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/).
REFERENCES 1 2
3 4 S 6
7 8 9
10 11 12 13
14 1S
16 17
18 19 20 21 22
23 24
II
Bimbaumer, Neuron (1994) 13: 505-6. Snutch, Neuron (1991) 7: 45-57. Tsien, Trends Pharmacol Sci (1991) 12.: 349-54. Starr, Proc Natl Acad Sci USA (1991) 88: 5621-5. Mori, Nature (1991) 350: 398-402. Perez-Reyes, J Biol Chem (1990) 2.65: 20430-6. Stea, Proc Natl Acad Sci USA (1994) 91: 10576-80. Dejongh, Proc Natl Acad Sci USA (1991) 88: 10778-82. Grove, Proc Natl Acad Sci USA (1991) 88: 6418-22. Nakayama, Proc Natl Acad Sci USA (1991) 88: 9203-7. Striessnig, Proc Natl Acad Sci USA (1991) 88: 10769-73. Lacerda, Nature (1991) 352.: 527-30. Sather, Neuron (1993) 11: 291-303. Ellinor, Nature (1993) 363: 455-8. Williams, Neuron (1992) 8: 71-84. Dubel, Proc Natl Acad Sci USA (1992) 89: 5058-62. Williams, Science (1992) 2.57: 389-95. Fujita, Neuron (1993) 10: 585-98. Mikami, Nature (1989) 340: 230-3. Biel, FEBS Lett (1990) 2.69: 409-12. Koch, J Biol Chem (1990) 2.65: 17786-91. Welling, Jpn J Pharmacol (1992) 58: P258-P262. Welling, Circ Res (1993) 73: 974-80. Bosse, EMBO J (1992) 11: 2033-38.
1'--_e_n_try_4_2
_
Gutierrez, TBioI Chern (1991) 266: 16387-94. Hw, Neuron (1991) 7: 35-44. 27 Seino, Proc Nati Acad Sci USA (1992) 89: 584-8. 28 Yaney, Mol Endocrinol (1992) 6: 2143-52. 29 Soong, Science (1993) 260: 1133-6. 30 Niidome, FEBS Lett (1992) 308: 7-13. 31 Williams,' BioI Chern (1994) 269: 22347-57. 32 Tanabe, Nature (1987) 328: 313-18. 33 Hofmann, Annu Rev Neurosci (1994) 17: 399-418. 34 Catterall, Science (1991) 253: 1499-500. 35 Catterall, Science (1988) 242: 50-61. 36 Jay, TBioI Chern (1991) 266: 3287-93. 37 DeJongh, , BioI Chern (1990) 265: 14738-41. 3B Ellis, Science (1988) 241: 1661-4. 39 Ahlijanian, Neuron (1990) 4: 819-32. 4lJ Kim, Proc Natl Acad Sci USA (1992) 89: 3251-5. 41 Dolphin, Exp Physiol (1995) 80: 1-36. 42 Singer, Science (1991) 253: 1553-7. 43 Stea, Neuropharmacology (1993) 32: 1103-16. 44 Caffrey, Science (1987) 236: 570-3. 4S Biel, Eur , Biochem (1991) 200: 81-8. 4IJ Perez-Reyes,' BioI Chem (1992) 267: 1792-7. 47 Berrow, , Physiol (1995) 482: 481-91. 4B Ruth, Science (1989) 245: 1115-18. 49 Pragnell, FEBS Lett (1991) 291: 253-8. so Collin, Circ Res (1993) 72: 1337-44. 51 Powers,' BioI Chem (1992) 267: 22967-72. 52 Hullin, EMBO '(1992) 11: 885-90. 53 Castellano, , BioI Chem (1993) 268: 3450-5. 54 Castellano, J BioI Chern (1993) 268: 12359-66. 55 Catterall, Ann NY Acad Sci (1989) 560: 1-14. S6 Burgess, Eur , Biochern (1988) 178: 527-33. 57 Jay, Science (1990) 248: 490-2. 58 Powers, , BioI Chem (1993) 268: 9275-9. 59 Wei, , BioI Chem (1991) 266: 21943-7. 60 Tsien, Trends Neurosci (1988) 11: 431-8. 61 Wheeler, Science (1994) 264: 107-11. 62 McEnery, Proc Natl Acad Sci USA (1991) 88: 11095-9. 63 Witcher, Science (1993) 261: 486-9. 64 Dewaard, , BioI Chem (1994) 269: 6716-24. 6S Llinas, Ann NY Acad Sci (1989) 560: 103-11. 66 Mintz, Nature (1992) 355: 827-9. 67 Llinas, Proc Natl Acad Sci USA (1989) 86: 1689-93. 6B Mintz, Neuron (1992) 9: 85-95. 69 Turner, Science (1992) 2.58: 310-13. 70 Zhang, Neuropharmacology (1993) 32.: 1075-88. 71 Coulter, J Physiol (1989) 414: 587-604. 72 Huguenard, J Neurosci (1992) 12: 3804-17. 73 Akaike, J Physiol (1989) 412.: 181-95. 2S
26
II
_L..-
Hagiwara, J Physiol (1988) 395: 233-53. Arnoult, Proc Natl Acad Sci (1996) 93: 13004-9. 76 Porzig, Rev Physiol Biochem Pharmacol (1990) 114: 209-62. 77 Rios, Nature (1987) 325: 717-20. 78 Knudson, J BioI Chem (1989) 264: 1345-8. 79 Adams, Nature (1990) 346: 569-72. 80 Tanabe, Nature (1988) 336: 134-9. 81 Betz, Science (1992) 255: 200-3. 82 Betz, J Neurosci (1992) 12: 363-75. 83 Reuter, Neuron (1995) 14: 773-9. 84 Takahashi, Nature (1993) 366: 156-8. 85 Dunlap, Science (1994) 266: 828-30. 86 Wheeler, Science (1994) 266: 830-31. 87 Zidanic, Biophys J (1995) 68: 1323-36. 88 Barry, J Neurosci (1995) 15: 274-83. 89 Rose, J Physiol (1992) 456: 267-84. 90 Shorofsky, Circ Res (1992) 70: 456-64. 91 Matsuda, J Physiol (1990) 427: 657-80. 92 Droogmans, J Physiol (1989) 419: 627-50. 93 Greenberg, J Pharmacol Exp Ther (1986) 328: 1021-27. 94 Messing, Biochem Biophys Res Commun (1986) 136: 1049-56. 95 Kunze, Mol Pharmacol (1987) 31: 401-9. 96 Akaike, J Physiol (1989) 416: 141-60. 97 Loirand, J Physiol (1989) 412: 333-49. 98 Mcdonald, Physiol Rev (1994) 74: 365-507. 99 Artalejo, Neuron (1992) 8: 85-95. 100 Passafaro, Neuron (1994) 12: 317-26. 101 Nishimura, FEBS Lett (1993) 324: 283-6. 102 Neely, Science (1993) 262: 575-8. 103 Wakamori, Biochem Biophys Res Commun (1993) 196: 1170-6. 104 Ma, J BioI Chem (1992) 267: 22728-32. 105 Ophoff, Cell (1996) 87: 543-52. 106 Liu, J Neurosci (1996) 16: 7557-65. 107 Spitzer, J Neurobiol (1991) 22: 659-73. 108 Morton, Neuron (1989) 2: 1499-506., 109 Malouf, Neuron (1992) 8: 899-906. 110 Diebold, Proc Natl Acad Sci USA (1992) 89: 1497-501. 111 Bading, Science (1993) 260: 181-6. 112 Marks, Circulation (1992) 86: 61-67. 113 Gonoi, J Physiol (1988) 401: 617-37. 114 Furukawa, Circ Res (1992) 71: 1242-53. 115 Lievano, Am J Physiol (1994) 267: C411-24. 116 Fletcher, Cell (1996) 87: 607-17. 117 Chin, FEBS Lett (1992) 299: 69-74. 118 Angelotti, FEBS Lett (1996) 397: 331-7. 119 Usowicz, J Physiol (1990) 426: 95-116. 120 Avidor, FEBS Lett (1994) 342: 209-13. 121 Tanabe, Nature (1990) 344: 451-3. 122 Adams, J Gen Physiol (1994) 104: 985-96. 74
