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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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
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"------------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...----
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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)
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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
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'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
-
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
_
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
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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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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
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_
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
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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
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(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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----"_
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
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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
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_
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 .
I
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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
II
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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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_
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
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_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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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 -----------
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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+
II
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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
_e_n_t_ry_4_4
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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•
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_
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
l_e_n_t_ry_45
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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.
1__e_n_t_ry_45
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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
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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.
_~
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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
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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"
~ ~
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(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.
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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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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
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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
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_6_1
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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1'--_e_n_t_ry_4_6
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
II
____________________ 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).
II
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
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_
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
II
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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
-
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
_ _ _ry_4_7 en t
_
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
II
_
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
II
_
...
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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_1....--
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.
II
_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).
II
----J_
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.
II
_L.-
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
II
1__e_n_t_ry_4_7
-----..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).
---J_
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).
II
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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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e_nt_ry_4_8_
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
='
t"'t
~ ~
00
_L-..
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.
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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
MTV
MSGEJA
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-----V
----A----·----
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--·-A·-·------
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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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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
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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)
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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).
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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, ,,-,
