THE ION CHANNEL FactsBook I
Extracellular Ligand-Gated Channels
THE ION CHANNEL FactsBook I
Extracellular Ligand-Gated Channels
Other books in the FactsBook Series:
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 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 Shirley Ayad, Ray Boot-Handford, Martin J. Humphries, Karl E. Kadler and C. Adrian Shuttleworth The Extracellular Matrix FactsBook Robin Hesketh The Oncogene FactsBook Grahame Hardie and Steven Hanks The Protein Kinase Factsbook
THE ION CHANNEL
FactsBook 1 Extracellular Ligand-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, UK with contributions from
William J. Brammar Department of Biochemistry, University of Leicester, UK
Academic Press Harcourt Brace & Company, Publishers LONDON SAN D I E G O NEW YORK BOSTON SYDNEY TOKYO TORONTO
This book is printed on acid-free paper ACADEMIC PRESS LIMITED 24-28 Oval Road LONDON NW1 7DX
United States Edition Published by ACADEMIC PRESS INC. San Diego, CA 92101 Copyright 9 1996 by ACADEMIC PRESS LIMITED
All rights reserved No part of this book may be reproduced in any form by photostat, microfilm, or by any other means, without written permission from the publishers A catalogue record for this book is available from the British Library ISBN 0-12-184450-1
Typeset by Alden Multimedia, Oxford and Northampton Printed and bound in Great Britain by WBC, Bridgend, Mid Glam.
Cumulative tables of contents for Volumes 1 to 4 (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)
VIII XII XIII XV XVII XXXIX
VOLUME I
ELG Key facts (entry 04)
Extracellular ligand-gated receptor-channels - key facts References
3 11
ELG C A T 5-HT3 (entry 05)
Extracellular 5-hydroxytryptamine-gated integral receptor-channels Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
12 12 14 18 19 23 27 31 33
ELG CAT ATP (entry 06)
Extracellular ATP-gated receptor-channels (P2xR) Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
36 36 40 47 49 53 63 70 72
ELG C A T GLU AMPA/KAIN (entry 07)
AMPA / kainate-selective (non-NMDA) glutamate receptor-channels Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
75 75 86 99 105 113 122 129 136
m
Contents
entry 01
(entry 08) N-Methyl-D-aspartate (NMDA)-selective glutamate receptor-channels ELG C A T GLU N M D A
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 09) Nicotinic acetylcholine-gated integral receptor-channels
140 140 146 170 172 191 198 220 226
ELG C A T nAChR
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 10) Inhibitory receptor-channels gated by extracellular gamma-aminobutyric acid
234 234 238 248 256 269 274 284 288
ELG CI GABAA
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 11) Inhibitory receptor-channels gated by extracellular glycine
293 293 299 309 310 321 329 350 361
ELG CI GLY
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
Feedback and access to the Cell-Signalling Network (entry 12) Feedback Guidelines on the types of feedback required The Cell-Signalling Network
m
366 366 371 378 380 388 391 394 397 403 403 404 404
entry 01
Rubrics (entry 13) Entry number rubric Field number rubric
Index
417 417 419 420
Note: A set of supporting appendices (Resources) and a cumulative subject index for Volumes I to IV appears at the end of Volume IV. An on-line glossary of terms marked with the dagger symbol (t) will be accessible from the Cell-Signalling Network 'home page' from mid-1996.
VII
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)
ELG Key facts (entry 04)
ELG CAT nAChR (entry 09)
Extracellular ligand-gated receptorchannels- key facts
Nicotinic acetylcholine-gated integral receptor-channels
ELG CAT 5-HT3 (entry 05)
ELG C1 GABAA (entry 10)
Extracellular 5-hydroxytryptaminegated integral receptor-channels ELG C A T ATP (entry 06)
Extracellular ATP-gated receptorchannels (P2xR) ELG CA T GLUAMPA/KAIN (entry 07)
AMPA / kainate-selective (nonNMDA) glutamate receptor-channels ELG C A T GLU NMDA (entry 08)
Inhibitory receptor-channels gated by extracellular gamma-aminobutyric acid ELG C! GL Y (entry 11)
Inhibitory receptor-channels gated by extracellular glycine Feedback and access to the CellSignalling Network (entry 12)
N-Methyl-D-aspartate (NMDA)selective glutamate receptor-channels
Rubrics (entry 13) Entry and field number rubrics
ILG K e y facts (entry 14)
ILG Ca Ca RyR-Caf (entry 17)
The intracellular ligand-gated channel group- key facts
Caffeine-sensitive Ca2+-release channels (ryanodine receptors,
ILG Ca AA-LTCa [native] (entry 15)
Native Ca ).+ channels gated by the arachidonic acid metabolite leukotriene C4 incorporating general properties of ion channel regulation by arachidonate metabolites
RyRI ILG Ca CSRC [native] (entry 18)
Candidate native intracellularligand-gated Ca)+-store repletion channels
ILG Ca Ca InsP4S [native] (entry 16)
ILG Ca InsP3 (entry 19)
Native Ca )`+ channels sensitive to inositol 1,3,4,5-tetrakisphosphate (InsPa)
Inositol 1,4,5-trisphosphatesensitive Ca~+-release channels (InsP~R)
entry O1
ILG CAT Ca [native] (entry 20) Native calcium-activated nonselective cation channels (NSca) 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
Cumulative contents
ILG CI Ca [native] (entry 25) Native calcium-activated chloride channels (C1ca) 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 CI ABC-CF (entry 23) ATP-binding and phosphorylationdependent C1-channels (CFTR)
ILG K Ca (entry 27) Intracellular calcium-activated K§ channels (Kca)
ILG C1 ABC-MDR/PG (entry 24) Volume-regulated C1--channels (multidrug-resistance P-glycoprotein)
ILG K Na [native] (entry 28) Native intracellular sodium-activated K§ channels (KNa)
INR K Key facts (entry 29) Inwardly-rectifying K* channelskey facts
JUN [connexins] (entry 35) Intercellular gap junction channels formed by connexin proteins
INR K A TP-i [native] (entry 30) Properties of intracellular ATPinhibited K§ channels in native cells INR K G/A Ch [native] (entry 31) Properties of muscarinic-activated K§ channels underlying /KACh in native cells INR K [native] (entry 32) Properties of 'classical' inward rectifyer 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 K/Na I~q [native] (entry 34) Hyperpolarization-activated cation channels underlying the inward currents if,/h, iq
MEC [mechanosensitive] (entry 36) Ion channels activated by mechanical stimuli MIT [mitochondrial] (entry 37) Survey of ioni 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)
IX
Cumulative contents
VOLUME IV
VOLTAGE-GATED CHANNELS
VLG K e y facts (entry 41) Voltage-gated c h a n n e l s - key facts VLG Ca (entry 42) Voltage-gated Ca ~+ channels VLG CI (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
(entry 46)
Vertebrate K § channel subunits related to Drosophila ether-~-go-
go (eag) VLG K Kv-beta
(entry 47)
Beta subunits associated with voltage-gated K+ channels VLG K K v l - S h a k
(entry 48)
Vertebrate K§ channel subunits related to Drosophila Shaker (subfamily 1) incorporating general
features of Kv channel expression in heterologous cells
Resource A
(entry 56)
G protein-linked receptors regulating ion channel activities
(alphabetical listing) Resource B
(entry 57)
'Generalized' electrical effects of endogeneous receptor agonists Resource C
(entry 58)
Compounds and proteins used in ion channel research
II
entry 01
VLG K Kv2-Shab
(entry 49)
Vertebrate K § channel subunits related to Drosophila Shab (subfamily 2) VLG K K v 3 - S h a w
(entry 50)
Vertebrate K§ channel subunits related to Drosophila Shaw (subfamily 3) VLG K Kv4-Shal (entry 51) Vertebrate K § channel subunits related to Drosophila Shal (subfamily 4) VLG K K v x (Kv5.1/Kv6.1)
(entry 52)
Features of the 'non-expressible' cDNAs 1K8 and K13 VLG K M-i [native]
(entry 53)
Properties of native 'muscarinicinhibited' K§ channels underlying IM VLG K m i n K
(entry 54)
'Minimal' subunits forming slowactivating voltage-gated K § channels VLG Na
(entry 55)
Voltage-gated Na § channels
Resource D
(entry 59)
'Diagnostic' tests Resource E
(entry 60)
Ion channel book references (sorted by year of publication) Resource F
(entry 61)
Supplementary ion channel reviews (listed by subject)
1
entry O1
Resource G (entry 62) Reported 'consensus sites' and 'motifs' in primary sequence of ion channels Resource H (entry 63) Listings of cell types Resource I (entry 64) Framework of cell-signalling molecule types (preliminary listing)
Resource J (entry 65) Search criteria & CSN development Resource K (entry 66) Framework for a multidisciplinary glossary Cumulative page index (for volumes
My)
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the coverage/contents can be sent to the e-mail feedback file
[email protected]. (see field 57 of most entries for
further details)
Ui
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 Allan Winter, Angela Baxter, Shelly Hundal, Phil Shelton and Sue Robinson for help with photocopying, 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: Stephen Ashcroft (Oxford), Eric Barnard (London), Dale Benos (Harvard), William Catterall (Washington), K. George Chandy (UC Irvine), Peter Cobbold (Liverpool), David Clapham (Mayo Foundation), Noel Davies (Leicester), Dario DiFrancesco (Milano), Ian Forsythe (Leicester), Sidney Fleischer (Vanderbilt), George Gutman (UC Irvine), Richard Haugland (Molecular Probes, Inc.), Bertil Hille (Washington), Michael Hollmann (G6ttingen), Anthony Hope (Dundee), Benjamin Kaupp (Jtilich), Jeremy Lambert (Dundee), Shigetada Nakanishi (Kyoto), Alan North (Glaxo Institute for Molecular Biology), John Peters (Dundee), Olaf Pongs (Hamburg), David Spray (Yeshiva), Kent Springer (Institute for Scientific Information), Steve Watson (Oxford), Paul Van Houlte (I.R.I.S.)and Steven Wertheim (Harvard). Thanks are also due to the Department of Pathology at the University of Leicester, Harcourt Brace, the Medical Research Council 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 and Anthony Campbell in Cardiff, Tony Buzan in Winton, Dorset and Richard Gregory in Bristol for help and inspiration, and would like to dedicate his contributions to Paula, Rebecca and Katharine for all their love and support over the past four years.
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~
Left: Edward Conley, Right: William Brammer
XII
Edward C. Conley
Entry 02
The Ion Channels 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 Channels 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.
A n e m p h a s i s on properties e m e r g e n t from ion c h a n n e l m o l e c u l a r f u n c t i o n s 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
Introduction
entry 02
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 c o m p o n e n t s - in this case the task t h e n will be to integrate these into what is already known (see also
description of Field number 06: Subtype classifications 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 world-wide: The Cell-Signalling Network (CSN) operating from mid-1996 under the World Wide Webt of the Internett has been designed to disseminate consensus properties of a wide range of molecules involved in cell signal transduction. While it may 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 around the world. On-line support for technical terms (glossary items, indicated by dagger symbols (t) throughout the
I entry 02
text) and reference to explanatory appendices (e.g. on associated signalling components such as G proteint-linked receptorst) are already supported for use with this book. Eventually, benefits could include (for instance) direct 'look-up' of graphical resources for protein structure, in situ and developmental gene expression atlasest, interactive molecular models for structure/function analysis, DNA/protein sequences linked to feature tables, gene mapping resources and other pictorial data. These developments (not all are presently supported)will use interactive electronic media for efficient browsing and maintenance. For a brief account of the Cell-Signalling Network, see Feedback & CSN access, entry 12. For a full specification, see Resource J - Search criteria & CSN development, entry 65.
l O W TO USE THE I O N C H A N N E L F A C T S B O 0 ~ C o m m o n formats within the entries A proposed organizational hierarchy for i n f o r m a t i o n about ion c h a n n e l 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 crossreferencing 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 & CSN access, entry 12). Establishment of this 'field' format has been designed so that every available 'fact' should have its logical 'place'. In the future, this arrangement may help to establish 'universally accepted' or '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.
I n d e p e n d e n t presentation of 'facts' and c o n v e n t i o n s 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 'from beginning to end', but each 'fact' is presented independently under the most pertinent fieldname. Independent citation of 'facts' may sometimes result in some repetition (redundancy)of general
Introduction
entry 02
1
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). 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 'electronically updated' 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 e x a m p l e - see m R N A distribution, under ELG C1 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 approximate runnin~ order based on physiological features such as mode of gatingr, ionic selectivityr, and agonistt 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 use serial entry numbers as 'electronic pointers' to appropriate files. Cross-references are frequently made to an on-line index of glossary items by dagger symbolst 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 is accessible in updated form over the Internett/World Wide Webt with suitable browsingt software (for details, see Feedback & CSN access, entry 12).
C o n t e x t u a l m a r k e r s and styles e m p l o y e d 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. 19- ~ InsP3 receptor-channels), 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 m a x i m u m flexibility of updating (both error-correction and consolidation with new information), cross-referencing and multi-authoring. The CSN specification (see entry 65) includes longer term plans to structure field-based information into convenient data-types which will be indexed by a zz numerical designation. 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
Introduction
entry 02
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 the electronically updated Resource C Compounds & proteins, also available via the 'home page' of the Cell-Signalling Network. Generally, highlighting of related subtopics emergent from the molecular properties ('facts') associated with the ion channel under description are indicated within a field by lettering in bold. All subtopics are cross-referenced by means of a large cumulative subject index (entry 66), which can permit retrieval of information by topic without requiring prior knowledge of ion channel properties. 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 'cloned' and 'native' 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 the electronically updated Resource F - Supplementary ion channel reviews accessible via the CSN (see Feedback & CSN access, entry 12) 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
IPDTMI. Wherever molecular subtype-specific data are quoted {such as the particular behaviour of a ion channel gene familyt member or i s o f o r m t ) a convention of using the underlined trivial or systematic name as a prefix has been a d o p t e d - e.g. mIRKI: ; RCKI: ; Kv3.1: etc.
GUIDE
TO THE PLACEMENT
Criteria for N O M E N C L A T U R E S
CRITERIA
FOR EACH FIELD
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 alphabetical 'sortcode' 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,
XVII
Introduction
entry 02
]
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 UEI) 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 J (in this entry) and for a full description, see Resource J - Search criteria & CSN development, entry 65.
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 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 activatione.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 ( O S M ) - 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, C1, 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 C] GABAA, entry 10 and ELG C] GLY, entry 11).