75
II
_ry_42_1
en_t
1"--_e_n_try_4_2
123 124 125 126 127 128 129
130 131 132 133 134 135 136 137 138 139
140 141 142 143 144 145 146 147 148 149
150 151 152 153 154 155 156 157 158 159
160 161 162 163
164 165 166 167 168 169
170 171
_
Lambert, Am J Physiol (1956) 187: 612-13. Leveque, Proc Natl Acad Sci USA (1992) 89: 3625-9. Fontaine, Nature Genet (1994) 6: 267-72. Jurkatt-Rott, Hum Mol Genet (1994) 3: 1415-19. Ptacek, Cell (1994) 77: 863-8. Sipos, J Physiol (1995) 483: 299-306. Cannon, Trends Neurosci (1996) 19: 3-10. Zhuchenko, Nature Genet (1997) 15: 62-69. Gregg, Biophys J (1995) 68: A372. Strube, Biophys J (1996) 71: 2531-43. Codignola, J Bioi Chem (1993) 268: 26240-7. Tsakiridou, J Neurosci (1995) 15: 3110-17. Stanley, Neuron (1993) 11: 1007-11. Kostyuk, J Physiol (1989) 409: 343-55. Delcour, J Neurosci (1993) 13: 181-94. Rosen, Neuron (1994) 12: 1207-21. Hirning, Science (1988) 239: 57-61. Komatsu, J Neurophysiol (1992) 67: 401-10. Kullmann, Neuron (1992) 9: 1175-83. Westenbroek, Nature (1990) 347: 281-4. Westenbroek, Neuron (1992) 9: 1099-115. Hell, J Cell Bioi (1993) 123: 949-62. Flucher, Neuron (1990) 5: 339-51. Jorgensen, J Cell Bioi (1989) 109: 409-16. Hillman, Proc Natl Acad Sci USA (1991) 88: 7076-80. Poo, Annu Rev Neurosci (1985) 8: 369-406. Magee, Science (1995) 268: 301-4. Kavalali, Neuron (1997) 18: 651-63. Robitaille, Neuron (1990) 5: 773-9. Lang, Prog Neuroendocrinol (1990) 3: 83-9. Ma, J Bioi Chem (1995) 270: 483-93. Schultz, Proc Natl Acad Sci USA (1993) 90: 6228-32. Gregg, Genomics (1993) 15: 107-12. TIes, Genomics (1994) 19: 561-3. Chin, Genomics (1991) 11: 914-19. Seino, Genomics (1992) 13: 1375-7. Diriong, Genomics (1995) 30: 605-9. Church, Nature Genet (1994) 6: 98-105. Powers, Genomics (1994) 19: 192-3. TIes, Hum Mol Genet (1994) 3: 969-75. TIes, Hum Mol Genet (1993) 2: 863-8. Gregg, Genomics (1993) 15: 185-7. Collin, Bur J Biochem (1994) 220: 257-62. TIes, Cytogenet Cell Genet (1993) 64: 227-30. Chin, Genomics (1995) 28: 592-5. Chin, Genomics (1992) 14: 1089-91. Nargeot, J Membr Bioi (1992) 126: 97-108. Perez-Reyes, Nature (1989) 340: 233-6. Lin, Neuron (1997) 18: 153-66.
II
____________ en_try_42
172 173 174 175 176 177
178 179 180 181 182 183 184 185 186 187 188 189 190
191 192 193 194 195 196 197 198 199
200 201 202
203 204 205
206 207
208 209
210 211 212 213 214 215 216 217 218 219 220
III
Hug, Biochem J (1993) 291: 329-43. Grabner, Proc Natl Acad Sci USA (1991) 88: 727-31. Home, Proc Natl Acad Sci USA (1993) 90: 3787-91. Snutch, Proc Natl Acad Sci USA (1990) 87: 3391-5. Snutch, Curr Opin Neurobiol (1992) 2: 247-53. Hardie, Neuron (1992) 8: 643-51. Phillips, Neuron (1992) 8: 631-42. Gundersen, Neuron (1992) 9: 527-37. Beam, Nature (1992) 360: 169-71. Schneider, Bur J Biochem (1988) 174: 369-75. Haase, Bur J Pharmacol (1991) 207: 51-9. Ferry, Bioehem J (1987) 243: 127-35. Chang, J Biol Chem (1988) 263: 18929-37. Flockerzi, Bur J Biochem (1986) 161: 217-24. Flockerzi, Nature (1986) 323: 66-8. Leung, J Biol Chem (1987) 262: 7943-6. Curtis, Biochemistry (1984) 23: 2113-18. Heginbotham, Science (1992) 258: 1152-5. Heinemann, Nature (1992) 356: 441-3. Catterall, Cell (1991) 64: 871-4. Catterall, Ann NY Acad Sci (1991) 625: 174-80. Catterall, Adv Bxp Med Biol (1989) 255: 101-9. Tanabe, Nature (1990) 346: 567-9. Kim, Biochemistry (1990) 29: 9281-9. Kass, J Gen Physiol (1991) 98: 63-75. Herbette, J Mol Cell Cardiol (1989) 21: 187-201. Tanabe, Nature (1991) 352: 800-3. Catterall, Trends Pharmacol Sci (1992) 13: 256-62. Regulla, BMBO J (1991) 10: 45-9. Tang, Neuron (1993) 11: 1013-21. Babitch, Nature (1990) 346: 321-2. de Leon, Science (1995) 270: 1502-6. Pragnell, Nature (1994) 368: 67-70. Kim, FBBS Lett (1993) 318: 145-8. Wei, J BioI Chem (1994) 269: 1635-40. Hescheler, J Physiol (1988) 404: 259-74. Ellinor, Nature (1994) 372: 272-5. StUhmer, Nature (1989) 339: 597-603. Papazian, Nature (1991) 349: 305-10. Leung, J Biol Chem (1988) 263: 994-1001. Sharp, J Biol Chem (1989) 264: 2816-25. Takahashi, Proe Natl Acad Sei USA (1987) 84: 5478-82. Gola, Neuron (1993) 10: 689-99. Benham, J Physiol (1989) 419: 689-701. Christie, J Physiol (1992) 445: 369-88. Bezprozvanny, Nature (1995) 378: 623-6. Bennett, Science (1992) 257: 255-9. Leveque, J Biol Chem (1994) 269: 6306-12. Sheng, Neuron (1994) 13: 1303-13.
