, 660, 661, 663, 681, 684-5, 691-2, 697 Nicardipine, 130, 213 Nickel (Ni 2+), 30, 102, 110, 130 Nicotinamide (NAD+), 682-3 Nicotinic acetylcholine receptor-channels, 678 Nifedipine, 101, 103, 110, 130, 494 Niflumic acid, 750, 759 Niguldipine, 106, 130 Niludipine, 130 Nimodipine, 82, 101, 103-4, 130 Nisoldipine, 29, 130 Nitrarine dihydrochloride, 817 Nitrate (N0 3 -), 181, 182, 186 Nitrendipine, 101-3, 104, 123, 130, 131 Nitric oxide, 261, 757-8 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB), 187-8, 192 S-Nitrosocysteine (SNOC), 738, 757-8 NMDA receptor-channels, 119, 376, 389 Nodes of Ranvier, 237, 243, 412-13, 414, 773, 786 Nomenclatures section VLG Ca, 22-39 VLG CI, 154-7 VLG K A-T [native], 196-200 VLG K DR [native], 226-31 VLG K eag/elk/erg, 275-84 VLG K Kv-beta, 327-32 VLG K Kv1-Shak, 374-83 VLG K Kv2-Shab, 524-8 VLG K Kv3-Shaw, 559-65 VLG K Kv4-Shal, 617-21 VLG K Kvx [unassigned], 647-51 VLG K M-i [native], 657-61 VLG (K) minK, 703-8 VLG Na, 768-72 Non-excitable cells, 3-4, 388 Non-NMDA receptor-channels, 119 Noradrenaline, 51-2, 99-100, 111-14, 127, 221, 678 N ordihydroguaiaretic acid, 683, 684 Noxiustoxin, 254, 394, 489, 496 N-type Ca2+ channels, 23, 24, 28, 36-7, 123, 125 autoantibodies, 45 blockers, 102, 107-8, 109-10 electrophysiology, 95, 99-100, 678 expression, 41-2, 52, 53, 55 modulators, Ill, 112-13, 114, lIS, 117, 118, 119-20, 121 neurotransmitter release, 51-2 purified, 67-8 selective antagonists, 130, 131 structure and function, 73, 76-8, 80, 82, 84 Nucleotide triphosphates, 657, 688 Nystatin, 502 17-0ctadecynoic acid, 218, 259 Octanol, 110 Oesophagus, 235 Oestradiol (E2), 711 Oestrogens, 128, 710, 710, 756

III

1.-.-

_

Okadaic acid (OA), 79, 81, 88, 247, 264, 668 1-0Ieoyl-2-acetyl-sn-glycerol (OAG), 740, 827 Olfactory bulb, 412, 779 Olfactory receptor neurones, 13, 806-7, 808 Oligodendrocyte progenitor (O-2A) cells, 241 Oligodendrocytes, 786-7 OPC-13340, 131 Openers field VLG Ca, 124 VLG CI, 188 VLG K eag/elk/erg, 320 VLG Na, 826-7 Opiates, 112, 121, 398, 695 Opioid receptors delta, 695 kappa, 266, 695 mu, 121,695 Opisthotonus (opt) mutation, 422 Organic anions, 156, 188 Osmolarity, ISS, 164, 173-4, 178, 736, 757 Osmosensitivity,218-19 Osteoclasts, 235, 260, 407, 409 Over-recovery phenomenon, 657-8, 664, 671, 682, 684, 687 Oxidative agents, 758 Oxidative stress, 327, 340 Oxidoreductive modulation, 364, 365, 371 Oximes, 108-9 8-0xoberberine, 218 Oxodipine, 130 Oxotremorine methiodide (M), 81, 679, 680, 681, 695 Oxygen, singlet, 262 Oxygen free radicals, 83, 262, 504 Oxygen sensors, 261, 366, 563-4, 608-9 Oxytocin receptors, 719 p34 (cdc2), 277, 285 Pancreas, 37, 709, 712 Pancreatic acinar cells, 203, 208 Pancreatic beta cells, 235, 407 Pancuronium, 770, 817 Pandinotoxins, 489 para gene, 785, 791 Paralysis, 785, 808-9 see also Periodic paralysis Paramyotonia congenita, 12, 768, 782-3, 789, 809 Parasympathetic ganglia, 658, 661 Parathyroid hormone (PTH), 112, 127, 266, 741, 760 Parvalbumin, 559, 568-9, 571 PCMBS (para-chloromercuribenzenesulphonate), 481-2 PD85639, 818 PDZ domains, 376, 389, 447, 451 Penfluridol, 110 Pentobarbital, 110 Pentylenetetrazole, 394 Peptidergic nerve terminals, 704, 709, 719 Periodic paralysis hyperkalaemic (hyperPP), 12, 768, 782-3, 789, 808-10, 812 hypokalaemic (hypoPP), 12, 23, 48-9, 56 Peripheral nerve sodium channel 3 (PN3), 788