Field number 03: Channel designation: This field should contain a shorthand designation for the ion channel m o l e c u l e - mostly of the form Xy or Xtv 1 where X denotes the major ionic permeabilitiest (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/x(v), /x,Y or/x-Y where X and Y are 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
entry 02
amino acid primary sequence homologyt, within gene familiest or gene superfamiliest. 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 treet derived by a primary sequence alignment algorithmt (see Resource D - 'Diagnostic' tests, entry 59) 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 similarities that are not easily discernible by pharmacological or electrophysiological criteria a l o n e - see, for example, the entries JUN (connexins), entry 35, and INR K (subunits), entry 33. Note, however, that some nativet 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 ILG Key facts, entry 14, ILG C A T cAMP, entry 21, and ILG C A T cGMP, 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 cDNAst, genest 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 BKc~ variants under ILG K Ca, entry 27). Distinct proteins resulting from transcription, 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 phenotypict 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 n u m b e r 18: Chromosomal location) can encompass all structural and functional variation, while being 'compatible' with efforts directed to identifying phenotypic and pathophysiologicalt roles of individual gene products (e.g. by gene-knockoutt, locus replacementt or disease-linked gene mappingt procedures - see Resource D - 'Diagnostic' tests, entry 59). Subtype classifications based on gene locus control can also incorporate the marked developmental changes which pertain to many ion channel genes (see Field n u m b e r 11: Developmental regulation) and can be implemented when the 'logic' underlying gene expression-controlt 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, H and l, entries 62, 63 and 64).
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Field number 07: Trivial names: This field should list commonly used names for the ion channel (or its conductancet). 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 & CSN access, entry 12). Criteria for E X P R E S S I O N s e c t i o n s 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 developmental loci ('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 'expression surveys', revealing properties of interacting sets of ion channels, pumps, transporters and associated receptors. Such review-type information is of importance when discussing the contribution of individual ion channel molecules to a complex electrophysiological phenotypet and/or overall function of the cell. For further references to 'cell-type-selective' reviews, see Resource H - Listings of cell types, entry 63 accessible via the CSN (see Feedback & CSN access, entry 12).
Problems and opportunities in listing ion channel molecules by cell type: Understanding the roles which individual ionic channels play in the complex electrophysiological phenotypes of nativet cells remains a significant challenge. The overwhelming range of studies covering aspects of ion channel expression in vertebrate cells offers unique problems when compiling 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 conductancest 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
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Introduction
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 difficult 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 definition. 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 nativet ionic currents can be induced or inhibited by agonistst that bind to co-expressed G proteint-coupled receptorst. Thus a difficulty arises in deciding whether channel currents can be unambiguously defined in terms of action at a separately encoded receptor protein. While it is valid to report that an a~onist-sensitive current is expressed in a defined cell type, the factors of crosstalk! 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, entry 56). 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 gatingt mechanism is also part of the assembled protein complex. Cell preparation m e t h o d s 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 phenotypet. The behaviour of channel-mediated ionic currents can be measured in nativet 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 linest are uncoupled from normal processes of cell proliferationf, differentiationt and apoptosist. Acutely dissociated cells from nativet tissue may provide cell-type-specific 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-controllT). The complexity of this task can extend to processes controlling, for example, developmental regulation, co-expressed protein subunit stoichiometries and subcellular localizations.
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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 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, entry 63. 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 & 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 abundancet). Otherwise, this field defines a 'positive control' preparation likely to contain messengerf RNAt encoding the channel. Preparations may express only specific subtypes of the channel and therefore T r eprobes l a(especially t e d PCR probes) may not work. Alternatively, a genomict 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 nativet tissues (or by other experimental interventions). Protein factors in transt or DNA structural motifst in cist which influence transcriptional activationt, transcriptional enhancementt or transcriptional silencingt 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 activationt or patterningt during vertebrate development.
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Introduction
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 screenst. 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 m a y be able to serve as unique gene isolation probes, dependent upon the libraryt size, target abundancet, screening stringencyt and other factors. Field number 13: m R N A 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 Northern hybridizationt, RNAase protectiont analysis, RT-PCRt or in situ expression assays. See also notes on expression at]ases 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$ (wild-type) genes. Phenotypict 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 S T R U C T U R E & F U N C T I O N S section for interpretation of site-directed mutagenesist procedures as well as Resource D - 'Diagnostic' tests, entry 59). 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 & 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 locust 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$ or apicalt face). Field number 17: Transcript size: This field should list the main RNA transcriptt sizes estimated (in numbers of ribonucleotides) by Northernt hybridization analysis. Multiple transcript sizes may indicate (i) alternative processing
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('splicingt') of a primary transcriptt, (ii)the use of alternative transcriptional start sitest, or (iii) the presence of 'pre-spliced' or 'incompletely 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 sequencer (exonict)probes are the most likely to identify all variants, while probes based on intronict sequences (where appropriate)will identify 'pre-splice' variants.
Criteria for S E Q U E N C E A N A L Y S E S s e c t i o ns 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 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 d i a g r a m m a t i c - 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. Notes on interactive linking to gene mapping database resources appear under an option of the Cell-Signalling Network 'home page' (see Feedback & CSN access, entry 12).
Field number 19: Encoding: This field should report open reading framer 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 20: Gene organization: This field should describe known introntand exont junctions within or outside the protein coding sequence, together with positional information on gene expression-controlt 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 21: 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 percenta~ge conservation values under Field number 28: Domain conservation). Isoformst* of a channel protein can exist
entry 02
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-to-individual (as a result of normal sequence polymorphismT 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 molecular constitutions and functional roles in specified cell tyl~es of closely related species. Comparative information on different gene familyl 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 nativet or heterologoust cell membranes). Data derived from nativet preparations generally includes the weight contribution f~om oligosaccharidet chains added durin~ post-translational protein glycosylation. In general, extracellular saccharide! 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 framest 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, ligandt-binding sites, transcription factort-binding sitest etc. N-glycosylationt motifs are sometimes indicated using the shorthand designation N-gly:. 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 hybridizationt procedures. Note that
entry 02
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nativet diploid somatict cells will generally maintain two copies of a given ion channel gene locust, but stablet heterologoust expression procedures may result in multiple locus insertiont. Multiple locus insertion can be quantitated in SouthernT 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 - 'Diagnostic' tests, entry 59, and the section describing heterologous ion channel gene expression under Resource H - Listings of ceil types, entry 63).
Criteria for S T R U C T U R E & F U N C T I O N S 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 mutagenesist 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 & CSN access, entry 12, and in Resource J Search criteria & CSN development, entry 65.
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 (~9 A)structural studies of the nicotinic acetylcholine receptor (nA ChR, see ELG CAT nAChR, entry 09) predict that only one membrane-spanning c~-helixt (likely to be M2, a pore-lining domain) is present per subunit, with the other hydrophobic regions being present as fl-sheetst (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?). 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~ are used.
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Field number 29: Domain functions (predicted): This field should indicate predicted functions of channel molecular subregions based on structural or functional d a t a e.g. regions affecting properties such as voltage-sensitivity, ionic selectivityt, channel gatingt or agonistibinding. Field number 30: Predicted protein topography: This field should include information on the stoichiometrict assemblytpatterns of protein subunits derived from the same or different genes. This field indicates whether channel monomers are likely to form homomultimers~, 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 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 & 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 (generall referred to as 'protein-protein' interactions), but may involve a messenger 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 heterologoust cell expression systems may require accessory subunit expression (e.g. see VLG K Kv-beta, entry 47). Common channelreceptor or G protein-channel interactions are described in principle under Resource A - G protein-linked receptors, Receptor~transducer interactions.
entry 56 and Field n u m b e r
49:
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 & proteins, entry 58 and Resource G - Reported 'Consensus sites' and 'motifs', entry 62 (both updatable via the CSN). 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.
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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 gatingt behaviour such as flickeringt, burstingt, activation latencyt 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 ligandt-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 conductance t, reversal potentialst and steepnesst of rectifyingt (non-ohmict) behaviour. The conventions used for labelling the axes of I-V relations for different charge carrierst are 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 timer, 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 coefficientt and Equilibrium dissociation constantt. 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 particular 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.
Introduction
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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 permeabilitiest under stated conditions by means of permeability ratiot and/or selectivitY,T ratiot parameters. The field may also corn?are measured reversal potentials in response to ionic equilibrium potentials 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. Field number 41: Single-channel data: This field should report exarnples of singlechannel current amplitudes and single-channel conductancest measured under stated conditions. In the absence of authentic single-channel data, estimates of channel conductancest derived from whole-cell recordingt and fluctuation analysist may be listed. Field number 42: Voltage sensitivity: This field should describe the behaviour of the channel in terms of parameters (e.g. P,,pent) which are directly dependent upon applied membrane voltage. A distinction should be made between 'voltage sensitivity' resulting from intrinsic voltage-gatingt phenomena (i.e. applicable to channels possessing integral voltage sensorst) and indirect effects of applied membrane voltage influencing general physical parameters such as electrochemical driving forcer. Criteria for P H A R M A C O L O G Y
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 messengerst 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 conductancet 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 appropriate. Where sites of block have been determined by site-directed mutagenesisT, 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 primaryt sequence (as determined by site-directed mutagenesist procedures)should be cross-referenced to Domain functions, field 29.
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J
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 constantt. Field number 46: Hill coefficient: This field records calculated Hill coefficientst of ligandt-activated processes. The Hill coefficient (n) generally estimates the m i n i m u m 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 radioligandst which have been used to investigate receptor-channel function and that are commercially available. Note that numbers of ligandt-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 timer of the channel in nativet 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, entry 56, accessible via the CSN). Types of 'receptor/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 G-Protein 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 portion of the molecule and thereby increase the open timer, open probability! or conductancet of the integral channel. Anta~onistst should be categorized as competitivet, non-competitiveT: or uncompetitivJ where this has been determined. Field number 51: Receptor antagonists (selective): This field should list agents that selectively bind to the ligandt 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 timer, open probabilityt or conductancet of the integral channel.
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Criteria for I N F O R M A T I O N
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 Internett or available on CD-ROM. A full discussion of the potential scope for integration of these resources with molecular-based entries appears in Resource J - Search criteria & C S N development, entry 65. Brief details are given in Feedback & CSN access, entry 12, in each volume.
Field number 53: D a t a b a s e 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) 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 pit:). 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 m a x i m u m 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 ID' - 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
Introduction
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]
will usually be allocated a new 'gim' number, but the old one will still be available under its UID from the ID 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 (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). See also entry 65. Alternatively or in addition, alignments can be performed according to need by dedicated sequence-manipulation software. Presently available compilations of sequences (e.g. the Entrez CD-ROM set or online equivalent, for example)can perform powerful 'neighbouringt analyses' based on pre-computed alignments of any sequence against the remainder of the existing database. Establishment of homologous alignmentst 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 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 serverst 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 Internett site. For local searches, alignment programs such as BLAST can also be retrieved by anonymous filetransfer protocolt 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 & 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 anonymoust FTPt). NCBIGenBank" can also be accessed over the World Wide Webt (http:// www.ncbi.nlm.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
[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:.
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Nucleic acid sequences from NCBI-GenBank 'I 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 /~/f/n F//~/F/"
[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 X X X X containing the accession number nnnnnn, you would send an e-mail message as follows:
[email protected] G ET X X X X ACCESSION nnnnnn General information on using these file serverst can be obtained using the above emailt 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 loci t using terms defined by a human genome mapping workshop (HGMW)T 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 & CSN access, entry 12). The opportunities for linking to a wide range of genetic information resources are discussed in Resource J - Search criteria & CSN development, entry 65.
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-gatedt channels within an entry describing non-selective cation channels gated by the same ligandt, 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.
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Introduction
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Field number 56: Related sources 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 could be linked to established on-line bibliographic resources appears in entries 12 and 65.
Field number 57: Feedback: Information supplementary to the entries but appearing after the publication deadlines will be accessible from the CMHT servert over the Internett using a World Wide Webt utility from mid-1996 (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 upto-date: Feedback from individual users is an essential part of this process. The Feedback field identifies the appropriate address for e-mailt 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 CellSignalling Network, see Feedback e,) CSN access, entry 12).
Field number ## (inserted at appropriate points): In-press updates: 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. The scope of the Cell-Signalling Network means that users (especially 'non-specialists')can 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-mailt notification where 're-interpretation' is justified (see Feedback & CSN access, entry 12, and Resource L entry 65). 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 'consensus' knowledge on ion channel molecular signalling.
Criteria for R E F E R E N C E S s e c t i o n s 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, entry 60, Resource F - Supplementary ion channel reviews, entry 61 and Resource H - Listings of cell types, entry 63. Plans for 'hyperlinking' to full bibliographic databases within the CSN framework are described in Resource J - Search criteria & CSN development, entry 65.
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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 'home page' of the Cell-Signalling Network from mid-1996 - for further details, see Feedback & CSN access, entry 12 and Resource J - Search criteria & CSN development, entry 65. 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 protein t molecules should form a framework of possible regulatory mechanisms 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 Devel-
opmental regulation, field 11, Protein interactions, field 31, Protein phosphorylation, field 32, Channel modulation, field 44, and Receptor~transducer interactions, field 49. Resource B - "Generalized' electrical effects of endogenous receptor agonists: This resource should present a tabulated summary of genera] patterns of agonisttinduced ionic current fluxes that have been reported across a large number of studies, predominantly in the central nervous system. The table may help to indicate whether receptort agonists tend to act in an excitatoryt or inhibitoryt fashion 'or both'. Resource C - Compounds & 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 an alphabetical 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
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Introduction
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channel 'molecular type' category. This appendix should therefore list these 'supplementary' sources, indexed by topic. Updated 're-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 & 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 (for details, see entry 65). Resource G - Reported 'Consensus sites' and "motifs': Based on extensive analysis of primaryt sequences and determination of substrate specificities for various enzymes~ a number of 'consensus' recognition sequences for post-translational modification 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 a subset of consensust sites from genomic DNA sequences associated with mechanisms of ion channel gene expression-controlt (e.g. in transt protein factors which act at DNA structural motifs_ t in cist, influencing transcriptional activationt, transcriptional enhancementt or transcriptional silencingt of ion channel genes#). Resource H - Listings of cell types: Studies of ion channels within the context of celltype function often reflect 'recruitment' of selected genes from the genomet in a celldevelopmental lineaget. 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 differentiatedt 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 kinasest or tcanS~rivPtiltngfaCtorl~tg na~egrt tg 'sense' 'activated' conditions which resembles a i phenomena commonly observed for ion channels. These modes of protein activationt probably have many features in common, and understanding their
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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 ratidnal 'connection' between activation of receptors, ion channels, enzymes and other effectort proteins, it is hoped that some general principles will emerge on the electrical- and ligandt-control of complex cell phenotypest (such as those affecting the cell cycler, cell proliferationt, cell differentiationt 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.