1
1......_e_n_try_4_2
221 222 223 224 225 226 227 228 229
230 231 232 233 234 235 236 237 238 239
240 241 242 243 244 245
246 247 248 249
250 251 252 253 254 255 256 257 258 259
260 261 262 263
264 265 266 267 268 269
_
Sheng, Nature (1996) 379: 451-4. David, FEBS Lett (1993) 326: 135-9. Wiser, EMBO J (1996) 15: 4100-10. Mochida, Neuron (1996) 17: 781-8. Hymel, Proc Natl Acad Sci USA (1988) 85: 4290-94. Nunoki, Proc Natl Acad Sci USA (1989) 86: 6816-20. Lai, J Biol Chem (1990) 265: 20839-48. Fischmeister, J Physiol (1986) 376: 183-202. Kameyama, Pflugers Arch (1986) 407: 123-8. Bean, Nature (1984) 307: 371-5. Yue, Proc Natl Acad Sci USA (1990) 87: 753-7. Haase, FEBS Lett (1993) 335: 217-22. Dolphin, Br J Pharmacol (1989) 97: 263-73. Gray, Nature (1987) 327: 620-2. Leighton, J Physiol (1994) 477P: 89P. Artalejo, Nature (1992) 358: 63-6. Sculptoreanu, Nature (1993) 364: 240-243. Rohrkasten, J Cardiovasc Pharmacol (1988) 12: S43-6. Rotman, J Biol Chem (1992) 267: 16100-5. Zong, Pflugers Arch-Eur J Physiol (1995) 430: 340-7. Perez-Reyes, FEBS Lett (1994) 342: 119-23. Frace, J Physiol (1993) 472: 305-26. Johnson, Proc Natl Acad Sci USA (1994) 91: 11492-6. Gao, Neuron (1997) 19: 185-96. Ahlijanian, J Biol Chem (1991) 266: 20192-7. Fournier, Pflugers Arch-Eur J Physiol (1993) 423: 173-80. Wakade, Naunyn-Schmiedeberg's Arch Pharmacol (1985) 331: 122-4. Malenka, Nature (1986) 321: 175-7. Shapira, Nature (1987) 325: 58-60. Rane, Proe Natl Aead Sci USA (1986) 83: 184-8. Rane, Neuron (1989) 3: 239-45. Diversepierluissi, Neuron (1993) 10: 753-60. Bley, Neuron (1990) 4: 379-91. Yang, Neuron (1993) 10: 127-36. Bernheim, Neuron (1991) 6: 859-67. Swartz, Nature (1993) 361: 165-8. O'Dell, J Physiol (1991) 436: 739-67. Navarro, J Biol Chem (1987) 262: 4649-52. Chang, J Biol Chem (1991) 266: 16395-400. Ma, Biophys J (1992) 63:639-47. Litten, Eur J Pharmacol (1987) 144: 185-91. Sperti, Mol Pharmacol (1987) 32: 37-42. Loirand, J Biol Chem (1992) 267: 4312-16. Dike, J Physiol (1992) 448: 133-52. Swartz, Neuron (1993) 11: 305-20. Zhu, J Neurophysiol (1994) 72: 1549-60. Stea, Neuron (1995) 15: 929-40. Bourinet, FEBS Lett (1992) 299: 5-9. Linden, J Physiol (1989) 419: 95-119.
II
_L..Keyser, Neuron (1990) 5: 545-53. Chik, J Neurosci (1995) 15: 3104-9. 272 Hell, EMBO J (1995) 14: 3036-44. 273 Hell, J BioI Chem (1994) 269: 7390-6. 274 Hell, J BioI Chem (1993) 268: 19451-7. 275 Lai, J Neurochem (1993) 61: 1333-9. 276 Kameyama, Pflugers Arch (1986) 407: 461-3. 277 Dolphin, Biochim Biophys Acta (1991) 1091: 68-80. 278 Cohen, Annu Rev Biochem (1989) 58: 453-508. 279 Armstrong, Trends Neurosci (1989) 12: 117-22. 280 Kostyuk, Pflugers Arch (1993) 424: 73-83. 281 Barish, J Physiol (1983) 342: 309-25. 282 Dascal, Science (1986) 231: 1147-50. 283 Leonard, J Neurosci (1987) 7: 875-81. 284 Moorman, Am J Physiol (1987) 253: H985-91. 285 Nakai, Proc Natl Acad Sci USA (1994) 91: 1014-18. 286 McDonald, J Biol Chem (1993) 268: 3889-96. 287 Isenberg, Pflugers Arch (1982) 395: 30-41. 288 Josephson, Circ Res (1984) 54: 144-56. 289 Cohen, J Gen Physiol (1992) 100: 703-28. 290 Richard, J Physiol (1993) 462: 197-228. 291 McCarthy, J Gen Physiol (1989) 94: 669-92. 292 Simard, Pflugers Arch (1991) 417: 528-36. 293 Francini, J Physiol (1992) 448: 633-53. 294 Delbono, J Physiol (1992) 451: 187-203. 295 Swandulla, J Gen Physiol (1988) 92: 197-218. 296 Chen, J Gen Physiol (1990) 96: 603-30. 297 Fenwick, J Physiol (1982) 331: 599-635. 298 Artalejo, Nature (1990) 348: 239-42. 299 Artalejo, J Physiol (1991) 444: 213-40. 300 Dolphin, Trends Neurosci (1996) 19: 35-43. 301 Kleppisch, EMBO J (1994) 13: 2502--7. 302 Hoshi, J Neurosci (1987) 7: 571-80. 303 Pietrobon, Nature (1990) 346: 651-S. 304 Sculptoreanu, Proc Nati Acad Sci USA (1993) 90: 10135-9. 305 Bourinet, EMBO J (1994) 13: 5032-9. 306 Stein, Biophys J (1988) 54: 357-63. 307 Smith, Nature (1985) 316: 446-9. 308 Smith, J Gen Physiol (1988) 92: 1-26. 309 Ma, Science (1988) 242: 99-102. 310 Ganitkevich, J Physiol (1990) 426: 19-42. 311 McDonald, Pflugers Arch (1986) 406: 437-48. 312 Cota, J Gen Physiol (1989) 94: 937-51. 313 Alvarez, J Gen Physiol (1992) 100: 519-45. 314 Cox, Am J Physiol (1992) 263: H453-63. 315 Aaroson, J Physiol (1988) 405: 57-75. 316 Hess, Nature (1984) 309: 453-6. 317 Almers, J Physiol (1984) 353: 585-608. 318 Campbell, J Physiol (1988) 403: 267-86. 270
271
_ry_42_1
en_t
II--_e_n_try_4_2