_ _ _ _ _11II Peroxides, 758 Pertussis toxin, Ill, 115, 118 Pervanadate, 458 pH external, 105-6, 180, 185, 501, 755-6 internal, 180, 185, 186-7, 218, 672, 755-6 Phaeochromocytoma PC12 cells Kvcx subunits, 387, 525, 535, 571, 573 M current, 693 VLGCa, 41,44,45, 46, 51, 68 VLG K DR [native], 230, 235, 248 VLG Na, 773, 774, 827 Pharmacology section VLG Ca, 101-31 VLG CI, 186-8 VLG K A-T [native], 211-19 VLG K DR [native], 252-67 VLG K eag/elk/erg, 313-21 VLG K Kv-beta, 363-6 VLG K Kvl-Shak, 484-507 VLG K Kv2-Shab, 550-4 VLG K Kv3-Shaw, 607-10 VLG K Kv4-Shal, 638-42 VLG K M-i [native], 679-99 VLG (K) minK, 750-60 VLG Na, 816-27 Phencyclidine, 257 Phenotypic expression field VLG Ca, 45-52 VLG CI, 159-65 VLG K A-T [native], 204, 207-9 VLG K DR [native], 243 VLG K eag/elk/erg, 287-91 VLG K Kv-beta, 339-42 VLG K Kvl-Shak, 404-8 VLG K Kv2-Shab, 534-5 VLG K Kv3-Shaw, 578-9 VLG K Kv4-Shal, 624-7 VLG K Kvx [unassigned], 652-3 VLC K M-i [native], 663-5 VLC (K) minK, 712-20 VLG Na, 782-4, 785 Phenylacetamide, 818 Phenylalkylamines, 22, 36, 101, 104-5, 130, 131 binding sites, 27, 70-1 dissociation constants, 122 structure, 103 Phenytoin, 105, 109, 110, 770, 818 PHMPS (p-hydroxymercuriphenylsulphonic acid), 262 Phorbol-12-myristate-13-acetate (PMA, TPA), 669 Ca2 + channels and, 80-1, 82 Kv channels and, 188, 239, 506 minK and, 736, 740 Phorbol dibutyrate (PDBu), 82, 83, 118, 164, 188, 668-9 Phorbol 12,13-didecanoate (PDD), 740 Phorbol esters, 80, 668-9 Phosphodiesterase inhibitors, 267 Phospholemman (PLM), 191, 735, 762-3 Phospholipase A2, 74, 500, 684 Phospholipase C, 691, 760 Phosphonoformate, 239