Resource J - Search criteria & 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. Their implementation outside the context of the CSN will be difficult unless contributing authors and journal editors acknowledge the benefits. If an alternative system is proposed and accepted by field consensus, then the CSN will move to adopt the system in the interests of simplifying search criteria on specific molecules or topics. The central principle of unique embedded identifiers is that they can 'automatically' find articles on topics of interest (in for example weekly 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. 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 'controversial' results, and identification of areas or highly focused topics requiring consolidation/extension of knowledge. The importance of standardized computer software compatible with Internett-mediated
entry 02
communication should be emphasized (see also Feedback & 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 xxyy-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 & CSN access, entry 12). Entry 65 contains a full specification of the CSN.
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 online 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 symbolt.
Cumulative subject index for 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, or table of contents 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 section). For location of information on ion channel molecules by miscellaneous related topics, the cumulative subject index should comprehensively list pertinent functional characteristics, concepts, compounds and proteins including those shown in bold text under the fieldnames, relating the topic to the six-figure index number. 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 J - Search criteria & CSN development, entry 65.
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to this introduction can be sent to the email feedback file
[email protected]. (see field 57 of most entries for
further details)
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For most abbreviations of compound names in use, refer to the Resource C Compounds & 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 0 C a 2+
5-HT 7TD A aa
AHP AP APD AV AVN
Ca2+-free solution 5-hydroxytryptamine; serotonin 7 transmembrane domains amperet amino acidt afterhyperpolarizationt action potentialt action potential durationt atrio-ventricular atrio-ventricular nodet (of heart)
BP bp
large ('big')-conductance calcium-activated K§ channels blood pressure base pairst
C C-terminal C/A or C-A Ca(mech) or Camech Cav cds CF CICR CI(Ca) or C1ca CNG CNS COOH CRC cRNA CTK Cx or Cxn
coulombt carboxylt terminalt (of protein) cell-attached~ (recgrding configuration) mechanosensitiveT Ca 2§ channel voltage-gatedt Ca 2+ channels codingt sequence (used in GenBankt ~"~'entries) cystic fibrosist calcium-induced-calcium-release calcium-activated chloride channel cyclic-nucleotide-gated Ichannels) central nervous system carboxyl groupt calcium release channels complementaryt RNA cytoplasmic tyrosine kinaset (cf. RTK) connexin
Da
DHPR DMD DPSP
daltons putative (consensust)site for dephosphorylationt by a specified enzyme, e.g. Dephos/PP-l: endogenous protein phosphatase-1; Dephos/PP-2A: protein phosphatase-2A dihydropyridine receptor Duchenne muscular dystrophyt depolarizing post-synaptic potentialt
E EAA E-C
potential differencet, inside relative to outside excitatory amino acidt excitation-contractiont
BKCa
Dephos/enzyme
entry 03
Erev
50% effective concentration equilibrium potentialt for K§ ions (analogous nomenclature for other ions) extracellular ligandt-gated (as used in FactsBook sortcode) membrane potentialt European Molecular Biology Laboratoryt electromotive forcet endiPlttet ~otentialt e c'ta ory post-synaptic potentialt endoplasmic reticulum reversal potentialt
F F fS
faradt Faraday's constantt femtosiemens (10-is Siemenst)
G g G/Gmax gb: gj, Gj or G(j)
conductancet conductance (unit- Siemenst, formerly reciprocal ohmst or mhot) peak conductancet designation for GenBank c"~accession numbert gap-junctional conductancet
HGMW HH h.p. HVA
Human Gene Mapping Workshopt after Hodgkint-Huxleyt holding potential high-voltage-activated Ca 2§ channels
I
IPSC IPSP
currentt subscript abbreviation for intracellular peakt currentt inside-outt (patcht, recording configurationt) concentration which gives 50% 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 1,4,5,-trisphosphate-sensitive receptorchannel inhibitoryt post-synaptic currentt inhibitoryt post-synaptic potentialt
JCC
junctional channel complex
k KA or K(A) kb KCa, Kca or K(Ca)
Boltzmann's constantt A-typet I~§ channels kilobases (kbp - kilobase pairs or bp x1031 calcium-activated K* channels
EC5o EK ELG Em EMBL EMF EPP EPSP ER
i
I/Imax I/O or I-O ICso ILG max
InsPlx1or InsPx
-
InsP3R or IP3R
]
entry 03
Kv
equilibrium dissociation constantt kilodaltonst (daltons • equilibrium dissociation constantt for an inhibitor for KATP channels, the ATP concentration (/~M) that produces half-maximal inhibition of channel activity inhibition constantt at zero voltaget inward rectifiert-type K§ channels shorthand designation for mechanosensitive K§ channels voltage-gated K§ channels (generally delayed rectifierst)
LTD LTP LVA
long-term depressiont long-term potentiationt low-voltaget-activated Ca ~§ channels
mAChR MARCKS Mb MEPC MDa MH
muscarinic acetylcholine receptor myristoylatedt, alanine-rich C-kinase substrate megabasest (Mbp - megabase pairs) miniature endplate currents megadaltonst (daltons • malignant hyperthermia relative molecular masst messenger RNAt millivolt (10 -3 V)
KD
kDa KI Ki ATP
/;i{01 KIR or K(IR) K(mech) or Kmech
Mr
mRNA mV N //
nAChR Nav N-gly: NH2 NSA NSC NSC(Ca) nt N-terminal O
O-gly OHC O/O or O-O PCa PCAP PDE
number of functional channels a l s o - Avogadro's numbert Hill coefficientt nicotinic acetylcholine receptor-channel shorthand designation for voltage-gated Na § channels predicted sites for N-linked glycosylationt (e.g. Ngly: 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) subscript abbreviation for extracellular O-linked glycosylationt outer hair cells outside-outt (patcht, recording configurationt) permeabilityt of Ca ~§ ions (analogous nomenclature for other ions, e.g. PK, PNa, PRB etc.) pituitary adenylyl cyclase-activating polypeptide phosphodiesteraset
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pS PSC PSP PSS
protein domain topology model; within the text, use of the abbreviation in square brackets denotes a positional feature illustrated on the model intracellular pHt Putativet (consensust) site for phosphorylationt by a specified enzyme, e.g. Phos/CaM kinase II- multifunctional (Ca2+/calmodulin)-dependent protein kinase II; Phos/CaseKII: casein kinase II; Phos/ GPK: glycogen phosphorylase kinase; Phos/MLCK: myosin light-chain kinase; Phos/PKA: cAMPdependent protein kinase (PKA); Phos/PKC: protein kinase C (PKC); Phos/PKG: cGMPdependent protein kinase; Phos/TyrK: tyrosine kinase (TyrK)subtypes post-injection Protein Identification Resourcet (protein sequence database) designation for Protein Identification Resourcet accession numberst peripheral nervous system t polyadenylation t (site) polyadenylated~ (mRNA).fraction of total cellular RNA channel open probability~ picosiemens (10-12 Siemenst) post-synaptic currentt post-synaptic potentialt porcine stress syndrome
Qlo
coefficientt for a ten-degree change in temperature
R
receptor resistancet (unit- ohmt), reciprocal of conductancet resting potentialt ribosomal RNAt receptor tyrosine kinaset (at plasma membrane, cf. CTK) ryanodine receptor-channel
[PDTM] pHi Phos/enzyme
p.i.
PIR pir: PNS poly(A) poly(A)+ Popen or Po
R r.p. rRNA RTK RyR
SAN SAPs s.c.a. s.c.c.
s.c.p. SCR SD SDS-PAGE SEM
Siemenst (unit of conductancet; reciprocal ohm t or mhot) sino-atrial nodet (of heart) signal-activated phospholipasest single-channel amplitudet single-channel conductancet (symbol, 7) single-channel permeabilityt single-channel recordingt standard deviation sodium dodecyl sulphate-polyacrylamide gel electrophoresis t (i) standard errort of the meanst or (ii)scanning electron microscopyt
entry 03
spike frequency adaptation t indicates the range of amino acids which form the signal peptide of a precursor protein (e.g. Sig: aal26); alternatively, the abbreviation indicates the actual cleavage sitet forming the signal peptidet and mature chain~ from the precursort protein substance P designation for SWISSPROT protein sequence database accession numbert sarcoplasmic reticulumt disulphide bondt; 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.
SFA Sig:
SP sp: SR S-S"
TM Tm
Tm TPeA+ TT
transmembrane melting temperaturet upper limit to the amount of material that carriermediated transport can move across a membrane tetrapentylammonium ions transverse tubulet
VDAC VDCC
voltt voltaget voltage-activated calcium channels; analogous nomenclature for other channels, e.g. VAC1C, VAKC, VANaC, VDAC voltage-dependentt anion channel voltage-dependentt calcium channel
W/C or W-C WCR
whole-cellt (recording configuration) whole-cellt recording
YAC
yeast artificial chromosomet
Y yj or ylj) ~A f~ co-CgTx
unitary (single-channel) conductance single-channel junctional conductancet microamp (10 -6 Amperes) ohmt, unit of electrical resistance t; reciprocal of conductance t omega-conotoxin
V V VACaC
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the abbreviations section can be sent to the e-mail feedback file
[email protected]. (see field 57 of most entries
for further details)
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EXTRACELLULAR LIGAND-GATED CHANNELS (ELG)
i/
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Edward C . Conley
n
Entry 04
N o t c Thc. 'Key facts' srPu!ronsnrc ~ r ~ t ~ n {or c l rrellrlrrs ~f unfamiliar w t h thc morc $encral rl
Extracellular Iigand-gated (ELG) receptor-channets rapidly transduce transmitter-hinding events into electrical signals
I n t e ~ r ~ ~ mnleculnr ted f~~ncrions of ELC: channels 04-01-01: Fast synaptic ncurntransmissinn, 170th excitatory nnrl lnhihitory, is mcdtatcd hy cxtmccllular IiganJ-gatcd [ELG) rcccptor channel.;. Thesc channcls comhinc on-sulvctivc function4 with thosc for agonist h i n d ~ n g nnrl signal trarnsductlon within a multi-sul~unit rnolcc~ilarasscmhly. In ~ c n u m l ,excitation from rcsting mcmhrnnc patcntials 1s asst~clntcrl with opcninr: of c:ltion-influx (rlcpr3lnrizin~l channels, w h ~ l c inhibition nf ncurona! firiny: 1s guncrally associated w ~ t h lncrcnscd chloritIc ion purmcahility and hypcrpolnrization. A n u m l ~ c rof chnnncl mt~luculnrtypc4 arc rusponsihlc for thcsc actions (Tohlc I ) .
Funcr~c~nnl diversity o f cxtmcelIulnr lignnd-gated receptor channels ELG rcccptor-channels which function in fast synaptic transm lsslon ~ncliidc channcls dircctly gatcd by thc neurotransrnittczs, ~nclutliny:L-~lutnmatc,acctylchol~nc, glycine, ATP, serotonin (5-hydroxytryptam~ncl,;-alnlnohutyric acrd and po~sihlyhistarn~nc(vcc Trrhle I ) . Thc cxcrt;ltory amino act(ls I -cvstcinc srllphinatc ant1 q u ~ n o l ~ n n tmay v also act a < cndogcnclns ncurotransmlttcrs i ~ a s(11~1vbrain rugions. Furthcrmoru, certain tastantst rnay d~rcct!y activatu ( u . ~ .L-arginincl or I~lock1e.g Fib nons1 apical non-sclcctzvc catirtn channcls whcrc thc tastnnt acts a s sn cxtracellular Iigand ( s r ~ r ,(rlso Reccp!or/!rrrnsdrrccr ;nrrrrrctions undcr JLC: CAT cdMIJ.21-49] In cxc~tahlccclls, ruccptor-opcratcd channcls may also scrvv ta dcpolarizu thc ccll to thc threshold of actlon potential generation. In nnn-cxcltahlc cclls, rcct'ptnr-opcratcrl channcls pvrmit a limltcd CA"-influx ciurin): thc prcscncu of an nRonlst (ccr nlco r,n!rics r13r.\urzhznEqthe in!rmcrllular h.y(~nil-sorf~i! illJ<;l (.!ii~rzrir~l~). 04-01-02:
Timc rutilr of ELC: chnrlr~elsixnnlling 04-01-03: Ruccptors c c i n t a ~ n ~ ni ng t c ~ r a !on l channels mcdintc r u t ~ t ~ v u rapid ly transductian events, nnd arc acttvatcd (In a millisccond timc sca te, w ~ r h tvpical lntcncyl t,f -< 1 in.;. This can I,c compared to rccuptors activat~ng C; prt~tcln-cr,upl.cdchanncls w h ~ c htypically npcratc in thc millisccond-tosecond rango folli~wingagonist rcccption. T h c risc-t~mcfor transmitter
Table 1. Examples of t h e extracellular ligand-gated i o n channel f a m i l y (From 04-01-01) Extracellular ligand
ELG channel subtype
Principal ionic selectivities'
Protein superfamily
Covered under
5-hydroxytryptamine ATP Glutamate Glutamate Acetylcholine Acetylcholine y-Aminobutyric acid Glycine
5-HT3
Na+, K' Ca2+,Na+, Mg2+ Na+, K+, [Ca2+) Na+, K-, Ca2+ Na+, K', Ca2+ Na+, K+, Ca2+ C1-, HCO, C1-, HCO,
Ia 2b Ib Ib Ia Ia Ia Ia
ELG ELG ELG ELG ELG ELG ELG ELG
PZX
non-NMDA NMDA nAChR [neuronal) nAChR [muscle) GABAA GlyR
I
CAT 5-HT3 CAT ATP CAT GLU AMPA/KAIN CAT GLU NMDA CAT nAChR CAT nAChR C1 GABAA C1 GLY
"At resting membrane potential, neuronal excitation is usually associated with influx of sodium ions while inhibition of firing generally results from activation of chloride and potassium conductances. For generalized effects of G protein-coupled receptor agonists on the activation and inhibition of ionic conductances expressed in central neuronal cells, see Ap p e n d i x A - I n d e x of G protein-linked receptors, en try 56, and Appendix B - I n d e x of generalized electrical effects of receptor agonism, entry 57. 'Determination of primary sequences for genes encoding P2x purinoceptors'p2 indicates a distinct structural motif for these receptor-channels consisting of two transmembrane domains per monomer and a pore-forming motif reminiscent of that proposed for potassium channels (see ELG CAT ATP, entry 06). CD
R 0
P
I
cntry 04
-
canccntrations to kvuls that activatc ELG channels I $ also rclativcly hricf (a typical diffusion distancc across thc synaptic clcft' h c i n ~ G 2 pm). Durations of synaptic curruntqt arc Rcncrally dctermincd hy thc intrinsic molecular properties nf thc rcccptor channcls ~nvnlvcrl ( c . ~ rcccptor . rfcsunsmzation 1 :and channel inactiv;itront].