319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340
341 342 343
344
345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364
365 366 367
_
Ganitkevich, J Physiol (1988) 399: 419-36. Coronado, J Gen Physiol (1986) 87: 933-53. McDonald, Annu Rev Physiol (1982) 44: 425-34. Hirano, Am J Physiol (1989) 256: H1478-92. Sims, Am J Physiol (1992) 262: G859-67. Welling, Am J Physiol (1992) 262: L351-9. Cognard, Proc Natl Acad Sci USA (1986) 83: 517-21. Amedee, J Physiol (1987) 392: 253-72. Ganitkevich, J Physiol (1987) 392: 431-49. Katzka, J Physiol (1989) 413: 175-97. Kass, J Mol Cardiol (1982) 14: 615-82. Irisawa, Jpn J Physiol (1984) 34: 375-88. Nakazawa, Pflugers Arch (1988) 411: 289-95. Klockner, Pflugers Arch (1985) 405: 340-8. Mejia-Alvarez, J Gen Physiol (1991) 97: 393-412. Eckert, J Physiol (1981) 314: 265-80. Fedida, J Physiol (1988) 405: 439-60. Bechem, Pflugers Arch (1985) 404: 10-20. Kalman, J Gen Physiol (1988) 92: 531-48. Hadley, J Physiol (1991) 444: 257-68. Mazzanti, Biophys J (1990) 58: 1059-65. Sherman, Biophys J (1990) 56: 1007-16. Hirano, Biophys J (1989) 56: 1007-16. Bonvallet, Pflugers Arch (1987) 408: 540-2. Zhang, Nature (1994) 372: 97-100. Klockner, Pflugers Arch (1988) 412: R82. Obejero-Paz, J Gen Physiol (1991) 98: 1127-40. Armstrong, Proc Natl Acad Sci USA (1987) 84: 2518-22. Eckert, Prog Biophys Mol BioI (1984) 44: 215-67. Belles, Pfiugers Arch (1988) 411: 353-60. Hescheler, Pflugers Arch (1988) 412: 248-52. Belles, Pflugers Arch (1988) 412: 554-6. Rich, Am J Physiol (1993) 264: C745-54. Nelson, J Physiol (1989) 412: 65-91. Huang, Biophys J (1989) 56: 1023-8. Elhamdani, Pflugers Arch-Bur J Physiol (1995) 430: 401-9. Chad, J Physiol (1986) 378: 31-51. Schouten, Pflugers Arch (1989) 415: 1-11. Beaumont, J BioI Chem (1994) 269: 20281-8. Hryshko, Am J Physiol (1990) 259: H951-61. Charnet, J Mol Cell Cardiol (1991) 23: 343-56. Bean, J Gen Physiol (1989) 94: 65-93. Hadley, J Gen Physiol (1991) 98: 265-85. Kostyuk, Neuroscience (1981) 6: 2431-7. Almers, J Physiol (1984) 353: 565-85. New, Pflugers Arch (1972) 334: 1-23. Ganitkevich, J Physiol (1991) 399: 419-36. Yue, J Gen Physiol (1990) 95: 911-39. Hess, Can J Physiol Pharmacol (1988) 66: 1218-23.
III
_1-368
369
370 371 372 373
374 375
376 377
378 379
380 381 382
383 384 385
386 387 388 389 390
391 392 393
394 395
396 397 398 399 400
401 402 403
404 405
406 407 408 409
410 411 412 413 414 415 416
II
e_ntry_42_1
Isenberg, TPhysiol (1985) 358: 60P. Rosenberg, Science (1986) 231: 1564-6. Hess, TGen Physiol (1986) 88: 293-319. Lansman, TGen Physiol (1986) 88: 321-47. Pietrobon, Nature (1988) 333: 373-6. McCleskey, Proc Natl Acad Sci USA (1985) 82: 7149-53. McCleskey, TGen Physiol (1985) 86: 22a. Coronado, Biophys T(1987) 51: 497-502. Lux, TPhysiol (1990) 430: 159-88. Lee, Nature (1982) 297: 498-501. Matsuda, TPhysiol (1984) 357: 553-73. Lee, TPhysiol (1984) 354: 253-72. Pelzer, Rev Physiol Biochem Pharmacol (1990) 114: 107-207. Yang, Nature (1993) 366: 158-61. Scamps, PflugeIs Arch-Eur TPhysiol (1993) 422: 465-71. Nelson, Am TPhysiol (1990) 259: C3-18. Nelson, Nature (1988) 336: 382-5. Quayle, Am TPhysiol (1993) 264: H470-8. Kamishima, Am TPhysiol (1992) 263: C69-77. Tomasic, Am TPhysiol (1992) 263: C106-13. Hess, Nature (1984) 311: 538-44. Nowycky, PIOC Natl Acad Sci USA (1985) 82: 2178-82. Balke, TPhysiol (1992) 456: 247-65,. Chen, TPhysiol (1987) 390: 80P. Lacerda, TGen Physiol (1989) 93: 1243-73. Kunze, TMembI BioI (1990) 118: 171-8. Bonev, Pflugers Arch-Etu TPhysiol (1992) 420: 219-22. Worley, Am TPhysiol (1990) 259: L468-80. Artalejo, TPhysiol (1991) 432.: 681-707. Kuo, Neuron (1992) 9: 515-26. Delcour, Science (1993) 2.59: 980-84. Delbono, TPhysiol (1993) 460: 173-83. Nathan, TGen Physiol (1988) 91: 549-72. Fox, TPhysiol (1987) 394: 149-72. Palade, Pflugers Arch (1985) 405: 91-101. Herrington, TNeurophysiol (1992) 68: 213-32. Bean, TGen Physiol (1985) 86: 1-30. Docherty, TPhysiol (1988) 398: 33-47. Campbell, TPhysiol (1988) 403: 239-66. Anderson, TGen Physiol (1971) 58: 322-39. Bean, eirc Res (1986) 59: 229-35. White, Science (1988) 239: 778-80. Agus, Am TPhysiol (1989) 256: C452-5. Bean, Proc Natl Acad Sci USA (1984) 81: 6388-92. Bean, Am THypertens (1991) 4: 4068-115. Regan, Neuron (1991) 6: 269-80. Yatani, eirc Res (1985) 56: 868-75. Wu, TPharmacol Exp Ther (1992) 260: 966-73. Striessnig, Proc Natl Acad Sci USA (1990) 87: 9108-112.