__

Photoreceptors, 235, 245, 259 Phototransduction, 66 Phylogenetic tree, 382, 387 pleIn, 191-2 Pinaverium, 130 Pineal cells, 83, 203, 235 Pituitary cells, 203, 212, 235 Pituitary GH3 cells, 110, 120, 396, 397, 402, 409 Pituitary GH4 cells, 248-9 Pituitary gland, 37, 47 Pituitary nerve terminals, 704, 709, 719 Pituitary pars intermedia, 217 PKA see Protein kinase A PKC see Protein kinase C PKC(19-31)' 82, 668-9 PKC(19-36), 81, 83 PKI, 78, 79 Plant inwardly rectifying K+ channel family, 10, 277, 281, 298 Platelet-derived growth factor (PDGF) receptors, 506 Platelets, 232 PN200-110, 100, 122 Polycyclic cations, 770, 817 Portal vein, 41, 248, 264, 265 Post-synaptic functions, 621, 625 Post-synaptic potentials, 88, 227, 243 Post-translational transport, 334 Potassium (K+) channel selectivity, 96, 251, 312, 377, 476, 548, 674 efflux, 340 extracellular, 625, 809-10 VLG K eag/elk/erg, 279, 311, 319-20 VLG K Kvl-Shak, 378, 472, 501-2 permeation pathway, 704, 716-17, 717, 738 transepithelial secretion, 715-16, 717-18 Potassium (K+) channels, 4, 374 ATP-inhibited, 661 inward rectifier, 15 muscarinic.. inhibited, 230, 657-702 plant inwardly rectifying, 10, 277, 281, 298 voltage-gated see Kv channels Potentiation, 88 P/Q-type Ca2+ channels, 24, 42, 80 mutations, 12, 23, 49-50 pharmacology, Ill, 112-13, 115-16, 121, 131 subunits, 27, 28, 58, 69 Prajmalium, 105 Predicted protein topography field VLG Ca, 73-4 VLG CI, 174-6 VLG K eag/elk/erg, 303 VLG K Kv-beta, 354 VLG K Kv I-Shak, 441-52 VLG K Kv2-Shab, 542 VLG K Kvx [unassigned], 654 VLG (K) minK, 732-3 VLG Na, 802 P-region (pore region) Kv channels, 377, 438-9, 440, 476, 477, 548 VLG Na, 796-7,800-1 Pregnanc~ 711, 719 Pre-synaptic inhibition, 24

'---------~---Pre-synaptic nerve terminals, 39-40, 52, 246, 580 Proadilen, 218,259 Procaine, 20, 816-1 7, 823 Prolactin, 693 Pronase, 211, 808 Propafenone, 105 Prostaglandin E1 , 663 Prostaglandin E2, 113,248 Proteases, 94, 333, 808 Protein distribution field VLG Ca, 52-3 VLG CI, 165 VLG K Kv-beta, 343, 344 VLG K Kv1-Shak, 411, 412-13 VLG K Kv2-Shab, 535 VLG K Kv3-Shaw, 579-80 VLG K Kv4-Shal, 627, 628 VLG K M-i [native], 665 VLG (K) minK, 720 VLG Na, 786 Protein domain topography models (PDTMs), 75, 175, 29~ 446, 729, 804-5 Protein interactions field VLG Ca, 74-8 VLG CI, 176-7 VLG K eag/elk/erg, 304-5 VLG K Kv-beta, 354-5, 358 VLG K Kv1-Shak, 447-50, 452-4 VLG K Kv2-Shab, 542-4 VLG K Kv3-Shaw, 595-8 VLG K Kv4-Shal, 633-4 VLG K Kvx [unassigned], 654-5 VLG K M-i [native], 665-6 VLG (K) minK, 734-7,738-9 VLG Na, 802-3 Protein kinase A (PKA), 690 VLGCa, 24, 68,78-80,83-4,88,114 VLG CI, 176, 177 VLG K DR [native], 247, 248 VLG K eag/elk/erg, 305 VLG K Kv-beta, 359 VLG K Kvl-Shak, 396,455-6,457,459,459-60, 467 VLG K Kv2-Shab, 526, 544 VLG K Kv3-Shaw, 562, 599, 600-1 VLG K Kv4-Shal, 634 VLG (K) minK, 719, 741, 760 VLG Na, 769, 794,794,803,806 Protein kinase A (PKA)-anchoring protein (AKAP), 79-80 Protein kinase C (PKC) VLG Ca, 38, 68, 74, 80-3, Ill, 117, 121 VLG CI, ISS, 156, 164, 176, 177, 188 VLG K A-T [native], 215 VLG K DR [native], 247, 248, 264 VLG K eag/elk/erg, 277, 305 VLG K Kv-beta, 359 VLG K Kvl-Shak, 457, 458, 459, 467 VLG K Kv2-Shab, 526, 544 VLG K Kv3-Shaw, 562, 598, 599, 600-1, 605 VLG K Kv4-Shal, 634 VLG K M-i [native], 668-9, 693, 695 VLG (K) minK, 719, 736, 740-1,743, 757