Distinctinn of EL[: chunnelx from receptor-rnodnlniedchnnnets 04-01-04: Ry definition, ELC; channcl gating1 is indupcndvnt nf any intracellular or mcmhranc.-diff119ihlc [actor, although phiaphorylation 1s a major mcchatiism tor rcp,uIaiing ~Iicirfunction' (see Jraicr srctirin). In contrast t o the ELC; channcl ~ n ~ u many p , ion chnnnuls ~ o u p l ~ttol scparatc rcccptors (via C, prntcins anti sccond rncwcngcrs) c m hc gatcd hy rncmhranc potcntial chnngcs In thc ahsencc of agonist. Thc majority (if thcsc c m hc considurc.d as receptor-mtdrrlrzred chunnelq, as neurotransmitters activatc nr block primary vtiltagc-dcpcnticnt rcsponscs. In order to distinguish thcsc parallcl signalling rcsponscs initiated hy single neurotransmrtteys, lrcccptnrs which couple tn I; prntcins arc often rcfcrrcd tu as rnetabotropic' receptors (we Rewtirr-e A - G protein-linker! rccrptors. c n t y S h ) , whilc rccuptox protein complcxcs fnrmrng intep-31 ionic channcls arc Ccntiwn ns ianatrnpict receptors. ELG channel genes are differentially expressed ~
Cn-ordirsnltnn nf rornplrx o w r l q p ' n c ypnt icms [if ELG ~ c n Cc X ~ T C S S I I ~ 04-01-05: Dcvchipmcntal gcnc-cxprcwion prnRratns co-ordinatc the activatwn and silcncingt o f ELC. ion clianncl Rcncs, prociiicinF: coinplcx pattcrns oh cxprcssion which unrlurfic fanctinnal specializntton (if individual ccll types. ncvclopiircnt~l rcgyrlatirm may he cffcctutl hy multiplc signals, inclutlinR growth f;ictor?t, inFraccll IIInr scctintl rnuwmgcrst, ccll t ypc-spcci ftc t mnct~. scnptton complc~xcs~, 'Finc-ttinrnaq'of ELC: chrnnrrcl ~ c n cxpre c n4-0.1-1okIn sdditicm to the spccification (if Appropriatu protcmn suhunits, cclls crnploy a variety of rncchaniqmq to modulatc ELG channcl Rent c'xprcssion, inc~uc~ing a~tcmativcspjicinRt of primary transcriptst, R N A cdttingt and activity-dcpundcnt control ( r w hrhw).
' D m v i n i m ' rynnptic in termlions in developinl: hrnin m ffect genc cxprcssion 04-01-07: Activity-dcpuntlcnt, ctmpctitivc synaptic intcractions, which stahilizc some axon hranchcs and dcndritcs whilc rcmoving others, ccntm!Iy involvc RIutamatc rcccptar-channcl cxprcwion'-" ((or (urthcr ifctirilf w e Dcvclopmcntirl rcg:trlmtion iinilrr Ef,C CAT CiLU R M P A I K A I N , 0 7 - 1 I . o r RrwFoprnrntrr! reayulatzonundrur E I X CAT GI.1 I N M l l A . OX- 11).
FLC: chnnncl expression
IS
srlmcerl
111
come c ~ l typcs l
04-01-08: Ccrtain ccll typcs cnn co-cxprcw multiplu ruccptrw channcl tvpcs (C.R. nt'uroncs), wlrilc othcr c c l l typcs d r i not cxprcss any fast Iiganil-gntcd
ch;inntl.; ;IF nll [ c . ~cprthclial . ccllsl.
entry 04
The ELG receptor-channela form an extended protein sequence ‘sriperEamilyJ
ELG channel gene evohtion 04-01-09: Similar hydropathy plotst and amino acid sequence motifst are ohserved at equivalent positions in subunits of diffcrcnt supcrfamilyt mcmhcrs (see FIR. I cxcmplificd for rcccptor-channels gated hy acetylchnlinc, GARA, glycinc and glutamatc). Rascd on closcncss of optirnnl amino acid sequcncc alipnmcnts, thc ELG superfamily has hccn further d i v i d ~ dinto ~ groups Ia and Ih (see Tuhle 1). Thus ELG channels activatcd by acctylcholinc,
y-arninohutyric acid, glycinc and S-hydroxytrypzamine are mnrc similar to cach othcr than thosc in group Jh {the ionntropicj glutamatc rcccptors). Thcsc structural and functional similarities can hc explained i f all mcmhcrs of thc gcnc supcrfamily originated by adapzivc radiatmnt from a common gcnc encoding a n ancestral channcl type. A striking cxccption to this pattcrn of ‘divergentt’ evolution is shown by the Emelprotein domain strtucture of ATP-gated cation channel^'.^ which exemplify the ’cnnvergcntt cvolutirm of similar protcin tunctians via markedly M c r c n t structural charactcristm (see ELG CAT ATP, entry 06) J
The rnoleciilnr hasis of functional similarities and differencesin the ELG super/Qmily
04-01-10: Partial conservation d sequences hctwecn gcnc families can cxplnin how ccrtain tcaturcs havc hccn rctaincd 1c.R. ionic sdcctwityt and suhconductancct levelsj while othcr fcaturcs h a w divcrsiticd bctwccn Renc family mcmhcrs (F.R. pharmacological scnsitivities). Gencrally, thc mnlccular hctcrogcncity of ELG channcfs shown by molecular clnning has hccn larger than t h a t cxpcctcd from pharmacnlopical stcrrlius of nativc rcccptors’.
ELG chnnnel suhunit stoichiomerrics support a stereotypicnl model for protein qrrnternory structure 04-01-1 1: investigations of ELG channel quaternaryt structurc based nn clcctron microscopy, suhunit cross-linking, electrophoretic and sedimentation analysis provide dircct support for a pentarnerjc arrangement of subunits in native receptor-channels. Probably all nf the superfamily mcmhcrs posscss a ’quasi-symmctrical‘ arrangement of subunits around the central pore. Suhunit stoichiomctrics of both cation- and anionselective ELG channels ( e . ~ see . ELC: CAT nAChR, entry 09. and EL<: CI GLY. cnrry 11) havc hcen dcduced as cnnforming tn a ‘3r : 2p’ arrang;ernmt nf scpnrntely tncodtd 2 and p subunits. This basic armn~:cmcnt has thcrcfnrc hccn proposcd ns thc basic quaternary structure of thc ELG supcrfamiEyY (we Pmlcin dornnin ! o p o g m p h y rnodcl.r within the entrics). The smichiomctry nnd nativc s t r u c t u r ~of ATP rcccptnr-channcls, which display a distinct transmcmhranc and porc-forming domain structure’’2, arc prcscntly unclear ( w p ELC; CAT ATP. entry Ml.
w
w
...
w
GIUR-K1 see ELG CAT Glu AMPNKAIN
b;;iyes
-----m
I
I
Hydrophobic regions (transmembrane domains)
s
ss
I i
IS
0Hydrophilic
regions (exposed segments)
S
S
S Positions of cyslelne residues
=
ss
Positions of conserved Cysteine domain
-.- - - -. s
y Positions
of potential N-glycosylation sites
Figure 1. Structural similarities between primary amino acid sequences for subunits of selected E L G receptor-channel proteins. Hydropathy analyses predict a common transmembrane topology which underlies functional relatedness. (Based on alignments from Betz ( I 990) Neuron 5: 383-92). (From 04-01-09)
cntry 04
L
ELG channel pmpertieq are largely determined hy the specification nf ca-expressed subunit types and their stoichiometriefi
'One-gene variant, one-subunit' 04-01-12 Extracehlarl y activa tcd reccptor-channcls form relativcly large
multi-suhunit complcxcs, cnch suhunit gcncrally hcing derived from the exprcssinn of a scparatc Rcnc or variant of a gcnc. The same gene product (prntcin suhunit) may hc ruprcscntcrl more than unct in an asscmhlcrl multi-suhunit cnmplcx [for clarification, scc the [PDTM] protein domain topography modcls within thc cntrics).
Mnleculnr intcgrrrtion of Jigand-hinding, sign:nal trnnsduction and ionic conduction 04-01-13 ELG channel gcnes encode proteins which structurally integratc the distinct functions of Iigand-hinding, signal transduction and ionic conductance with in single macromol ecul ar cnmplcxes. The prntein-protein interactinns necessary for mnnomcric components to cn-asscmhlu into ahcsc macromolccular complcxcs arc intrinsic tn parts of thc prntcin prirnatyt sequence ( w e hclow).
Recruitment and assembly of ELC: channel suhunit proteins 04-01-14: Following 'recruitment' of particular comhinatinns nf subunits a t thc ccllular level, specific asscmhlics of receptor channels with unique functiona! charactcnstlcs may depend on amino acid sequences which rncdiatc inter-subunit contacts and post-translational rnndificationst of componcnt suhunits (see also S E C t l O R on phosphorylntion, hefow).
Transmembrane topography QE the ELG receptor-channels is difficultto predict alone from aminn arid sequence and mutagenesis studies _ I _I
Conclusions frrorn high-resnlution protcin irnnging 04-01-15: In gcncral, ELG channcls display four predicted mcmhranc-spanning regions (Ml-M4] on the hasis of hydrophohcityt plots. Howcver, structural studtcs of the nicotinic rcccptor at hi$ resolution"' (-9 A] predict that only
one mcmbranc-spanning q-helixt (prcsumcd to hc M2, as a pore-lining domain) i s present per suhunit, with the other hydrophobic regions being prcsent as Ifsheetst. Rccausc of these factors, all f i ~ r c c sprcscntcd as "protcin domain topngraphy models' within the entries should hc considcrcd as diagrammatic. Fn particular, the relative p o d i o n s of motifs, domain shapcs and sizes arc suhicct tci rc-intcrprctation in thc IiRht of hcttcr stmctuml data. EtG inn channel proteins are invariably regulated by post-translational modifications 1
ELG channel proteins nrc suhstrates for LI vnriety of protein kinnses 04-01-16: E X channels such a s thc nicotinic acctylchnlinc receptor (see ELG
C A T nAChR, entry 09) can he phosphorylated hy a family of endngenous protein kinases including CAMP-dependent protein kinase, protein kinase U, protein tyrosinc kinasc and calcium/calmodulin-dependent protein kinasc".
''"
An example of functiond modulation hy phosphorylation - receptor
desensitization
04-01-17: Reconstitution into liposomes has permitted functnonal studies of subunit phosphorylation. Phnsplinrylation nf the nAChR reconstituted into liposnmcs accelerates receptor desensitization, a process hy which membrane receptors do not transduce signals evcn in thc continued presence of agonist. Desensitization processes are a result of agonistinduced ctlnfnrrnationaE changes that close the ion channel rather than
activating lopening) it in thc presence of agonist. In addition to nczirotransmitters, hormoncs and othcr first and second mcsscngcrs can regulate phosphorylation statcs of ELG channcls, thus acting 3s physiological rogulatnrs. Subunit phosphorylation status may also regulate ELG channel assembly processes. (See n l w ~the Protein phnTpphorylatmn field in the FLG entries nnd Rewurce C: - Reported 'Consensus sites' nnd 'motifs', entry 62.)
I
Glutamate is the major excitatory? agonist in the vertebrate brain
Ubiquity of gluiomnte ngnnism 04-01-18: Receptor4 for the neurotransmitter glutamatc (GluRs)arc cxprcsscd on virtually every neumnal cell and some glial cells in the CNS. Ionotropict GluRs (possessing integral channels) have major roles in fast synaptic transmissinn and are participants in the establishment of synaptic networks, processing of associativcJscnsory information and co-ordinatinn of rnntnr functions".
Suhclosses of glutamate tccepmrs with integral ion chnnnels 04-01-19: GluRs have been initially classified into thrcc separate populations,
cach dcfincd by sclcctivc activation with different structural analogues of glutamatc'" I s . Thus, broad categories of channels gated hy N-methyl-oaspartatc (NMDAF, r-amino-,~-hydroxy-fi-methyl-4-isoxazolepr~piona~e [ AMPA] and kainrc acid (kainate) have heen dcscrihed extcnslvely (thc lattur two catcgoiics arc collcctivcly refcrred tn as the 'non-NMDA receptors').Thc large numhcr (if GluR protein suhtypes [and the complexity of their potential arrangements in functional channcls] indicatcs that prcscnt classifications arc not ahsrilutc and therefore may he inadequate for dcscrihing the range nf GIuR expression ohsorvod m V I V O (for detailed nomcnclrrttirc w t h m these fnmrlies, sec E C G GAT GLU AMPAIKAIN, entry 07. rind ELC: CAT C:LrI NMDA. m t r y OR).
Glutomnte toxicity nnd neurodegenerntjon 04-01-20 In addition to its normal function as an excitatory ncuro-
transmittcr, glutamatc can kill neurons by prolonged receptor-mcdiatcd dcpoIarization, resulting tn irrcvcrsihlc disturhances in ionic hnmeostasis'". ' L ' R .
entry 04
Glutamate toxicity has been implicated in the death of neurones following ischaemia, epilepsy, and newodegenerative disorders such as Alzheimer's, Huntington's and Parkinson's diseases (for further details, see Phenotypic expression under the ELG CAT GLU entries). Glycine and GABA ate principal agonists for inhibitory receptorchannels
GABA- and glycine-gatedchloride channels
04-01-21: En the spinal cord, most post-synaptic inhibition is mediated hy glycine (see ELG CI GLY, entry 11) whcruas the vast majority of post-
synaptic inhibition in the rest of the brain uses GABA (gamma-aminohutyric acid - see ELG Cl GARAA, entry TO\.Both amino acids activate integral receptor-channels to increase CI- conductance. Selective hlockade of different populations of inhibitory response can be induced with strychnine {for ionotropic glycine rcccptors), picrotoxin (for ionotropic GARA receptors] and hicuculline {for G protein-linked GARAn receptors).
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For m a p r rcvlcws o n thc inrlivitlual typcs of uxtr~cullular ligand-~atutl channels, see thr, Relclted fourcrs d rrvlewc fruM rn thc ELG cntrres. A classification of rncrnhranc reccptnr classes (including transmitter-gated ion channclsl hascd on structural and functional critcraa has appeared in rcf.". Ruv~cwsources on tht. icmr,trc,pict glutnmatc rcccptors which havc hccn qucltuti ahuvu inclutlc rcfsF3. 17"Y.