1'--_e_n_try_4_2
417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441
442 443
444 445 446 447 448 449
450 451 452 453 454 455 456 457 458 459
460 461 462 463 464 465
_
Hescheler, Pflugers Arch (1982) 393: 287-91. Lee, Nature (1983) 302: 790-4. Caretta, TMembr Biol (1991) 122: 203-13. Glossmann, Rev Physiol Biochem Pharmacol (1990) 114: 1-105. Godfraind, Pharmacol Rev (1986) 38: 321-416. Kokubun, Mol Pharmacol (1986) 30: 571-84. Porzig, Mol Pharmacol (1988) 34: 172-9. Schreibmayer, Br TPharmacol (1992) 106: 151-6. Tytgat, Naunyn-Schmiedeberg's Arch Pharmacol (1988) 337: 690-2. Barbour, Nature (1989) 342: 918-20. Rampe, TPharmacol Bxp Ther (1989) 248: 164-9. Qar, Mol Pharmacol (1988) 33: 363-9. Rampe, T Cardiovasc Pharmacol (1989) 13: 547-56. Stanton, T Cardiovasc Pharmacol (1988) 11: 387-95. Boer, Bur TPharmacol (1989) 172: 131-46. Rampe, Trends Pharmacol Sci (1990) 11: 112-15. Satoh, Tpn Heart T (1982) 23: 57-9. Yatani, Tpn TPhysiol (1983) 33: 403-15. Prod'hom, Nature (1987) 329: 243-6. Tytgat, T Gen Physiol (1990) 96: 973-90. Brown, Circ Res (1987) 61 (Suppl I): 6-9. King, TBiol Chem (1988) 263: 2238-44. Aosaki, Pflugers Arch (1989) 414: 150-6. Uchitel, Proc Natl Acad Sci USA (1992) 89: 3330-3. Galizzi, Proc Natl Acad Sci USA (1986) 83: 7513-17. Enyeart, Am TPhysiol (1987) 253: C162-70. Flaim, Proc Natl Acad Sci USA (1985) 82: 1237-41. Gould, Proc Natl Acad Sci USA (1983) 80: 5122-25. Qar, Bur TPharmacal (1987) 141: 261-8. Lang, , Physial (1991) 433: 1-24. Coulombe, , Mol Cell Cardial (1990) 22: 921-32. Chapman, Pflugers Arch-Bur' Physial (1993) 422: 325-31. Twombly, , Pharmacal Bxp Ther (1988) 246: 189-95. De Weille, Prac Natl Acad Sci USA (1991) 88: 2437-40. Merritt, Biachem' (1990) 271: 515-22. Netzer, Bur' Pharmacal (1993) 238: 209-16. Coulter, Ann Neural (1989) 25: 582-93. Enyeart, Mal Pharmacol (1992) 42: 364-72. Shearman, Pharmacal Rev (1989) 41: 211-37. Hille, Trends Neurasci (1994) 17: 531-6. Dunlap, Nature (1997) 385: 394-6. Shapiro, Neuron (1994) 12: 1319-29. Beech, Neuron (1992) 8: 97-106. Bernheim, Proc Natl Acad Sci USA (1992) 89: 9544-48. Hescheler, BMBO T (1988) 7: 619-24. Kojima, FBBS Lett (1986) 204: 347-51. Cohen, Proc Natl Acad Sci USA (1988) 85: 2412-16. Pang, Prac Natl Acad Sci USA (1990) 87: 623-7. Kostyuk, Pflugers Archiv-Bur TPhysiol (1992) 420: 146-52.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _e_n_try_4_----..J1 2
466 467 468 469 470
471 472 473 474 475 476 477 478 479 480
481 482 483 484 485 486 487 488 489 490
491 492 493 494 495 496 497 498 499
500 501 502 503 504
505 506 507
508 509
510
511
II
Zhu, Neuron (1994) 13: 657-69. Nah, Prc Natl Acad Sci USA (1995) 92: 8739-43. Wang, Am J Physiol (1993) 264: C1473-9. Yatani, Science (1987) 238: 1288-92. Imoto, Am J Physiol (1988) 25: H722-8. Hamilton, J Biol Chem (1991) 266: 19528-35. Cachelin, Nature (1983) 304: 462-4. Elmslie, Neuron (1990) 5: 75-80. Kasai, Proc Natl Acad Sci USA (1991) 88: 8855-9. Lopez, Neuron (1991) 7: 1061-8. Ikeda, J Physiol (1991) 439: 181-214. Dolphin, Eur J Neurosci (1990) 2: 104-8. Ewald, Proc Natl Acad Sci USA (1988) 85: 3633-7. Hescheler, Nature (1987) 325: 445--7. Harris-Warrick, Neuron (1988) 1: 27-32. Taussig, Neuron (1992) 8: 799-809. VanDongen, Science (1988) 242: 1433-7. Mcenery, J Biol Chem (1994) 269: 5-8. Sweeney, FEBS Lett (1992) 310: 66--70. Herlitze, Nature (1996) 380: 258-62. Ikeda, Nature (1996) 380: 255-8. De Waard, Nature (1997) 385: 446--50. De Waard, J Biol Chem (1995) 270: 12056-64. De Waard, FEBS Lett (1996) 380: 272-6. Zamponi, Nature (1997) 385: 442-6. Mogul, Neuron (1993) 10: 327-34. Isenberg, Circ Res (1984) 55: 309-25. Cerbai, Am J Physiol (1988) 255: H872-8. Belardinelli, J Physio (1988) 405: 615-33. Alvarez, Pflugers Arch (1990) 416: 189-95. Gupta, Circ Res (1993) 72: 65-74. Callewaert, Science (1989) 243: 66(~-6. Homaidan, Proc Natl Acad Sci US.A (1991) 88: 8744-8. Palazzi, J Neurochem (1991) 56: 739-47. Schmidt, J Biol Chem (1991) 266: 18025-33. Kalkbrenner, EMBO J (1995) 14: 4728-37. Mackie, Proc Natl Acad Sci USA (1992) 89: 3825-9. Shimada, J Gen Physiol (1992) 100: 27-44. Zeilhofer, Neuron (1993) 10: 879-87. Dewaard, Neuron (1992) 9: 497-50.3. Leslie, J Pharmacol Exp Ther (1983) 225: 571-5. Mullikinkilpatrick, Eur J Pharmacol Environ Toxicol Pharmacol Section (1994) 270: 17-25. Hawthorn, J Aliment Pharmacol (1988) 2: 101-18. Boland, J Neurosci (1993) 13: 516-33. Rosenthal, In Handbook of Experirnental Pharmacology: GTPases in Biology (eds B.F. Dickey and L. Birnbaumer) (1993) Springer, Berlin, pp.447-69. Gollasch, Proc Natl Acad Sci USA (1993) 90: 6265-9.