VLG Na, 769, 795, 803, 806, 827 Protein kinase G (PKG, cGMP-dependent), 83, 84, 634 Protein molecular weight (calc.) field VLG Ca, 62-4, 68 VLG CI, 168, 171 VLG K eag/elk/erg, 297 VLG K Kv-beta, 351 VLG K Kv1-Shak, 432, 432 VLG K Kv2-Shab, 540 VLG K Kv3-Shaw, 589 VLG K Kv4-Shal, 632 VLG (K) minK, 725 VLG Na, 793, 794 Protein molecular weight (purified) field VLG Ca, 66-8 VLG CI, 171 VLG K Kv-beta, 348-51,349-50 VLG K Kv1-Shak, 431-2 VLG K Kv2-Shab, 540 VLG K Kv4-Shal, 630-2 VLG (K) minK, 725 VLG Na, 793-4 Protein phosphatase I, 79, 803 Protein phosphatase 2A, 264, 803 Protein phosphatase 2B see Calcineurin Protein phosphatases, 84, 94 Protein phosphorylation field VLG Ca, 78-84 VLG CI, 176, 177 VLG K DR [native], 246, 247-9 VLG K eag/elk/erg, 305-6 VLG K Kv-beta, 355-9 VLG K Kv1-Shak, 454-60, 455-9 VLG K Kv2-Shab, 544 VLG K Kv3-Shaw, 598-9, 600-1 VLG K Kv4-Shal, 634 VLG K M-i [native], 666-9 VLG (K) minK, 740-2, 743 VLG Na, 803-6 Proteolysis, 93 Protons see pH PSD-95, 376, 389, 414, 447, 451 P-type Ca2+ channels, 23, 37 expression, 40, 42, 53 pharmacology, 102, 108, 120, 123 Pulmonary artery smooth muscle, 261, 364-6 Pulmonary vasoconstriction, hypoxic, 261 Purinoceptors P2,266 P2U, 691-2 P2Y, 128 Pyrethrins, IS, 821, 826 Pyrethroids, IS, 770, 801-2, 821, 823, 824, 826 Pyridines, 363 Q-type Ca2+ channels, 37-8, 40, 42, 76, 83 pharmacology, 102, 118 Quaternary ammonium (QA) derivatives, 255,608 see also Tetraethylammonium (TEA) ions Quinidine, lOS, 278, 313, 318, 320, 753 Quinine, 318, 607 Quivering (qv) phenotype, 420 QX-314, 817, 823

'---------'-------Ranvier, nodes of, 237, 243, 412-13, 414, 773, 786 Ray electric see Torpedo marine, 60-1 RBAT, 738, 757 Rbe gene, 58, 69 Receptor agonists (selective) field, VLG Ca, 128-9 Receptor antagonists (selective) field, VLG Ca, 129-31 Receptors, channel modulation by, 20-1 Receptor/transducer interactions field VLG Ca, 124-8 VLG K A-T [native], 214-19 VLG K DR [native], 263-7, 264-7 VLG K eag/elk/erg, 320-1 VLG K Kv-beta, 366 VLG K Kvl-Shak, 496-507, 505-6 VLG K Kv2-Shab, 553-4 VLG K Kv3-Shaw, 610 VLG K M-i [native], 685-99, 691-7 VLG (K) minK, 759-60 VLG Na, 827 Recombination, genetic, 376, 418, 439 Rectification, definition, 231 Redox status, 218-19, 364, 365, 371, 378, 503-4, 625 References section VLG Ca, 140-53 VLG CI, 193-5 VLG K A-T [native], 223-5 VLG K DR [native], 269-74 VLG K eag/elk/erg, 323-6 VLG K Kv-beta, 371-3 VLG K Kvl-Shak, 513-23 VLG K Kv2-Shab, 556-8 VLG K Kv3-Shaw, 614-16 VLG K Kv4-Shal, 644-6 VLG K Kvx [unassigned], 656 VLG K M-i [native], 699-702 VLG (K) minK, 765-7 VLG Na, 831-8 Refractory period, 228, 243, 253, 578 Reissner's membrane, 713, 716 Related sources and reviews field VLG Ca, 139-40 VLG CI, 192 VLG K A-T [native], 219-22 VLG K DR [native], 268 VLG K eag/elk/erg, 322-3 VLG K Kv-beta, 368-71 VLG K Kvl-Shak, 511-12 VLG K Kv2-Shab, 556 VLG K Kv3-Shaw, 613-14 VLG K Kv4-Shal, 644 VLG K Kvx [unassigned], 655 VLG K M-i [native], 699 VLG (K) minK, 764 VLG Na, 830 Re04-, 187 Repolarization, 86 action potentials, 227, 287 fast transient (spike), 207 heart, 253, 287, 565