Yalcra, N r ~ f ~ i[I51041 rr 371: 516-19 R r ~ k c ,I Y t ~ t i i r(1494) ~ 3JE.519-2.3. " Swtlpu, FASER J ( 19921 6 . 2.514-2,1. "iptcln, Trends N C I I ~ O(~I OLX'9I) 12: 265-70. Collingridgc, Trcnrls l'hrirrnncol Scl 4 1990) 11: 290-6. 1311ss, N ~ ~ i ~[l993) z r c 361:3l-9. ftarnsrd, Trends Riochern Sci ( 1992) 17: 36H-74. ' D~nglcd~nc, FASEIF (1900)4: 2 6 3 W S . Langnwh, Prou N n t l Aclrd Sci [JSA (19881 85:7,394-8. I n Unwin, Mnl Rzol [ I Y0.3) 229: 1 101-24. II H u ~ a n l r ,Crrt H r v Nlochcm Mol fllr~l( l9K91 24. 183-21 5. I2 Hugantr, Nrrlrrjn [ I 9903 5 : 555-67. Caslc, Annlr K r v lJhvszol (10921 5 4 : 507-.3h. Maycr, I'rr)#qN~1~rol)tol l( 1987) 28: 197-276. Macncrmntt, Trrl~rlsNrltlroccr ( 1987) 10: 280-4. 1fr Snmmcs, T r ~ n d IJhrtrrnrtc*ol s Scr [ 1992) 13: 29 1-6. 17 Nnkanlshi, Sclcnihc( I 9921 258: 597-60.3. Olncv, Exp Krl~mIIrs (15171)14: 61-76, " Visdcn, Curr Opjn Ntwrol)iol ( 1 99.31 3: 291-8.
'
" " '' ''
Edward C. Conley
Entry 05
Abstractlgeneral description 05-01-01: 5-Hydmxytryptmnine (serotonin, 5-HT) is a hiogcnic aminc that functions as a neurotransmitter', a rnitogcn' and a hormonc. The large
nurnher of hiological functions for 5-MT i s reflected hy thc cxistencc of An extraordinarily largc family of rcccptnr suhtypcs fnr 5-HT (see Suhtype G ~ ~ S S I ~ F C ~ ~ F 0.5-M). O ~ S , T h e grcat majority of 5-HT rcccptors coziplc to scparatc cffcctor mnlccuPes thrnugh C protcins (see RcceptorJtransducer Intermtions, 05-4Y nnd Rcsoorce A - G protein-linked receptors, entry 56). Howcvcr, the receptor subtype 5 H T 3 lfor convcnicncc designated as 5-HT.lR within thls entry) has a rapidly activating, 5-HT ligand-gated, non-selective cation channel intcgral to its primary structure. 05-01-02: The genes encoding 5-HT.lrcccptor-channcls form part of the cxtra, 04) cellular ligand-gated channcl gcnc supcrfarnily (see ELG Key f ~ c t s entry and functional fi-MTA rcccptors display a nurnhur of rnolccular, pharma-
cological and physiological sirnilantics to othcr supcrhmily mumhers. Notshly, 5-HT 1 receptor Rcncs prc.wnrly show much less structural drvcrsity than thnsc encnding other typcs of ELG channel, a l t h o u ~ hRNA splice variants of a single 5-HT7R Rcnc havc hccn chnractcrizcd. fi-HT-+ receptors in diffcrent species and preparations often show great variation in electaophysiological and pharmacoloRical propcrtics. 05-01.03: In addition to expression nn native central and peripheral ncuroncs, 5HT, reccptcirs arc also cxprcsscd a t high density in several ncurnne-derived clonal ccll lines. In native tissues, post-synaptlc fi-HT1 receptors display hricf, excitatory post-synaptic currents' in rusponsc to synaptically rcleascd 5-HT. Prc-synaptic 5-tdT;r rcccptors mediate the release of several neurntransmitters. 5-HT,
FI-HT,~R~ display fast dcsensitization kinetics m the continued presence of agonists, and show cooperative interactions hctween ligantl-hindinp, sites. Under physiological conditions, the 5-HT3R ion channcl is cqually permeable to N a and K' ( E ~ . ~ ~ = O O VS-HT~RS I. in some cell typcs art' pcrmcnhlu tn ca7 ions. 05-01-04: Like othcr ELG receptor-channels, the
+
1
Category (aartcode) 0.542-01: ELG C A T 5-HT,3r i x . cxtraccllular ligand-gated cation channcls activatcd by 5-hydroxytryptaminc. Thc tcrm '5-HT.3 receptnr' was originally introduccd tri maintain consistency with thc 5-HTl and S-HTz G protcincoupled rcccptors dcfincd initially by ligand hinding. Thc suwcstcd electronic retrieval code (unique embedded idcntificr or UEI) for 'taming' of ncw articles
cntry 05
of rclevancc to the contents of this entty is UEI: SHT3-NAT (for reports or rcviews on nativct channcl properties) and UEI: SHT.7-HET {for reports or rcviews on channcl propcrtics applicable to hetemlogously~ expressed rccomhinantt subunits cncodcd hy C D N A S ~or genest]. For a djscu.wim of the advantups of UEIs and guidelines on therr imphWnlOFiQn,see the section on Resource J under Intmdricrron eh Icryont, entry 02, nnd for further details. see Rcsourcc - Search criteria etlGb?N development, entry (75.
Channel designation 05-03-01: 5-HT3R; Gaddum‘s ’M’ rcccptor; 5-HT-M receptors, as originally designated by Gaddum and Picarelli’ as mcdiatinE an cxcitatory (cnntractilc) rcsponsc in minea-pig ileum via enteric nelrronal receptors, as
inhihitcd hy Morphanc.
Designation of cloned splice varionts 05-03-02Properties according tn the first cloned suhtype’ are designated wtthin this cntry hy the prefix S-HTAR-AL. The ’L’ subscript indicates a longer form of thc two picscntly known splice vanants of the same S-I-€T3R gent (.see Gene orpmizotmn, 0.5-20). T h e prefix 5-HTJR-A.s indicates data collated for the putative short-splice form’, which lacks a short stretch of amino acids (GSDLLP) in the M,3-M4 intracellular loop (see IPDTM], Fig 1 ) . Note Fieldnames without subtype prefixes indicate data applicable to native S-HT.3R isoforms cxpressed in specified cclF types.
Current designation 05-04-01: Usually of the form laRc,n,qt, i.e. 15.rrr-tor I,5.HT*4,.
Gene family 5-HTa3Rgenes encode proreins sharing hasfc properties with other ELG IECt?pKT-ChQnIldS. 05-05-01: Thc prototypc gcnc encoding the S-HT3 receptor-channel forms part nf the extracellular ligtnd-gatcd gcnc superfamily (see ELG Key facts. entry 134). The S-HT,
Subtype classifications Relufionship of 5-HT3 receptor-channels to other (G protein-linked) 5-HT r e c e p m s 05-06-01: Rascd on pharmacohgkal (‘~perationat’)~ and functional [’transductlonal’)‘ propcrtics, the large number of known serotonin receptors have hccn placed intci sewn malor classes In interim classifications: 5-HT1, 5 - H T 2 , 5-HT,r, S-HT1, 5-ht,, 5-ht,, 5-ht, (scc o l refs5.‘, ~ Receptorhransducer
entry 05
mternctions, 05-49nnd Rcsotrrce A - G protein-lmked receptors. entry 56).The 5-HTI class (fivcrcccptnr subtypes),5-HTzclass (three receptor suhtyFcsj and 5Ha4 class consist of C; protein-linked receptors (see Resnurce A , entry St,)while only 5-HTq subtypes mediatc rapid excitatory responses in ncurones through an intcgral ion channcl’. Note. For dcvelopmcnts in 5-HT rtccptor nomcnclnturu, rcfcr to thc latest IUPHAR NomuncIaturc Cmnmit tcc rcctimmcndations via thc CSN (see Fcedhrrclr d CSN nccess. entry 12) 05-06-QT:Variahilitics in mcasured channcl cnnductances, pharmacnlo~icat sclcctivitics and othcr propcrtic~’~’ suggest thc existence of multqdc suhtypcs of thc 5-HT3 rcccptnr, although rctativcly fcw suhunit variants dotn. 05-#1, nnd h a w bccn cloncd to datc (see nlso Sin&chonnel Receptor nnto,qonists. 0.5-51).
Trivial names 05-07-01: The serotonin-gated rcccptorxhanncl; the 5-HT-activated rcceptorchanncl.
CeH-type expression index 05-OR-01:f;-HT,3rcccptors are widcly distrihutcd in central and pcriphcral ncurones. For cxamplc, virtually a11 mammarian autonomic neumnes possess 5-HT1 reccptnrs, and thew may he co-expressed with othcr ELC. channcl types. Thc information in this entry largcly rcfcrs tn wcll-charactcrizcd ncurnnal types, including: olfactory
bulb
dorsal root ganglia am ygdala multiple nuclci of trigembnal ncrvc spinal tract hypothalamus brainstem motor neuroncs superior cervical ganglion cells PCI 2 phaenchrnmocytnrna cells nodose ganglion ncuroncs N 1 E- 1 1.5 neurohlastoma c c h (,we below) N I8 neurohlastoma cells NG108-15 hyhncl neurones (see helow) submucous plcxus ncuroncs coeliac ganglion ncurnnes
Availability of neuronnl cell lines expressing 5-HT3R 05-08-02: In addition to cxprcssion on nativc ccntrd and pcriphcral ncurones, 5-HT,3rcccptors arc alsn cxprcsscd at high dcnsity in several neuronc-dcrivcd clonal ccll lines’, c . g NlE-I 15 nciirohlastoma cellsF” ‘ I , NCB-20 cells‘‘ ( w p Clonmg resource. 115- 1 O), NC; OX- 15 ncuronal hyhridoma cells’2 and N 18
cells‘”.
Cloning resource Q5-10-01: S-HTJR-AL:Isolatc S-WT,4R-A(now dcsignatcd 5-HTIR-AL,we GI Channcl dcsr4iyntion.0s-03)was expression-clnned'in XcnopuT oocytcs from sn NCR-20 mousc hybrid (ncurohlastomalChincse hamstcr cmhryonic brain cell) cDNA library'. T h e w cclls were sclcctct! for their ahility to cxprcss IarRc, rtihust 5-HT,r rcccptrir-mcdiatcd rcspnnscs. 05-10-02: 5-HT.7R-hs: RT-PCR' has hccn usud to isolatc a 'short-splicc' variantn5 n f t h c 5-HT1R from mRNA of thc rtidunt ncwohlastoma linc NEE1 15' (src Chiinnel deT!,enorrnn, 05-03), Rat supcrior cchvical gan~lioncDNA lihrarics havc hccn uscil as resources for isolatinn of cloncs cncadmg 5-HTlR (see Dfltahirse Irsljngs, 05-53].Note. On thc basis of many conscrvcd clcctmphy~ialngicaland pharmacnlogical pmpcrtics, it wr)uld hc cxpcctcd t h a t cithcr ccl I lincs cxprcwng S-FIT,3R (.rcc Ccll-type cxprcwcm jndrx, OS-LW] cnuld ~ l s scrvc o as chining rcsourccs.
Developmental regulation Induction of 5 - h T 3 R cxprcssion Isy
R.CTV.L:growth
fnclor
cind CAMP
clcrivnt ives 05-11-01: Applicntion of nerve growth factor (NGF) or PI-homo-cyclicadcnosinc mnnophosphatc (8-Rr-CAMP]to PCI 2 cells fnr long periods (-10 rlsys) cxprcsscs 5-HT7-tvpc rcccptors, which arc of low ahundancc in iintrcatcd cc!Is". 05-1 1-02: Thc diffcrcntiatim status of ncuronnl hyhrirlcuna NG 1 OX- IS cclls ;iltua'i chinncl condiictancc and dcscnsitizatirm propcrtics of thc S-HT3R'5 ( v r . Tirhlr 2).
Isolation probe 05-12-01: S-HTlR-At: As n o molcculsr prnhc was availahlc, t h e S-HT7R-AL isofiirm was ~stilatcd' using rxprewion-cloning' tcchniqucs rn Xenoprrs oocytcs by micrmnicction nf cRNA pools from an NCR-20 ccll cDNA cxprcssinn (,we Clrmrnayr c w i i r w . 05- 10) ~~
mRNA distrihutfon tnHNA rJisrrihuiion wilhin hroin 05-13-nI:Usinc ratlidal~cllcttprolies spcciftc for thc S-HTaII-ALd o r m , Iahcllcd cclts a r t d~scrvctlthrtiirRhoiit thc cortical regions (c.g. pirifurm, ciii~ulatcand cntnrhinal arcas]. nisttnct lahcllmg is wen in thc postvcntral hippocattipus (within tha lacunosum r n u l c c u l ~ rlsvcr (if C A I , ctintaming Inhihtnrv nciiriint's). Thcw sturlics'' nlsn shnwcd 5-HT3 m R N A to hc
prcscnt in the olfactory hulh, dorsal mot ganglia, amygdala and rnultipk nuclci of trigcminal ncrvc spinal tract, hypnthalamus and hrainstcm m r m r ncuroncs.
Implications of hrnin m R N A distribution potterns 05-13-02 Distaihutions of WIT3 mRNA studied in mouse brain by in situ hybridization tcchniqucsf6 arc consistent with the roles for the 5-HT7 rcccptor in cognition, cranial motor neamne activity, sensory processing and modulation of affect.
Distribution ond npparenr hias for exprcssinn of rnKN.4 splice vuriun t s 05-13-03.: S-HT2R-AI,/S-HT.~R-AT:Clrmcd isolatc 5-HT3R-AI, mRNA is distnbutcd in hrain, sprnal ctml and hcart tissue‘. Approximately HO% of transcripts from mRNA populations dcrivcd frnm NGln8 cells and native cortcx/hrainstcm” cncodc the ’short fnrm’ of the S-HT.4receptor.
Phenotypic expression Distinct roles of pre- and post-synaptic5-HTk7receptor-channels 05-14-01: Endogennus rclcasc of serotonin (for exnmplc in thc lntcral amygdala] has hccn shown to mediatc rapid excitatory post-synaptic currents‘ in response tn synaptically rcleascrl 5-HT. Characteristically, synaptic potentials are of hrid duration (tens of milliseconds-set. Actrvritton. 05-3;7.rind Inrrctivrir ion, 05-37) and can hc mimickcd by 5-HT, potentiated hy a 5-HT uptake inhihitor, and hlnckcd hy selcctivc 5-HT3 rcceptor antagonists. The anti-emetic, anxiolytic, and possthly antipqychotic actinns of S-HT.
A-HT.? receptor-chnnnels also rnedinse neurotmnsmitter release in
the periphery
05-14-03 At locations in the periphery’ [e.g. enteric, sympathetic and parasympathetic autonomic and primary sensory neurones] S-HT.3-mediatcd excitation may wake neumttansmitter release’.”’ and play dircct roles in iaaitiatinn of intestinal muscular contraction. Rccausc ot its divcrsc rolcs in mcdiazing brain function and hchavinur, dcfccts in scrotoncrgic signallinfi systems may contrihutc to thc a e t i o l q y of pain reception in scnsory ncrvc fihrcs”, migraine, dcpruwivc condition.; and oI~scssivc-
compulsive huhaviours.