1__e_n_try_4_2
512 513 514 515 516 517 518 519
520 521 522 523 524 525 526 527 528 529
530 531 532
533 534
535 536 537
538 539
540 541 542
543 544
545 546 547
548 549
550 551 552 553
554 555
556 557
558 559
560
----"_
Scott, Neuroscience (1990) 38: 285-94. Kobrinsky, Neuroscience (1994) 58: 539-52. Bourinet, Proc Natl Acad Sci USA (1996) 93: 1486-91. Ohori, Neuroscience (1993) 54: 1043-50. Hillyard, Neuron (1992) 9: 69-77. Suman-Chauhan, Bur TPharmacal (1993) 244: 293-301. Gee, TBiol Chem (1996) 271: 5768-76. Dolphin, Annu Rev Physiol (1990) 52: 243-55. Trautwein, Annu Rev Physiol (1990) 52: 257-74. Schultz, Annu Rev Physiol (1990) 52: 275-92. Ewald, Neuron (1989) 2: 1185-93. Menon-Johansson, Pflugers Arch-Bur TPhysiol (1993) 425: 335-43. MacFadzean, Neuron (1989) 3: 177-82. Kleuss, Nature (1991) 353: 43-8. Campbell, TPhysiol (1993) 470: 1-11. Campbell, TPhysiol (1995) 485: 365-72. Allen, Circ Res (1988) 62: 524-34. Le Grand, Circ Res (1991) 69: 292-300. Ohya, Circ Res (1991) 68: 763-71. Ono, Nature (1989) 340: 721-4. Ohno, TPhysiol (1991) 436: 195-217. Ohmura, Br J Pharmacol (1990) 100: 27-30. Nakajima, Pflugers Arch-Bur TPhysiol (1991) 419: 644-50. Nakazawa, Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346: 515-22. Goto, Proc Natl Acad Sci USA (1989) 86: 3915-18. Inoue, TPhysiol (1990) 423: 171-91. Mery, Nature (1990) 345: 158-61. Hescheler, Pflugers Arch (1987) 410: 23-9. Muramatsu, Bur TPharmacal (1987) 138: 269-72. Levi, TPharmacal Exp Ther (1988) 246: 377-83. Millar, Am TPhysiol (1991) 261: HI727-33. Hartzell, Nature (1991) 351: 573-6. Wang, Bur TPharmacol (1990) 183: 1242-43. Wang, FBBS Lett (1991) 282: 331-4. Rampe, Am TPhysiol (1991) 261: HI945-50. Ouadid, Mol Pharmacol (1992) 41: 346-51. Worley, Am TPhysiol (1991) 261: HI951-60. Markwardt, Pflugers Arch (1988) 412: 668-70. Markwardt, Pflugers Arch (1990) 415: 547-53. Kleuss, Science (1993) 259: 832-4. Zhang, Neuron (1996) 17: 991-1003. Patil, Biophys T(1996) 71: 2509-21. Currie, Neuron (1996) 16: 1027-36. Spedding, Cardiovasc Drug Ther (1992) 6: 35-9. Curtis, Biochemistry (1986) 25: 3077-83. Schwartz, Nature (1985) 314: 747-51. Knaus, TBiol Chem (1990) 265: 11156-66. Olivera, Science (1990) 249: 257-63. Skeen, Mol Pharmacal (1993) 44: 443-50.
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e_n_t _ry_4_2_1
Villalobos, FASEB T(1992) 6: 2742--7. Alvarez, FASEB T(1992) 6: 786-92. 563 Kokubun, Proc Natl Acad Sci USA (1984) 81: 4824-27. 564 Franckowiak, Eur TPharmacol (1985) 114: 223-6. 565 Kass, Circ Res (1987) 61 (Suppl I): 1-1-1-5. 566 Zheng, Mol Pharmacol (1991) 40: 734-41. 567 Rampe, Mol Pharmacol (1992) 41: 599-602. 568 Qar, FEBS Lett (1986) 202: 331-6. 569 Hamilton, Science (1985) 229: 182--5. 570 Lacerda, Biophys T(1986) 49: 174a. 571 Rojas, TBiol Chem (1990) 265: 21207-15. 572 Burns, Ann NY Acad Sci (1991) 635: 450-1. 573 Pollard, Biophys T(1992) 62: 15-18. 574 Bean, Annu Rev Physiol (1989) 51: 367-84. 575 Miller, TBiol Chem (1992) 267: 1403-6. 576 Sheng, Curr Opin Neurobiol (1996) 6: 602-8. 577 Dunlap, Trends Neurosci (1995) 18: 89-98. 578 Zucker, Neuron (1996) 17: 1049-5S. 579 Catterall, Ann NY Acad Sci (1993) 681: 342-55. 580 Miller, Trends Neurosci (1997) 20: 189-92. 581 Hosey, TMembr Biol (1988) 104: 81-105. 582 Hosey, Ann NY Acad Sci (1989) 560: 27-38. 583 Rosenthal, FASEB T (1988) 2: 2784--90. 584 Rosenthal, Adv Second Messenger Phosphoprotein Res (1990) 24: 89-94. 585 Clapham, Curr Biol (1996) 6: 814-16. 586 Hartzell, Trends Pharmacol Sci (1992) 13: 380-5. 587 McCleskey, Curr Opin Neurobiol (1994) 4: 304-12. 588 Sather, Curr Opin Neurobiol (1994) 4: 313-23. 589 Catterall, TBiol Chem (1988) 263: l3533-8. 590 Vehara, TCardiovasc Pharmacol (1992) 20: 4048-415. 591 Yawo, Nature (1993) 365: 256-8. 592 Holz, Nature (1986) 319: 670-2. 593 Lipscombe, Proc Natl Acad Sci USA (1988) 85: 2398-402. 594 Gross, Proc Natl Acad Sci USA (1990) 87: 7025-9. 595 Seward, Proc R Soc Lond [Biol] (1991) 244: 129-35. 596 Dolphin, TPhysiol (1987) 386: 1-17. 597 Cottrell, TPhysiol (1987) 392: 32P. 598 Wanke, Proc Natl Acad Sci USA (1987) 84: 4313-17. 599 Caulfield, Br TPharmacol (1991) 104: 39-44. 600 Wanke, Eur TNeurosci (1994) 6: 381-91. 601 Mintz, Neuron (1993) 10: 889-98. 602 Dolphin, TPhysiol (1986) 373: 47-61. 603 Madison, Biophys T(1987) 51: 30a. 604 Ikeda, TPhysiol (1992) 458: 339-59. 605 Luini, TNeurosci (1986) 6: 3128-32. 606 Lewis, Proc Natl Acad Sci USA (1986) 83: 9035-9. 607 Shapiro, Neuron (1993) 10: 11-20. 608 Walker, TNeurosci (1988) 8: 2438-46. 609 Randall, Soc Neurosci Abs (1993) 1.9: 1478. 561
562
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1'--_e_n_t_ry_4_2
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620 621 622 623 624 625
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Galizzi, TBioI Chem (1987) 262: 6947-50. Striessnig, Naunyn Schmiedebergs Arch Physiol (1988) 337: 331-40. Barhanin, Biochem Biophys Res Commun (1988) 150: 1051-62. Ferry, FEBS Lett (1984) 169: 112-18. Striessnig, Eur TBiochem (1986) 161: 603-9. Striessnig, Biochem T(1988) 253: 37-46. Ferry, Naunyn Schmiedebergs Arch Phrmacol (1984) 325: 186-9. Glossmann, Naunyn Schmeidebergs Arch Physiol (1988) 338: R20. Goll, FEBS Lett (1984) 176: 371-7. Ruth, Eur TBiochem (1985) 150: 313-22. Striessnig, FEBS Lett (1987) 212: 247-53. Striessnig, Biochem T (1988) 253: 37-46. Glossmann, FEBS Lett (1983) 160: 226-32. Galizzi, T BioI Chem (1986) 261: 1393-7. Schoemaker, Eur TPharmacol (1985) 111: 273-7. King, T BioI Chem (1989) 264: 5633-41.