Resiniferatoxin, 494 Resting potential, 237, 407, 647 REST (RE-l-silencing transcription factor), 790 Retinal (ganglion) neurones, 536-7, 709, 712 Retinal pigment epithelial cells, 202, 206, 232, 240 Reversal potential, 96 Riluzole, 817-18 Riodipine, 130 RO 7-2956, 267 Rod bipolar cells, 235, 245, 415 Rod photoreceptor inner segments, 661, 664, 665-6, 679-80 Rohon-Beard cells, 392, 401 Rose bengal, 262, 504 RP 58866, 317 R-type Ca2+ channels, 38, 53-5, 88 Rubidium (Rb+), 262, 478, 478-9, 674 Rundown field VLG Ca, 94-5 VLG K eag/elk/erg, 311 VLG K M-i [native], 672-3 Ryanodine, 260

SI-S6+H5 transmembrane domain, 277, 298, 647 S4 transmembrane segment, 14,73 Kv channels, 302, 306, 310, 442, 484,550 VLG Na, 798-9, 815-16 Sadopine, 122 Salicylaldoxime, 213 Salivary glands, 709, 712, 720 SAP97, 376, 389, 414, 447 Saxitoxin (STx), 770, 793, 816, 820, 825 mutations affecting binding, 785, 800-1 structure, 822 S-channels, 678 Schwann cells, 235, 245, 256-7, 412, 413, 529 Sciatic nerve, 256, 812, 813 SCN4A gene, 776-7, 789, 789-90 mutations, 768, 782-3, 789, 808-9 SeN5A gene, 776-7, 789 mutations, 768, 782, 789, 809 Sen8a gene, 790-1 mutations, 768, 778, 782, 783-4, 789 Sen10a gene, 791 Scorpion toxins, 770, 801, 808, 820, 821-3 SDZ 207-180, 70 Sea anemone toxins, 770, 783, 808, 820, 824 Secretory epithelia, 231, 709, 712-18, 717, 720-2, 743-4 Secretory glands, 22 Secretory vesicles, 4 sei mutations, 785 Seizures, 394, 621, 664, 785 Selectivity field VLG Ca, 95-7 VLG CI, 181-2, 182 VLG K A-T [native], 211 VLG K DR [native], 251-2 VLG K eag/elk/erg, 311, 312 VLG K Kvl-Shak, 476-9, 477-8 VLG K Kv2-Shab, 548, 549-50 VLG K Kv3-Shaw, 605-6 VLG K Kv4-Shal, 638

--------Selectivity field (Cant.) VLC K M-i [native], 673, 674-5 VLC (K) minK, 746-7, 748-9 VLC Na, 812-14 Sematilide, 278, 313, 315, 320 Sensory nerve sodium channel (SNS), 788 Sensory neurones, 107, 772, 781 Sequence analyses section VLC Ca, 55-69 VLC CI, 166-73 VLC K eag/elk/erg, 291-8 VLC K Kv-beta, 346-52 VLC K Kv1-Shak, 417-36 VLC K Kv2-Shab, 537-41 VLC K Kv3-Shaw, 582-91 VLC K Kv4-Shal, 629-32 VLC K Kvx [unassigned], 653-4 VLC (K) minK, 723-8 VLC Na, 788-96 Sequence motifs field VLC Ca, 68 VLC CI, 171-2,172 VLC K eag/elk/erg, 297-8 VLC K Kv-beta, 351-2 VLC K Kv1-Shak, 432-6, 433-4 VLC K Kv2-Shab, 541 VLC K Kv3-Shaw, 589, 596 VLC K Kv4-Shal, 632 VLC (K) minK, 725-8 VLC Na, 794,794-5,795 Serotonin (5-HT), 50, 127, 457, 505 Serotonin receptors 5-HT1A,633 5-HT1c, 506, 610 5-HT2, 505-6, 760 Sevdinedione, 81 7 Shab gene/channels, 524, 525, 529, 534, 626-7 Shab-related (Kv2) lX subunit subfamily, 524-58 Shaker K+ channels, 374, 410, 432, 436, 626-7 alternative splicing, 376, 429 electrophysiology, 460, 480-1, 482 mutants resembling Ca2+ channels, 70, 96-7 Shaker-related (Kv1) lX subunit subfamily, 355, 374-523 Shaker/waltzer phenotype, 704, 712, 713 Shal-related (Kv4) lX subunit subfamily, 617-46 Shaw-related (Kv3) lX subunit subfamily, 559-616, 626-7 Shielding effects, 89, 98 Shiverer mutant mice, 772-3 Sialidation, 483-4 Silencer elements, 375, 397, 790 Silicon neurones, 251, 772 Silver ions, 476 Single-channel data field VLC Ca, 97-100 VLC CI, 182-4, 183 VLC K A-T [native], 211 VLC K DR [native], 252 VLC K eag/elk/erg, 311 VLG K Kv1-Shak, 479, 480 VLG K M-i [native], 673-8, 676-7 VLC (K) minK, 747 VLG Na, 814-15