Tnchycardic responses diie t o 5-HT,qR-linkerl mnsrnilter releaxe 05-1 4-04: S-HT.4rcccptor stimulation resulting in tachycardia’ and inatrapic’ actions is mediated by noradrcnalinc rclcasc from thc postgnnglionic cardiac
sympathetic wrves. This rcspcinsc is rnimickcd hy 5-HT agcinists and in somc c a w s can he hlncked by sutnnnmic antagonists (c.g. prop~-anolol)and 5-HT3 antagonists [c.g.MDLJ2222 nr ICSZOSr),TO\ (sce Rcucptcir nntri.qponist.<. 0.5 -5 I 1.
Rat striaturn and nucleus accum'hens
Rclcasc of cholecystokinin Kclvimsc of noradrenaline Rckase of GARA Rclcasc rrf 5-HT
1g-21
Ccrchral cortex and nucleus scumhcns Hippocampus
22
Hippocampiis
2r
Frontal cortex and h ippocamplls
Ucprccssrcin ot uvokcd rclcnsu of noradrenaline
Rat hvpnthnlamic
Dcprcssrnn nf cvnkcd rclcasc of acetylcholine
Ccrchral cnrtex nr synaptosamcs from ccrchral cortex
27
25. Zh
27.2R
SllCCS 29-31
hut scc
32
Retlcx hradpcnrdic respmxcs due to S-HT,R-linkedlrnnsrnirt er release 05-14-05: In the cerdinvascular system, the main initial rcspansc to a halus of S-MT rs R short intcnsc bradycardia' and hypotension, mcdiatcd via a Rczold-
Jarisch-like rcflcx', initeatcd hy stimulntion of S-HT? rccuptors pruscnt on nffcrcnt vagnl ncrvc cndinFs. Intmvcnous, intrasnronary or local cpicardial adminrstmtinn of 5-HT, 1-phunylhig:unnidc or 2-mcthyl-5-WT clicits the transicnt hmdyc;lrdia, which I F cffuctrvcly antagonizcd by 5-HTl-sckctivc hlockcrS35''". lScr n h i Kcocprrir/trrIn.rdFIct.r I ~ ~ ~ P T M ! ~ I Jiindrr T I S 1Nli K C / ACh. 3 r-4'Y.j
Protein distribution .S-HT7 rcceptnrs (IPC uhfquitoiis in rhc CNS nnd PNS 05-15-01: On thc basis of functional a n d radiciligand-hintinr: studies, 5-HTl rcccptw pmtcins appear tn he ubiqiiitonsly distributed thrnughout thc peripheral and central nervous systrms. In ccntral ncrvous tissue, autoradiographic mapping (if 5-MTl shows cxprcssicin nswcintcd wrth cortical, Iim'nrc and hrainstcm structirres. In the periphery, 5-HT1 rcccptnrq arc found on cnturic, autonomic (syinpathctic a n d parasympathuticl and primary sensory neumnes. Fuiictional 5-HTq-hindingsitcs have hccn found in scvcrat mammalian cell Iincs, inclurlin~,ncurohlasmrnas ( w c C r l I - ~ y w
expression index, 05-88), A full description of receptor distribution pattcms has appeared in a hook dedicated tn central and peripheral 5-HT1R [See Lnporte, in Hamon. 1982, p p . 157-87, under Related snurces d reviews,
0s-56). 05-15-02: The anti-emetic+ properties of 5-HTqantagonists lsee Receptor nntfigonists. 05-51)may uorrclatc with blockadc of rcccptors in the area postrema and solitary tract nuclcus nf thc brainstem, areas which display high densities of thc S-HT3 subtype. Blockadc of pcriphcral rcccptors present on v a g d afferents’ may also play a rolc.
Snhcellular locations 5-HTL3receptors are located on both pte- nnd post-synaptic mem bran ex 05-16-01: Both pre-synaptic and post-qynaptic 5-HT3 rcccptors have heen
studied in hram slice preparations, e.g those containing the nucleus tractus solitarius (NTS)”’. Generally, the pre-synapti c 5-HT.3 channels replate neurotransmitter release, whilc the post-synaptic channels regulate ionotropic neurotransmission (see Phenotypic expression, 05-14, nnd ReccpiorJiransdncer interactions, 05-49).
Encoding 05-18-01: Open reading frame’ sizes fnr cloned suhunit cDNAs are listed under Do tahase liTtings/prirnnry sequence drscussron, 0.5-53.
Gene organization
n
A putotive splice vorjnnt of the 5-HT.qR carrying a six amino acid deletion 05-20-01: _5-HT3R-As: _ A shoncr form of the prototypic S-HT.lR has been isolated from thc ncnrohlastoma ccll line N 1E- 115 using reversc transcriptasc polymerase chain reaction {RT-PCR’)techniques3. The S-HT~R-AL and S HTxlR-Ac forms sharc -98% amino acid sequence identity, but a contipinus s i x amino acid segment (GSDCZP) is deleted in S-HT,?R-AF(Fig. 1). The high degree of ~ d c n t i t yand the fact that both variants can he detected in the NlE-115, NCR-20 and NG108-15 hyhridoma cclls” indicate thc transcripts arise 3s altcmativc RNA transcripts from a singlc gcncRS (see also mRNA distribution, 05-13,rrnd Receptor mRnniTtT, 0s-SO].
Homologous isoforms 05-21-01: A 461 amino acid 5-HT3R variant3’ isolatcd horn a rat supcrior cervical ganglion cDNA lihrary shows -95% sequencc homnlogy with thc mouse 5-HT3R-Ac,and represents a spccics h o m o l o p c t of thc samc splicc
variant.
entry 05
Protein molecular weight (purified) P ~ i ~ i f i S-HT,?l< ed complexes drsplsiv mdectilrtr r t i ~ s s r stypicdl of ELG ch(1 RRC 1s 05-22-01: Palyacrylarnidc gcl clcctrclphorcsis of piirificd S-HT,3 rcccptor from NIE-I IS ncurohlastoma cclls rcvcnls a single protein band of 54 7 f I .A kDa". Thc ollgomcric form of thc S-HT{ rcccptor soluhilizcd from NGIOX-15 cells has hccn estimated as -hOOkDa by gel frltratlnn'". This value is cnnsistcnt with thc rangc rcpwtctl h r thc 5-HTIR complcx of rnhhit small howcl (44,7-h(1'l kDnl4' although rcccptr)r/dcturgcnt complcxcs and siicrosc dcnsity Kmdicnt tcchniqucs cstimatu a n M, nf -3OU kDaQmn4244. When the cffccts of dctcrgcnt hrnding arc takcn into account, A v s l ~ i c(if 249 ktln (morc typical of o t h t r incmhcrs of thc ELG channcl h m i l y ) can hc tlcrivctI".
Evldence for hcreto,yeneuusmolecular sizes of .5-H&R 05-22-02: .On thc basis of SDS-PAGE' analysis, 3 numhcr of studics sumest heterogeneity nf rccuptor suhtnit sizes tnakinR up thc S-FW3R complex ( t y p ~ a l l y In thu rangcs -50-54 kDa and -.3h-.38 k h ) . Thc smallcr component size iiiatchcs that of a sitc lahcllcd by I'HI-zxopridc in NGIOHI gJ" and nativc ccrcImI cortcx of rat45.
1
Protein molecular weight (cnlc.)
05-23-01:S-HT3R-hr _ _ -. /.5-HT.3 R - h r : Thc mousc' NCR-20 ctll linu rcccptor c D N h sequcncc" prcdicts a molccular wcight of t h t protcin of SS46hD.1 tncluding the signal' pcptidc, or 5.7 509 Da as a mature prcitcin. Thc cDNA scqucncc of t h c short-splicc form isrilatuc! from thc mousc ncurohlastorna cell ltnc NI E- 1 15 prcdicts ii rnnlccular wcight of 5 7 1 J X inclurlinE thc sign31 pc p t sdc ?"
Sequence motifs 05-24-01: 5-HTlR-A: - _.lsolatc S-HT3R-A2displays a signa! puptidc clcavagc mcitif hctwccn rcsiducs 23 and 24 of the prccilrsnx prntcm' chain. Motifs for N-Rlycosylation arc prcscnt ;It cxtracclIular amino acids 108, 174 and 1YO (positions 86, 152 and I68 in the mature 5-MT,x-R-AFprotcln] ([PIITM], FIJ 1 ) . A disulph~dchontl motif predicts thc fnrrnation of an S-S lmk
hctwccn aminn acids I h l and 175 (pmitinns 139 and 15'2 in thc mature protcinl ( I r w reIrriwc posrirms. scc [PIITMJ, FIX. 1 ) .
n
m4
.
_.
~
(b) Rentarneric arrangement (putative) -
Channel symbol
Na. K ,C a
,v,I iL .1
5-HT
v
Figure 1. Monomeric protein domain topography model for the murine 5-hydroxytryptamtne-gated receptor-chonneI (5-HT3R-A) (long splice form). Note: AU relative positions of motifs,domain shapes and sizes are diagrammatic and ore subject to m-interpretation. (From Q5-24-01]
Amino acid composition 05-26-01: 5-HTlR-A: Mydrophohicity' analysis of thu prototypic isolatc 5WT,,K-A' rcveals a pattcrn of h y d r t q h h i c domains (MI-M4) typical of the ligand-gated ion chsnncI supcrfamily ( I P D T M ] , Fi.r. I) (cornprm other
II'IITMv) oi ELL: entrirac).
Domain arrangement Structural hornnloRics with su buni t nss e m b1ien
of her
ELG chmnels sqqgest pentameric
05-27-01: In cornparism to thu homologous domain structure nf the nicotinic rcccptors (ser E M ; CAT nAChR, m t r y W ) , it is prcdictcd that the intact rcccptor may hc asscrnldcd from five monoiricr suhunits (see /PDTM], FIR. rp, with thc y-hclix' of each M2 region lining the channcl: porcQ4 (..ice Selectivii y . 05-40).
M n n y propcrties of nntive S-HT.?,R arc repmduced hornornult imerrc 5- NT<7 R -A
OR
expression of
05-27-02: Nntahly, thc S-HT,iR-A homomcric' ~solatc' displays inany of thc physiological and pharrnacnlngicaT prnpcrtrcs of native 5-HT rcccptrm In V I V O {q. in cell linus ustt! as thc mRNA snurccs for cDNA lihrary constructinn) Ry camparison with othcr rncrnhers of the superfamily, different suhiinit compositions of S-HT? may he expectcd (see ofher ELG entrws), hut B comparahlc suhunit diversity has not htcn found (to the end of 199.3 - see updates on this cntry, acccssihlu via thc 'hrimc pagc' of thc CSN, a s dew-ihcd in F r r d h u k 01 C S N ~ I C C C S . ~entry , 12 and Rcsourct. J Scnrch critcria A CSN dcvclopmcnt, entry 6.51.
Domain conservation Amino acid sequence identities wrtb other ELG receptor-channels 05-28-01: -S-HT~?I-AI _ _ : Thu dtduccd amino acid sequence nf the 5-HTlR-A is oI a t cz (.$ er 13n I~i h ( I YC InI I n . r ~ l p rimnry req L]e n c P disr I] F F on, 0.5- 5 3) Shnws 30% scqucncc idcntity with tlic T suhunit of chick hrain nAChR ( w r ELC: CAT riAChR. entrv (19) and both consist (if four putative membrane dornasns Scrlwncc consvrvntirm i s grcatcst in thc N-tcrminal ( n A C h R Iigand-binding) and M2 (nAChR porc-lming] domains. The 5-HTlR-A isofom shares -22% amino acid identity with GARA, suhunit (see E L G CI C:ARAA, enrry FO) and thc 48kDn subunit nf glycrnc rcceptor-channcls (see E L G Cl GLY, r n i r y 1 1 ) .
5 - H T 2 R rcsidricc conserved ffl rn ily mem hers
(it
equivnleni positions ~n other E L G Rctw
05-28-02:5-HT3R-AL:T h c rdativc positions nf twn cystrine residues arc conserved in thc S-HTaR, CARA,R and nAChR. Thcsc re~irluesare likely tn fnrm part r d a small disidphide bond loop which may hc impnrtant for thc tcrtrary' tot din^: of thc proturn ( w e /Ps)TM]. FIR. 1). A hydrophohlc lcuctnc rcsiduc (Ccu2R63 within thc M2 domain nf the S-HT.$R-AISOIR~C' is ctinscrvctf within all nicotinic, glyanc and C.ARAA rcccptor pmtcins"".
cntry 05
n
Evidence for subunit heterogeneity amongst native 5-HT.3 receptnr complexes 05-28-03: Native S-HT,3 complexes vary greatly in thcir single-channel conductance (see Single-channel hntn, R5-411, sumcsting that hctcrogcnaity exists amonR thcir suhunit asscmhlics (src olso llomnin fifnctwns.05-29).
Domain functions (predicted) Domain functions likely to he common across ELG superfnmily
memhers 05-29-01: As shown in the [PDTMl (Fig. I ) , a typical 5-HTIR rnmomcric suhunit consists nf fnur hydrophobic transmcmhranc rcgions (M1-M4), a
long cytoplasmic lonp hetwccn MA and M4, and n rulativcly largc Nterminal domain, Ry analogy with othcr ELG chnnncls, thc N-terminal domain probably forms part of thc agonist-binding site and thc sitc for some antagonists, whilc thc M3-M4 h o p contains pntcntial sites for channel regulation with thc M2 rcginn lining thc channel pare.
Direct evidence for struciurol and Jzinctionnl division of receptor ond channel dornnins 05-29-02 Chimacric rcccptcir mchcculcs, consisting nf the N-tcrminal domain of thc nicotinic ncctylcholinu rcccptor subunit 17 (see ELC: CAT nAChR, cntry 091 and the putative transmcmhranc and C-tcrminaE rcgions of 5-HTqR-A have heen cnnstructcdQ7. Chirnacras display receptor prnpcrties chnractcristic of thc nAChR and chonncl prtlpcrtics similar to thnsc of thc S-HT*4R whcn cxprcsscd a s homoiniiltimcrs in Xennprrs oncytts47. Thcsc studics rcprcscnt direct evidcncc for 5-HT agonist-binding sites hcinR forrncd hy asscmhlics coinpsising parts of thc Iiyrlrriphilic Ntcrminnl rlmnain.