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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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 30 31
Fisher, Genomics (1995) 29: 598-606. Kieferle, Proc Natl Acad Sci USA (1994) 91: 6943-7. Steinmeyer, T Bioi Chem (1995) 270: 31172-7. Miller, Ann NY Acad Sci (1980) 341: 534-51. Woodbury, Biophys T (1990) 58: 833-9. Goldberg, TMembrane Bioi (1991) 124: 199-206. Oneill, Biochim Biophys Acta (1991) 1129: 131-4. Middleton, Biochemistry (1994) 33: 13189-98. Jentsch, Nature (1990) 348: 510-14. Steinmeyer, Nature (1991) 354: 301-4. van Slegtenhorst, Hum Mol Genet (1994) 3: 547-52. Fujita, Nucleic Acids Res (1994) 22: 1637-9. Huang, TMol Bioi (1994) 242: 595-8. Wischmeyer, Neuromusc Disord (1993) 3: 267-74. Bardouille, FEBS Lett (1996) 396: 177-80. Klocke, T Bioi Chem (1994) 269: 27635-9. Uchida, T Bioi Chem (1993) 268: 3821-4. Cid, Hum Mol Genet (1995) 4: 407-13. Thiemann, Nature (1992) 356: 57-60. Kawasaki, Neuron (1994) 12: 597-604. Jentsch, TPhysiol (1995) 482P: SI9-25. Adachi, TBioi Chem (1994) 269: 17677-83. Steinmeyer, Nature (1991) 354: 304-8. Gronemeier, TBioi Chem (1994) 269: 5963-7. Steinmeyer, EMBO T (1994) 13: 737-43. Thomsen, Arch Psychiatr Nervenkrankh (1876) 6: 702-18. Pusch, Neuron (1995) 15: 1455-63. George, Nature Genet (1993) 3: 305-10. Koch, Science (1992) 257: 797-800. Lorenz, Hum Mol Genet (1994) 3: 941-6. Meyerkleine, Hum Mol Genet (1994) 3: 1015-16.
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Voltage-gated CI- channels
32 33 34
35 36
37
38 39
40
41 42 43
44 45
46 47 48
49 50
51
52 53 54
55 56 57 58 59 60
61 62 63 64
65 66
67 68 69
70
71 72 73
74 75
76 77 78
79 80
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entry 43
Heine, Hum Mol Genet (1994) 3: 1123-8. Fahlke, Neuron (1995) 15: 463-72. Bryant, TPhysiol (1971) 219: 367-83. Bryant, Pfluger's Arch (1991) 417: 605-10. Grunder, Nature (1992) 360: 759-62. Kunzelmann, Pfluger's Arch (1989) 415: 172-82. Ackerman, TGen Physiol (1994) 103: 153-79. Tseng, Am TPhysiol (1992) 262: C1056-68. Sorota, Circ Res (1992) 70: 679-87. Lewis, TGen Physiol (1993) 101: 801-26. Doroshenko, TPhysiol (1992) 449: 197-218. Pook, Hum Mol Genet (1993) 2: 2129-34. Fisher, Hum Molec Genet (1994) 3: 2053-9. Greene, Mol Gen Genet (1993) 241: 542-53. Gurnett, TBiol Chem (1995) 270: 9035-8. Jentsch, Ann NY Acad Sci (1993) 707: 285-93. Jockusch, Mouse Genome (1990) 86: 216. Abdalla, Am THum Genet (1992) 51: 579-84. Huang, TMol Biol (1994) 242: 595-8. Armstrong, T Gen Physiol (1977) 70:: 567-90. Hoshi, Science (1990) 250: 533-8. Zagotta, Science (1990) 250: 568-71. Astill, Biochim Biophys Acta (1996,1 1280: 178-86. Fahlke, TPhysiol (1995) 484: 355-68. Ackerman, T Gen Physiol (1994) 103: 153-79. Bauer, Proc Natl Acad Sci USA (1991) 88: 11052-6. Pusch, Nature (1995) 373: 527-31. Palade, T Gen Physiol (1977) 69: 325-42. Rychkov, TPhysiol (1996) 497.2: 423-35. Pusch, Biophys T (1994) 66: 149-52. Miller, Philos Trans R Soc London Ser B (1982) 299: 401-11. Fahlke, Biophys (1996) 71: 695-706. Ghadiri, Nature (1994) 369: 301-4. Isozaki, Kidney Int (1991) 40: Sl13-·18. Hanke, T Gen Physiol (1983) 82: 25-·45. Miller, Proc Natl Acad Sci USA (1984) 81: 2772-5. Richard, Science (1990) 247: 1208-10. Vaughan, Pfluger's Arch (1991) 419: 522-8. White, TGen Physiol (1981) 78: 1-19. Palade, T Gen Physiol (1977) 69: 879-96. Kwiecinski, Muscle Nerve (1988) 11: 576-81. Bretag, Physiol Rev (1987) 67: 618-724. Schlatter, Pfluger's Arch (1983) 396: 210-17. Wittner, Pfluger's Arch (1986) 407: 611-14. Wangemann, Pfluger's Arch (1986) 407, Supplement 2: S128-41. Gray, Am TPhysiol (1993) 264: C591-602. Cabantchik, Am TPhysiol (1992) 262: C803-27. Tricarico, Pfluger's Arch (1991) 418: 500-3. Moorman, TBioI Chem (1992) 267: 14551-4.
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.