filii

l1li

Single-file, multi-ion pore model, 95 Single-transmembrane domain proteins, 728, 762-4 SK&F 96365, 109 SKCa, 647 Skeletal muscle native Kv channels, 202, 233, 243 VLCCa, 25,36,39,43,44, 50,53, 91 VLG CI, 154, 160, 165 VLG K Kv2-Shab, 525, 532, 534 VLG K Kv3-Shaw, 559, 560, 573, 573, 578 VLG (K) minK, 709, 712 VLG Na, 768, 772, 776-8, 779-80, 791, 793, 814 Small cell lung carcinoma (SCLC) cells, 40,50, 77, 563-4, 608-9 Smooth muscle, 160, 202 VLG Ca, 22, 38, 41, 91 VLG K DR [native], 233-4, 245, 248, 264, 266 VLG K Kv1-Shak, 400, 401, 402, 405 SNAP25, 76-7 Sodium dithionite, 261 Sodium (Na+) channel selectivity, 95-6, 110, 251-2, 312, 675, 769-70, 812-13 influx, stimulating VLC Ca, 74-6 intracellular, 210, 212-13, 254, 545-7 Sodium (Na+) channels, voltage-gated, 4, 395, 678, 768-838 accessory (p) subunits, 368, 769, 796, 802-3 blockers, lOS, 816-21 lX subunits, 7-8, 769 mutations, 12-13,293, 782-4, 785 Sodium/potassium (Na+/K+)-ATPase pump, 716, 717 Sol 8 cells, 390-1 Somatodendritic localization, 621, 625, 627, 628 Somatostatin Ca2 + channel modulation, 113, lIS, 125 Kv channel modulation, 221, 249, 266,568 M-current modulation, 683-4, 690 Somatostatin receptors, 125, 219 Sotalol, 278, 313, 315, 320 Southerns field VLG Ca, 69 VLG CI, 172-3 VLG K Kv1-Shak, 436 VLG K Kv2-Shab, 541 VLG K Kv3-Shaw, 591 VLG (K) minK, 728 VLG Na, 796 Spermatozoa, 38 Spinal cord, 46, 47, 775, 776, 779, 780-1, 781-2 Spinal neurones, 248, 392, 529, 530 Spinobulbar muscular atrophy, 423 Spinocerebellar ataxia type 1 (SCA1), 423 type 2 (SCA2), 423 Spiral ganglion, 714 Spleen, 47 Spontaneous transient outward current (STOC), 200, 216 Squid giant axon Kv channels, 227, 231, 249, 251, 262 VLG Na, 812, 813