Single amino acid substitutions nffectinR desensitizntim properties of the 5-HT.7X 05-29-03: Rccomhinant 5-HTIA rccuptor-channul s cxprcsscd in Xunnpirs oncytcs (at -6OmV holding potcntinl) conduct an inward currcnt which dcclincs during thc continucd pscscncc of 5-HT with a half-timc of ahont 2 s; this desensitization' is -20 timcs slowcr in calcium-fruc solutiondH. Dcscnsitizatinn is markedly laster in channclq whcn Luu2X6 (ncar thc middle of thc M2 segment) is changcd to Phc, Tyr or Ala. Dcsensitizatian i s slower with Thr. Changcs at eqiiivalcnt pnsitians of the nicntinic acctylchalinc rcccptor h a w similar cffccts nn dcscnsitizatirm, sumesting that thc underlying protcin tnnfnrrnatiand change might hc a common fcnturc of
Fip;,?nd-gatcdchanncls".
Chemical rnodificntion of exsrucelhilnr tryptophm residues rrffccts axon i s t h tagonist binding 05-23-04: Prc-trcatmcnt of NG 1OR- 15 rncmhrancs with N-hrornosuccinimide (a sclcctivc cixirlnnt of tryptophan scsid~icslRrcatSy rcduccs thc rEcnsity of sites lahullcd with thc 5-HT,l rcccptnr antagonist 1'Hl-zacnprideuv. Thc cffccts of Nhromnsuccinimidc mtirlification can hc prcvcntcd l ~ yprc-mcuhation o r co-
incuhat~onwlth somc 5-HT, rcccptor a ~ o n i s t srtr antagonists. Othcr agents which rnod~fyaspartatc, cyqtcanc, cyFtInc sntl glutnmntc rcsidi~cshave l ~ t t l c cffcct on lrganii h~nding,whalc thosc sclcctivc for arghninc, hrqtidinc and tyroslnc rcsirl~~ux have only inotlcratc inhihitory ctfccts on I 4 ~ I - z a c r ~ p r ~ r l u Iahcll~n~~".
Predicted protein topography I'redlcted mnlrrmcnu r~sscm hly pnt t e m s 05-30-01: In common with other mcmhcrs of thc uxtracc.lliilar Ilgand-gatcd cation channel supcrfamily" thc 5-HT, rcccptor-channul 1 4 likely tn he formctl from f ~ v crnonnmcrq that contain from two to four hnrnr)logul~s s ~ ~ t n ~ n l t (.tcr, s ~ " ' /I'ISTM/. Fig. I ) . nlrcct uv~dcncc for hctcromultimcnc nsscm1~lic.shas not hccn rcporturl trl dntu. Electron tnicro~copicuxnm~natinnnf 5-HT, rcccptor c h ~ n n c l sruvcal* 'rosette stmcturtrs' nf X-9 nrn rli~mcturwith stain-fillcd ccntral rcglonq of -2 nrn diarnctcrJ4. Thiq 'rowttu' arrangcrnunt I S similar tcl that c~hscrvcdIn sim~lnrstudies c ~ thc f T o r p ~ d onAChR ruccptor. Ion-se1er.rrvc plrr \isr prr~cliu!rd b y modclllny: s~ udlus 05-30-02: Morlcl!rnK stzlrlrcs h:ivu s u ~ c s t c d that 5-HT1-gntcd rcccptorch,anncls arc 1 3 1 wntcr-fiII~r1 ~ ~ porc?, s t r ~ ~ c t ~ i r aal nl vd o g o ~ to i ~ thc nicotinic .~cctylcholinc rcccptclr of t h c ncurtimu~cular i u n c t ~ o n L\ec E l l : CAT nAChli, c b t ~ f r j00). r Mot!cll~nr:thc channcl nq s simple cy!lnrlcrs' suggests a m ~ n i m u mpore size of 7.6A, which cr,nnp:lrcs to cstitr~atcsrd 7.4A for thc nicotinic rcccptor LlwnE s i m ~ l n mcthotls r
0
n
Protein interactions 05-31-01: 5-HT,R-A: .-- .. - RNA trimscripts xynthcstzcd in vitro from thc 5-HTaR14 c I ) N A arc quff~cicntfor f u n c t ~ o n uxprcssion ~l o f 5-HT-gatcd inn channcl.;
with
Protein phosphorylation 05-32-01: Motif< for phosphr~rylntir~n hv protcin k ~ n a s cA, tyrrwnc kinaw and c:iscln k ~ n ; ~ s11carc prcqcnt in t h e nintno acftl s c q ~ ~ c n c of c s clnncd 5-HTaH s ~ h ~ ~ n l t ZEIC ~ ~ "rclntivc ; pcls~tic~iisof thcsv sitcs arc ~ll~tstratcrlin thc lPl3TMl (Fln. I ) .
paflxad:1@19:rk~ c a m l Activation 5-HT,
vat ion r ~ n ddcxcft cit rzml ion
05-33-01: T h c rorponr;c of Intact cclls to 5-I-IT ha4 a very short latencyi, tvp~cal of s r ~cxtmccllular li~nnd-gaacdrcccptor-channul"'"'" MrtfiodoI(\yrr.r~l r , o l r b Suhct:inti;ll rcccptor tlcscnsltlzntlont hchnviour (Tor dulrl~lr. Y C ~ II ~ I I ~ I C ~ I ~ 05-.77) ~ ~ ~ H nccuwt;ltcs I ~ I rapltl mcthotl.; of :1gonist application ~f 5-I-ITconcrntr;ition-cffcct rcli~tionships;ire t r hc ~ tlc.turm~ncd;~ccurntcly.
entry 05
Current type 05-34-01: 5-HT3R-At: Thc S-HT,3R-A-typc ion channels2 display inward currents which R a w a divalent cation-mediated ncgativc slope conductance
in Xenopis oocytes. This pattcm is similar to that dcscrihed for thc NMDA suhtypc of glutamntc r c c c p r (see ELC: CAT GLlJ N M D A , entry 08).
Current-voltage reIation 05-35-01: Most studies of cloncd and nativet 5-HT1R (reviewed in ref.") show currcnt rcspnnscs to 5-HT rcvcrsc polarity a t potcntials close to 0 mV, yiclding nutward current a t positive holding potentials and inword Current at ncgativc holding potcntials (ex. .we rpl''], In a number of indcpcndcnt studies nn native and imrnortalizcdt ncuronal cclls, the pcak singlc channel I-V rclation for thc 5-HT3rcccptor shnws rnodcst inward rectification t h a t is f d l y devclopcd within <2 rns nf thc applicd voltagc step (e.g.
re f,q9.
11- 1 3 .
54-55
E.
Dose-response Evidence for co-operative intermtion of agonist-hindr'nnqsites 05-36-01: A voltage-clamp analysis of thc crJnccntration-cffcct rclaticmship in NlE-3 15 cells (using rapid applications of 5-HT) has dcmonstratcd t h a t maximal and half-maximal inward currents arc cvokcd hy 10p1 and 2 p ~
5-HT rcspcctivcly. The steepness of thc dose-response curvc (slopc = 2.8) indicatcs ca-operative interaction hctwccn 5-HT-hinding sitcs, requiring at Iuast two aEonist mdecwlcs (see 05-36-02In ligand-binding assays lwhurc 5 H T 3 rccngnition sitcs in NIE-115 ncurohlilstorna cclls arc Lhcllcd with ratEialahcllctf antagonists], compctitinn curves nhtaincd with the agonwts 5-HT and 2-methyl-5-HT have also shown slopes greatcr than unity, further indicating co-nperativity within the 5-HT2 receptor. Pattcms d co-aperativity arc also characteristic of hetcrologously cxprcsscd homo-oligomcric S-HT~ rcccptors2. ' v J H .
Inactivation 5-HT,3receptors desensitize in cantrnued presence of ngonist 05-37-01: Synaptic dcpolarizatinns evokcd by mntophoretict applications of 5-HT show rapid desensitization1 hchaviriur when the agnnist i s continually applicd for longer than 100 ms8*'',51r5R. Furthcrmorc, 170th
-
synaptic dcpnlarizatinns and trnnsicnt responses to iontopharesist of 5-HT are hlockcd whcn agonists arc addcd to sLipurftrsing soluticins'. ' H - 5 ~ s R. The differcntiation status of ncurona! hyhridrima NC. 1 OX- F 5 cclls filters the dcscnsitization kinetics nf their cndogcnous S-FIT"-+R''. For a dcscription of amino acid suhstitutions influcncbng the rate of dcvclnpmcnt nf dcwnsitization, scc DO???flinGOFJSCrVflli#n. 05-28,
Phnrmacnhgicol modifiers of recepf or d .sensif ~ izn tion kinetics 05-37-02Cn-application nf 10 .7-10 M tetraethylammonium inns {TEA') with 5-HT is capahIc of pruvunting dcscnsitizatinn in vo'EtaKu-clampcd N1 E-
'
entry 05
1 15 neurd~lastnm3cclls, hut is incapahlc of rcvcrsinR dcscnsmzatiun oncc it has become cstahlishud". 5-Hydroxyindole also slows dcscnsitmtion of thc 5-HT3 rcccptor in thccic cclls"" antl can nvcrcome 5-HT receptor desensitization in the cnntinucil :ipplication nf anonist.
A mdinlnhelled ntynnist thni hincJs t n desensitized conformntions of
the 5-HT\$ 05-37-03:The Imding charsctcriszics of a radinlahelled 5-HTl rcccptar agonist, [ 'H1-mcto-chlornphenylhiguInide (rnCPRGF have revealed two populations cif binding sitcs in mum hrnnes of N 1 E-1 15 ncurr~hlastornacells, with K,, = 0.0.3 -t M I n~ mil 4.4 t 1.2 I ~ M and H,,,, = I1.9+4.2 and 897.9 184.7 fmol/mgprotein rcspectivtly". Competition data sumest that [ 'MI-rnCPRC. lahcls hi~h-affrnitydcscnsitazcd statcs of thc rcccptor.
*
Kinetic model 05-38-01: A numhcr of qtudles (r.g. re~~'"n'2*62 1, have shnwn that the kinetic pmperties of thc 5-HT3 receptor-mcdiarcd ionic current can only he dcscrihed by a complex, to-operative model (we nnce-rrTponsc, 05-3h).
Rundown 05-39-01:S-HT? rcccptor-channcls in ncurons of guincn-pig submucous plcxusH cxhihit high stability. Channtl activities arc rcproducihly evoked hy rcpcatcd applications nf 5-HT cvcn up to 5 h following cxcision of nirtsitlc-outi patchcs, sugjicsting that ncithcr a C. prtitcin nor a ciiffusihlc cytoplasmic mcsscngcr InrictivritIrm, 0.5-.7 7 )
1s
ncccswry tnr thcir gating o r modulation, ( w e nlco
Selectivity 5-HT\? reccptorf (ire prednminnntly No'lK'-sclcutrve under p h ys I oJO~TI ca I u on dit in 11s 05-40-01: Thc ohscrvct! E5 iIT values clnsc ti, 0 mV (see Current-vdtngr w l o t ~ m ,05-.151 arc consistcnt with opcning of nnn-sclectivc cationpcrmeahlc channcls [with Na'/K' at apprr)ximatcly cqual pcrmcahilityl. Under physiologtcal cnnditmns, rcspnnsw to 5-HT would hc prednminantlv carried hp inward movcmcnt of N a ' ions. Selectivity chamctcriqtics of native channels are rctaincd frd lowing homomeric cxprcssinn of cDNAs encoding 5-HTJI antl foll/)w thc wcrik sclcctivity scqucncc amangst mrmovalcnt ions of Cs' > K' > Ci' > Na' > R h ' .
~ - H Trrceptnrs , mc Co2+-permeaIrlein some cell types 05-40-132In addtticin to providing a dcpolarizing stimulus to activatc voltagcgatud Ca" channcls (srr V L G Ca. enfry 421, S-MT4 rcccptor stimulation might rcwlt in Ca"-influx thrnugh thc 5-HT3 rcccptnr itsdf. The 5-HT7 rcccptnr-channel conducts Cn" and other divalent cations in some cell tvpcs qturhud [c.g. for w p ~ r i o rccrvacal ganglion CCIIS" I~c-.,/~~N.l is -O.55, whilc in N 1R ncurohlastoma c ~ l l s Pt ~ ~3/J)N,I , is - 1 12). Thc N E 8 ccll 5-HT1 rcccptnr-channcf 15 cquipcrmcnhle tn Ca", Mg" and Ra" 5 5 . R y using
organic catinnic molecules as probes, it has hecn shown that perrneahility is inversely related to the Eeometric mean diameter of the permcant molecules (rcvicwcd in rcf.") (see n1.w Rlockers, 05-43),
Ionic p e r r n e d d i t y rotios nnd
POKC
si2e
05-40-03: Ion-substitution cxperimcnts in a variety of prcparations (supcrior cervical ganglion cells, N1 E-1 15 neurohlastnma cells, nodnse Ranglion neuroncs and N 18 neurohPastoma cclls) confirm that the S-HT
'.
Single-channel data 05-&I-01:There i s considerahlc variation in rcpnrtcd conductances for 5-HT1 receptor-channels in different cell typcs (SEC Table 21. Recordings have hecn madc under a numhcr of diffcrcnt configurations and thc stnglc-channct prnpcrtics arc complex (reviewEd in re{."7). Altcmativcly, the variahle cstimatcs of channel conductances might suggest thc existencc of several 5-HT1 molecular suhtypcs and/or devclnpmcntally rcgulatcd isohrms [as in NG108-15 hyhrid ncurnncs - see Development regululion, 05-1 I].
Tahle 2. Vnrialiility in reprirtcri' chnnne! crmductonces for .5-HT,~-pited rec t'pi or+ 11I I Rn els ( F r o rn 0.5-4I -0I Cell typc/recnrding mcthnd
Estimated channel conductance Ips)
Refs
Coclic ganglion ncuroncs, guinea-pig: WCR/
10
fk?
SCR
Ncurohlastoma (N181; WCRJFA Nciirohlastema (NlE-I 151; WCRJFA Neuronal hybridnma NGIOX- 15, diffcrcntiatcd cells; WCRJFA Ncuronnl hyhaidnrna NGlOX- 15, undiffcrentiated cells; FA/SCR Nodose ganglion cclls, rahhit; WCR/SCR (chord conductance1 Nndosc ganglion ccIIs, rahhit; WCR/SCR [slnpc conductancu) Suhmucous plcxus ncuroncs, guinca-pig; WCRJ SCR Supcrior cervical ganglion cclls, rat; SCR Supcrior ccrvical ganglinn cuIIs, rat; WCR/FA
0.59 0.,71 3.64.4
S.5
7.2-12.0
15
Z# 15
16.5
M
19.3
M
1S.O/ 19.2
R
11.1
1.7
2.6
1.1
WCR, whole-cell recording; FA, fluctuation analysis; SCR, single-channcl recording.
cntry 05
Consrdcrui ion
,
Z -
rat supcrlclr curvical gangl~rlnncuronq yicltl< an cstlrnatu for single-channel (SCJ conductance that is -4 t~incssmallcr than that nhscrvcd in direct outstdc-out mcrnhranc patchcs. Onc possihlc cxplanatlc~nof such variability has hccn suMcstt.rlJ3 to hc hctcrngencity nf 5-HT.3 rcccptors In thcsu culls 05-41-03: Rccnrdings frotr~ cxc~sctl [ I I ~ I ~ F I ~ u - o ~ ~ rnumllrnnc ~F patchcs from ncurcjncs of gulnc:i-pij: ~ l h ) ! n u c o ~plcx~ts' t~. show two 111st1nct 5-HT unitary currcnts of contl~~ctanccs -15 pS ;in11 -9 pS. Channcls opcncrl 11y 5-HT show chnmctoristtc faat c ~ p c n ~ nand ~ s bursts1 of r*pcnings, supcrfsc~ally s ~ m i l a rto the fratrtrcu of n~cc~tinrc ch;lnncls in othcr ;rutononrrc ncttronus (,F I , C CAT !rAChR, rTn!rrr09).