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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:
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REFERENCES 1 2
3 4
5 6 7
8 9
10 11
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13 14
15 16
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Connor, T Physiol (1971) 213: 21-30. Connor, T Physiol (1971) 213: 1-19. Sheng, Nature (1993) 365: 72-5. Sheng, Neuron (1992) 9: 271-84. Villarroel, TNeurosci (1996) 16: 2522-31. Ser6dio, T Neurophysiol (1996) 75: 2174-79. Ser6dio, T Neurophysiol (1994) 72: 1516-29. Boyett, Cardiovasc Res (1996) 32: 455-81. Tseng, Pfliigers Arch (1990) 415: 414-24. Hagiwara, T Physiol (1961) 155: 470-89. Neher, T Gen Physiol (1971) 58: 36-53. Rettig, EMBO T (1992) 11: 2473-86. Covarrubias, Neuron (1991) 7: 763-73. Rudy, Neuroscience (1988) 25: 729-49. Solc, T Gen Physiol (1990) 96: 135-65. Rogowski, Trends Neurosci (1985) 8: 214-19. Mlinar, T Gen Physiol (1993) 102: 239-55. Roy, T Neurochem (1995) 64: 1576-84. Nuss, T Physiol (1994) 479: 265-79. Simurda, Pfliigers Arch (1989) 415: 244-6. Apkon, Proc Natl Acad Sci USA (1988) 85: 8756-60. Dukes, J Physiol (1991) 435: 395-420. Clark, T Physiol (1988) 405: 147-68. Murrow, TPhysiol (1994) 480: 247-59. Takeda, Pfliigers Arch (1987) 410: 385-93. Watsky, TMembr Bioi (1992) 128: 123-32. Yamashita, T Physiol (1992) 450: 143-62. Segawa, TPhysiol (1994) 476: 41-53. Nilius, TPhysiol (1992) 445: 537-48. Vogalis, Pfliigers Arch-Eur TPhysiol (1994) 429: 160-4. Kamouchi, TPharmacol Exp Ther (1991) 259: 1396-403. Noack, Pfliigers Arch (1990) 416: 467-9. Hisada, Pfliigers Arch (1990) 416: 151-61. Watkins, Pflugers Arch-Eur T Physiol (1994) 428: 209-216. Yoshimura, TPhysiol (1996) 494: 1-16. Stefani, Synapse (1995) 20: 131-136. Ximoy, TPhysiol (1995) 486: 15-31. Halliwell, TPhysiol (1995) 487: 421-440. Moak, Am TPhysiol (1993) 265: H1596-602. Yoshimura, TNeurophysiol (1996) 76: 2508-2521. Zhou, TNeurophysiol (1996) 76: 668-682.
_L..-
,
en_t_ry_4_4_1
Wang, J Physiol (1996) 490: 63-77. Bardoni, Neurosci Lett (1996) 210: 95-98. 44 Pearce, Neuroscience (1994) 63: 1041-56. 45 Numann, J Physiol (1987) 393: 331-53. 46 Ficker, J Physiol (1992) 445: 431-55. 47 Miyazaki, Brain Res (1996) 743: 1-10. 48 Carrier, Brain Res (1995) 701: 1-12. 49 Turrigiano, J Neurosci (1995) 15: 3640-52. 50 Huang, Proc Natl Acad Sci USA (1993) 90: 11806-10. 51 Lipton, J Physiol (1987) 385: 361-91. 52 Robbins, Pfliigers Arch (1990) 416: 130-7. 53 Thorn, Pfliigers Arch-Eur J Physiol (1994) 428: 288-95. 54 Aguayo, J Physiol (1988) 405: 397-419. 55 Kehl, J Physiol (1989) 411: 457-68. 56 Oxford, J Physiol (1989) 410: 587-612. 57 Schneggenburger, Pfliigers Arch-Eur J Physiol (1992) 420: 302-12. 58 Davidson, Can J Physiol Pharmacol (1995) 73: 36-42. 59 Mei, J Physiol (1995) 489: 431-442. 60 Kusano, Proc Natl Acad Sci USA (1995) 92: 3918-3922. 61 Chen, J Neurophysiol (1996) 75: 820-831. 62 Corotto, J Exp Biol (1996) 199: 1115-1126. 63 Griguer, J Neurophysiol (1996) 75: 508-513. 64 Xu, Am J Physiol (1996) 40: H2190-H2196. 65 Guo, Cardiovasc Res (1997) 33: 139-146. 66 Wu, J Neurosci (1994) 14: 1677-87. 67 Klee, J Neurophysiol (1995) 74: 1982-1995. 68 Dourado, J Neurosci (1994) 14: 3156-65. 69 Sanchezchapula, Am J Physiol (1994) 266: HI184-94. 70 Guo, Cardiovasc Res (1996) 32: 524-533. 71 Teneick, FASEB J (1992) 6: 2568-80. 72 Potreau, Cardiovasc Res (1995) 30: 440-448. 73 Segal, J Neurosci (1984) 4: 604-9. 74 Storm, J Physiol (1987) 385: 733-59. 75 Kaang, Proc Natl Acad Sci USA (1992) 89: 1133-7. 76 Shimahara, Neurosci Lett (1981) 24: 139-42. 77 Jan, Proc Roy Soc Lond Ser B (1977) 198: 87-108. 78 Crow, Trends Neurosci (1988) 13: 415-19. 79 Nelson, Science (1990) 247: 1479-83. 80 Mahowald, Nature (1991) 354: 515-18. 81 Adams, Adv Neurol (1986) 44: 137-70. 82 Irisawa, Experientia (1987) 43: 1131·-5. 83 Irisawa, Physiol Rev (1993) 73: 197-·227. 84 Hiraoka, JAPANESE HEART JOURNAL (1996) 37: 693-707. 85 Amos, J Physiol (1996) 491: 31-50. 86 Walsh, Neuroscience (1995) 69: 915-929. 87 Forsythe, J Physiol (1992) 451: 553-83. 88 Stansfeld, Neuroscience (1987) 23: 893-902. 89 Solc, Science (1987) 236: 1094-8. 90 Matteson, Biophys J (1988) 53: 641-5.
42
43
1L...-_e_n_try_4_4
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136
---'_
Kawa, T Physiol (1987) 385: 189-205. Taylor, T Physiol (1987) 388: 437-47. Rudy, Neuron (1988) 1: 649-58. Kato, Am TPhysiol (1991) 261: C218-23. Gotoh, Am TPhysiol (1991) 260: H1737-42. Mlinar, Mol Pharmacol (1994) 46: 743-9. Lefevre, TPhysiol (1991) 432: 65-80. Su, Eur TPharmacol (1994) 254: 141-50. Karhu, Eur T Pharmacol (1995) 279: 7-13. Doggrell, Clin Exp Pharmacol Physiol (1994) 21: 833-43. Martin, Biochem T(1994) 304: 51-6. Villarroel, FEBS Lett (1993) 335: 184-8. Damron, TBiol Chem (1993) 268: 27335-44. Poling, Molec Pharmacol (1995) 47: 381-390. Wisgirda, Pfliigers Arch-Eur TPhysiol (1993) 423: 418-26. Carignani, TMembr Biol (1991) 122: 259-65. Inoue, Am T Physiol (1993) 264: C1434-8. Mayer, T Physiol (1988) 396: 41 7-33. Watkins, Pfliigers Arch-Eur TPhysiol (1994) 428: 209-16. Xu, Am T Phys