__-------J_~

__-------J

SR33557, 130 v-Src, 458 Staurosporine, 215, 669 minK and, 741, 757 VLG Ca and, 44, 81, 82, 121 Stomach, 46, 160, 709, 712 StreptozotoCll1, 682 Streptozotocll1-ll1duced diabetes mellitus, 201-4, 218 Stress responses, 396 Striatum, 569, 571, 775 Stria vascularis, 714, 722 Strontium (Sr2+), 89, 95, 502, 606 Structure-function analyses, 11-12 Structure and functions section VLG Ca, 69-84 VLG CI, 173-7 VLG K DR [native], 246-9 VLG K eag/elk/erg, 298-306 VLG K Kv-beta, 352-9 VLG K Kv1-Shak, 436-60 VLG K Kv2-Shab, 541-4 VLG K Kv3-Shaw, 591-9 VLG K Kv4-Shal, 632-4 VLG K Kvx [unassigned], 654-5 VLG K M-i [native], 665-9 VLG (K) mll1K, 728-43 VLG Na, 796-806 Subcellular locations field VLG Ca, 53-5 VLG CI, 165 VLG K A-T [native], 204 VLG K DR [native], 243-6 VLG K Kv-beta, 343-6 VLG K Kv1-Shak, 411-15 VLG K Kv2-Shab, 535-7 VLG K Kv3-Shaw, 580-1 VLG K Kv4-Shal, 627-9 VLG (K) mlllK, 720-2 VLG Na, 786-7 Substance P, 113, 657, 672-3, 690, 692, 695 Subtype classification field VLG Ca, 25, 36-8 VLG K A-T [native], 200 VLG K DR [native], 230-1 VLG K eag/elk/erg, 281-4 VLG K Kv-beta, 330-2 VLG K Kv1-Shak, 383 VLG Na, 771 Subunit associations, compatibility, 13-14 Sulphonamide derivatives, 187 Sulphydryl group-specific reagents, 262 Superior cervical ganglion neurones, 251-2 M channels, 670-1, 673, 687, 691, 694, 695-6, 698 VLG Ca, 60, 81, 114 Superior olivary complex, 246 Superoxide dismutase (SOD), 83 Suppression clonlllg, 66 Suprachiasmatic nucleus, 208-9, 217 Supraoptic neurosecretory cells, 203, 221 Swellll1g-ll1duced CI- currents, 164, 191-2 Sympathetic ganglia, 339, 399, 623, 658, 661, 662

Sympathetic neurones VLG Ca, 51-2, 82, 99-100, lIS, 124 VLG K Kv4-Shal, 618, 623, 639 VLG K M-i [native], 657-8, 661, 663-4, 666-70, 673-8, 682, 690-9 Synapse-associated protell1 90 (SAP90), 412, 581 Synapse-associated protell1 97 (SAP97), 376, 389, 414,447 Synapse development, 768, 774 Synaptic core complex, 37, 77-8 Synaptic plasticity, 618-19, 625 Synaptic transmission, 40, 80, 83, 306, 531, 560, 580 Synaptic vesicles, 76-7 Synaptotagmll1, 45, 76, 77 Synexin, 139 Syntaxll1, 76-7 Systemic lupus erythematosus, 579

~

T1 domall1s/subdomall1s, 448-9 Taicatoxlll, 106-7 Taste receptor cells, 203, 208, 236, 265, 529, 554 Taurll1e, 219, 263, 763 Tedisamil, 213-14, 493 Temperature dependence, 754 Terfenadllle, 317, 494 Terikalant, 317 Testis, 47, 236 12-0-TetradecanoylphorboI13-acetate (TPA) see Phorbol-12-myristate-13-acetate Tetraethylammonium (TEA) ions, 20, 314, 751 Kv/X subunits and, 469, 494-5, 498-9, 552-3, 563,607-8 VLG Ca and, 84, 96 VLG K DR [native] and, 244, 253, 255, 263 ~(9)- Tetrahydrocannabll1ol, 492 Tetrandll1e, 107 Tetrapentylammonium, 551 Tetrodotoxlll (TTx), 768, 770, 785, 811, 816, 825 bll1ding site, 800-1, 819, 820 structure, 822 Thalamus, 571, 575, 580, 599, 624, 628 Thaliporphll1e, 213 Theophyllll1e, 267 Thiazin dyes, 770, 817 Thiocyanate (SCN-), 186, 187 Thomsen's disease, 12, ISS, 162-3, 182 THP-1 human leukaemia cells, 239 Thrombll1, 127, 506 Thymocytes, 375, 390, 395 Thyroid cells, 236 Thyrotropin-releasll1g hormone (TRH), 120, 396, 525, 531, 693 Tipamil, 130 tip-E mutations, 785 Tityustoxlll-K alpha, 254, 489, 496 T K+ channels, 198 T lymphocytes, 85-6 VLG K DR [native], 232, 241, 242 VLG K Kv1-Shak, 390, 394-5, 402, 409 VLG K Kv3-Shaw, 559, 565, 569, 571, 579 VLG (K) mll1K, 708, 709,719-20, 751 Tonic inhibition, 114 Torasemide, 187

I......---

Torpedo (electric ray), 66, ISS, 157-8, 159-61, 165 Torsade de pointes, 279, 288, 313, 320 Tottering (tg) mouse, 12, 23, 49-50 Toxin I (black mamba venom), 212 Tracheal epithelium, 709, 718, 720 Transcriptional inhibitors, 240 Transcript size field VLG Ca, 54, 55 VLG CI, 165, 166 VLG K eag/elk/erg, 291 VLG K Kv-beta, 346 VLG K Kv1-Shak, 416-17, 417 VLG K Kv2-Shab, 537, 537 VLG K Kv3-Shaw, 581-2, 582 VLG K Kv4-Shal, 629 VLG K Kvx [unassigned], 653 VLG (K) minK, 721, 722,722 VLG Na, 787, 787 Transient K+ currents/channels, 196-7, 199 Transient outward K+ currents, 200 Trifluoperazine, 742 Trinucleotide repeat motifs, expanded, 423 Trisomy 12p syndrome, 421 Trisomy 21 marker, 704, 716, 723 Trivial names field VLG Ca, 25-39 VLG CI, 157 VLG K A-T [native], 200 VLG K DR [native], 231 VLG K eag/elk/erg, 284 VLG K Kv-beta, 332 VLG K Kv1-Shak, 383 VLG K Kv3-Shaw, 565 VLG K Kv4-Shal, 621 VLG K M-i [native], 661 VLG (K) minK, 707-8 VLG Na, 772 trp gene, Drosophila, 66 Trypsin, 93,333, 808 T-type Ca2+ channels, 23, 25-39, 38, 44, 98 activation, 85-6, 87, 88, 100 expression, 41, 42, 53-5 inactivation, 92 I-V relations, 89, 90 note added in proof, 153 pharmacology, 102, 105-6, 109, 110, 120-1, 131 in rat model of absence epilepsy, 51 selectivity, 95, 96 d-Tubocurarine, 679 202-791, 71, 128-9 Tyrosine kinase (TyrK), 456, 457, 458, 460, 634 Tytiustoxin (tityustoxin K alpha), 254, 489, 496

U-50,488H, 695 U0 2 2+, 187 Uterus, 46, 777, 780 minK channels, 709, 710-11, 712, 719, 720-2

II

~--UTP,695 Utricle hair cells, 232 Vascular smooth muscle cells, 248, 256, 260, 265 Vasoactive intestinal peptide (VIP), 113, 266 Vasopressin, 113, 267 Ventricular hypertrophy, 206 Ventricular myocytes Kv channels, 215, 216, 247, 267, 307, 628 VLG Ca, 41, 44, 91, 100, 121 Verapamil, 103, 104, 130, 257, 819-20 Veratridine, 820, 822, 824, 826 Veratroylzygadenine, 826 Vestibular dark cells, 715, 718, 747, 755-6, 759 Viral immediate-early/early genes, 239 Vitronectin, 285 Voltage-gated channel gene superfamily,S, 6-8, 25 Voltage sensitivity field VLG Ca, 100 VLG CI, 184-5 VLG K DR [native], 252 VLG K eag/elk/erg, 311-12 VLG K Kv1-Shak, 480-4 VLG K Kv2-Shab, 550 VLG K Kv3-Shaw, 606 VLG K Kv4-Shal, 638 VLG (K) minK, 747 VLG Na, 815-16 Voltage sensor, 14-15 Kv channels, 302, 306 VLGCa, 23,36,39,66-7, 73,88 VLG Na, 769, 798-9, 810 Volume regulation, 164, 405, 406 W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide), 304, 688, 742 Walsh peptide, 544 Wasted (wst) mutation, 538 WIN 55, 212-2, 118 WIN 61773-2, 278, 316 WIPTIDE, 120 Wortmannin, 668 Xenopus Kv1 channels, 375, 392, 393-4 Kv2 channels, 529, 530-1 myocytes, 774 Xenopus oocytes endogenous channels, 83, 84-5, 177, 530 minK/lsK expression, 703, 70S, 711 non-expressible Kv clones, 648, 652

Yeast, CI- channel, 158, 165, 167, 168, 171 Zinc (Zn2+), 187, 207, 217, 502-3, 679, 819, 825