Voltage sensitivity 05-42-01: Thv rnalority o f studies of n a t ~ v cand hctcroln~auslycxprcsscd rcct~rnbln;lnt5-HT,R currcnts show modcratc inward rectification4 hut thc ~ T C E I S Crnuchanisrn undcrlylng this is unclcar [sr*rCurrr,tlt-vol!r~~c rrlr~t~on, 05-,751.
Blockers
' (+)-Tubocumrinehlocknde shows nrurkcd speclcs dr/fercnc~s
I
05-43-01: 5-HT,?-gatcd channcl currents arc potuntly rcduccd hy (+Ituhacuaarine (ht~rqer, rllco Eld[: C A T AT[), ELC; CAT nAChK, nnd YLC: Cn. r'ntr1r.r 00. 09 c ~ r ~ 411 r l T h c htgh scnsitivttv typical of thc c!onuif 5-HTJII-A tsoforln t o ( t )-tl~hocur;jrlnc~ ~ B Shucn ohsurvctl i l l ,I nurnhcr of n;lKlVC t i s s ~ ~ c[nod(l~t' s gnnglion nciironcr sntl superior ccrvical ganglion cclls) ant1 cll~n;ll ccll 11nt.s". In gcncml, 5-HT, r c c c ~ ~ t r , r - i i i c d ~ : ~rcspotlscs tt'~I show mnrkctl interspecies diffcrcnccs - for cxamplc, thc conccntratlnn of 1-tl-tlll~ocurarinc rctlriirctl for 511"L inhihition can v:iry t ~ vrip to -10000-foIt1 acroFs nclrlosc ~ , ~ n g l i occlls n of mtjusc, r:ih!at nnd g l i ~ n c a - ~ i ~ "In. k c c p ~ n gwith t h c ~ cnhscrvations, mollsc sntl rat hornologucs of thc 5-HT,?R-A, lwhich only havc l h amino acid rliffuaunucsl cxhillit :I 100-fold rliffcrcncc in 1-t-l-tuhocurarinc s c n s i t ~ v i t" ~~'
Supprcsq~nnnC inwnrd 5-HT-~nduced curront hv extr~~cc!lu?nr cntions 05-43-02: Divalent cations such a s C;I" and M ~ ? cxcrt ' 3 prontl~tncctl rnodulatclrv influunct. on 4-HT I-inctlintcd rcsponscq. Roth nativct 5 5 snd ~I~IIIc~"-HT~ rcccptrtr-chnnnals show supprcsslon of 5-HT,,-clicltctl ~nwnrrEcurrent in the prcwncc. of thc divnlcnt catirms ~ n and " MR" in thc cxtr;~ccllul;lr motlilim at plivsirdogical cnnccnzratlons. IlctIuct~on in
<'.
cxtcrna! C:;I'' corlccrltration auKintnts thc :~rnplttudc nf dcpolarizlng rcsponscq 'tu 5-HT 111 NG 10%-15hyhrld cull';, whilc hrttlr ~ n ?ant1 ' M ~ ' ' (as physioloxacal conccntration~l modulatc arnplrtildo nntl c1rrratir)n of 5-HT~ntlrrccrlcvrrcnts ( r x ~ v ~ r w cEn ~ rrr>T.'~. l
entry 05
An apparent difference hetween cloned ond natrve S-HT3 receptor-
channels 05-43-02 The blockinp: and/or modulatory effccts of extracellular divalent cations have been shown to he voltagc-scnsitivc (lor hoth cloned' and chirnaeric 5-Srr, receptors"'j, with Ca" and Mg2+ introducing a region of negative slope conductancc into the I-V relationship upon hyperpolarizatim'. For notive 5-HT.3 receptors, divalcnt cation blockade appears to be voltage-in sensitives4*".
Channel modulation Potentiation of S-HT,? current responses 05-44-01: Electrical responscs elicited by S-HTA receptnr activation can he potentiated fmm suhmaximal responses hy ethanol and the dissociative anaesthetic ketamine. Trichloroethanol enhances current responses through both nativc'**67 and cloned receptor-channek
1
Equilibrium dissaciation constant 05-45-01: Saturation analyses of S-HT3 receptors from NlE-115 neuroblastoma cclls with [%]-GR67.130 ligand shows high-affinity binding to homogeneous populations nf sites in hoth soluhilizctl ( K J = 0.05 f 0.02 n ~ ) and purlfwd (& = 0.10 f 0.0411~)preparations. Cnmpetition experiments indicate that the soluhilized and purified receptor prcparations retain thc characteristics ohserved in N 1 E- 1 15 cclls in Y I Y O ~ ' ~ .
Ligands 05-47-0 1: 5-Hydroxytryptarnine (5-HT; serotonin 1.
Synaptic potentials medlatcd hy 5-HThave been recorded from hrain sliccs in sitv". Fast ncurotransmissinn in mammalian brain can therefore he mediated by amines as well as amino acids (see ELG CAT GLU,entries QJ und 0s).
05-47-02: Available radioligands for thc 5-HT3rcccptnrxhanncl include [%I]GR65630, [?H]-ICS205930, ['Hkzacopride, IJA]-GR65630, [3A]-quipazine, [ 'H]-granisetrnn, [ 3H]-LY2JR5Rd and I'HF-meta-chEomphenyl'biRuanide[['HImCBRGJ.
Receptor/transducer interactions Role of S-HT>qchnnnels in moddoted neurotronsmitter release 05-49-01: Thcrc is no indication that S-HTAreceptors modulate adcnylatc cyclasc activity, although therc is evidence that thcy moclulatc thc rclcasc of a varicty of CNS ncurotransmittcrs including acetylcholine", cholecystokinin", dopamine", GAR.4'" and noradrenaline (see also Bhenotvpic rxpression, 05-14). An analysis of rclaxant And contractile effects on ccrchmvascutaturc mcdiated by 5-)-IT rcccptors has been rcviewedmx. A '5-HTt-likej receptor which mediates a nnn-dcscnsitizing inhibition of rat medial pre-frontal cortical neurnns in vivo and coupling t o phospholipase C has heen repnncd (crted in ref.s7).
cntry 05
Compclnsnn of roles for 5 - H T nt differen!clnsscs of receptor 05-47-02 Fast aminc nc~rrt,transinissionvia 5HT1rccuptor-channels can bc cnntrastcd with thc s l ~ wm h i h i t m y svnnptic potontinls rncdiatcd hy 5-HT agnnism through G protcin-linked siEnalling pathways. Thus the malority ot known rcccptcx molcculcs fnr 5-MT cnuplc t o cffcctors (rncluding ion channcls) t h m u ~ hC: protcins. C. Protein-linked 5-HT receptors inchdc thc suhtypcs S-HT,,, 5-HTl13(cquwalcnt to -5-HT,,,,], 5-HTl,- (now 5HTlc-1, 5-HTl1,,, S-HTI,,,~, S-HTlr, S-HT,,, 5-HT,, 5-HT,, [now 5-HT313),5HT4, S-htGv,5-ht;,!, S-htr, and 5-ht, (Tor d/ww p i i l h w c i y ~ .we Rcsnurce A , entry 56,rind rcfcr t o t hr I i i t r \ / I t JI’HA I< Norncrrclol i i w Comtriirtuc rccmtirncndntionq V I I I thr C S N - scc Fccdlwrk mnd CSN I I C L ‘ P F S .cntry 12).
L
Receptor agonists (selective) Present S-HT,?R ngonists (ire som~tjrnesnon-selective ond hove vorioble Efficncy M ~ F Sspecies 05-50-01: Derivatives of thc endaRcnous ncurotmnsrnit tcr/agonist 5-hydroxytryptaminc such a s 2-melhyl-5-hydmxytryptamine act 3s partial agonists a t other sites and sgonists such as 1-phenylbiguanide (PRGJand rneta-chlorophenvlhiguanide ( r n C P K Jarc ot varinhlc ctficacy in diftcrcnt spccics5’. 7’. Thc ctmpnzinrl SR 57227A has hccn ruportcd tci act RS n h l l and 5-HT1sclcctivc n ~ o n i ~ t ~Inforinatirin ”. ahorrt rts cfficacy (in a widc rnngo of prcp;irations was nnt availahlc. a t thc tinic id compilatinn.
Receptor antagonists (selective) 0 vervi c w (I/ 5 - HT,?- s cIC c t I V P (int iI,coR I.< t s 05-51-01: r h u ‘nperational definition‘ of 5-MT1 rccuptors i s hascd upnn (11 rcsistancc t o antagonism hy cr~mpounrlsacting at ruccptor classcs 5-HT,, 5-HT7and 5-FITt, whilc. [ i i ) rcsponws clicitcrl hy 5-HT should hc mimickcti hy agonistq ‘sclcctivc’ for t h c 5 - H T I rcccptor Ins wvcral prcscntfv usud aRoni
S-HTqiantagonist and altcrnativo namu MDL72222 {Remestran) ICS 205-930 (Tmpisrtmn)
Prnpcrtics
Refs
cntry 05
a
Cantto!
fz !k OR 87330
OR a7330
/LAlornv
/L
111
b
ICS 206-930
Control
ICS 206-930
i*-
c
/
y
o
r
l Smv 1s
d Response to 5-HT
Synmpzlc potential
,
40 m@
Wash
(1 pM)
1100 nM)
.-I
1"
Wmnh
--#
,
o
/
y
;
o
oa eras
OR era30
C 0
-= -
GR 38032F
rrC
0
1
10
100
1000
loow
Antagonist concentration (nM)
QR 38032F
1
10 100 1000 AntagonlBt concentratton InM)
ExflmpIcs o f reversible hlockade .of synnptic potenlials hy 5-HT3 nn trr,yonlxts. (n) RIncknde hy GR(i7330 of synopirc responses m the Ifiierrrl n m y ~ y d f l l ~cvokcd lR Fry electric01 srirnrrlr' consrstms of folrr raprd pl~lsesfrom n hold in,^ potent In1 o f -90mV. (h) Rlockode b y ICS205-930of dcpofnrizution evoked hy ionlophorct ic tnpplicntrtln o f 5-HT. The second r~ndthird records were ttnkcn IOmm Jollowrn~thc change tcr drug-con!nining snllltion, whrk ~ h rfnurth : wns nhtnined followins 20m1n woshrn,r. (c) Redrlrtron of ~ynr~ptir potcntinl following oppIE'carion o f 5-HT7 nnmgonlxtq GRh7,730. ICS, CiR.78W2F. (cl) licdr~ction o f rpsponsc to rontophorclicolly applicd 5-HT lollowrng nppJfcntion o f 5-HG itntn~onisss.(Reproduced with perrninrnn lrom Sugitn ut al. (1992) Ncuron 8.199-2W.)(From 0.5-51-04) Figure 2.
Many antagonists havc bucn dcscrihcd (for review, see K m g , in Jones et nl.. 1994, pp. 1 4 4 , under Rclated .wurces el rewicws, 8.5-56)and suvcrnl ncw compounds are prcscntl y hcing cvaluatcd.
Tlrerrrpetltic applications of 5-HTh7 receptor nnt(~,qonists 05-51-02: 5-MT,R antagonists hilvc sclcctivc anti-emetic properties, hoing ufficacinus in thc cantml of nausea and vnmiting rcactlnns to canccr chcmnthcrapy and radiotherapy. Cr~tical rcvicws on the therapeutic potential and ncuropharmacnlogy nf 5-HT? rcccptor antagonists havc apFcarcd7', including their use as anti-emetic drugs and, in triajs, ss antipsychotic, anxioly tic and antinociceptivc agents7"74-7'.
A n ! n ~ o n ~ sbinding r c l t r c ond ~ p r c i rd~ffcrrnccs ~ 05-51-03: Thc dcvclopmcnt o f cclcct~vcantagonists for 5-HTI rvcuptrlrq hns rcvcnlcrl hinding sitcs for t h ~ qsuhtypc In autcmninlc ncuroncs and nlqo In thc CNS (cr,r1I : r l l - t r ~ ~c.xprrc\;on ~r. ~ridr~s. 05-081. Not,ihl y , 5-HT, rcccptrlrq in gulnca-pig ~ l c u r n , col(~n ant! vnKu.; ncrvc preparations chow l ~ t t t c
hctcrngcncity rn 5-HT, antngoni.;t ntl~nitiu\. In comparl'oll, ~ ~ i ~ I a trat cd vagus ncrvc prcpnmtions show ,iffinit~c.;w h ~ c harc -10- t o 100-h1ld h~ghcr 7 7 7H thdn gulncn-p~g Slgnlficnnt .;pccic\ iliffcrcnccs .Ire ~ u m n ~ ~ r l zinc tr! ~ f . ~ ~
Reversrhle hlouknsfe of synopt 14. pot rnt la].< b y .'5-HT.?(nntcl,qrmict Y 05-51-04: Some cxainplcs of rcvcrs~l,lc hlockatlc by 5-HT1 AntAgonlYts Arc ~ l l ~ ~ s t r a tinu tFir: l 2
),~lx*,:~,l,iw~ [,0,:4*#
4
! I , w;,
Datahase listfngs/primary sequence discussion 05-53-01: Thr, rr.lr-vi~ntdntr~llriroI.\ ~ n d mtcd ~ c Ilv rhu kowcr crric prchx (r,fi g!) ) which \ / T O I I no! ~ ( ! /?tt y p ~ d( ~ L ' Pf ~ ~ t r o ( j z ~ c~r4~7hoiry~o ~ ~n{t r,tl!rw$,pntry 1121 Do t r ~ l ~ i q Ioc*ur r* nmnlcc o ~ l drrLccr rlon nurnhcrs I rn~ncrl~r;!cly lollow the
colon Notr {hot ( 1 i - o r n ~ ~ r e ! ~ (!I\! , r rn.y t ~ ~o/ ~ ~ (~! / I~ ( I V O I I ~ / ~ I Lliccc\won ~ nrlrn??rri ~r r~rpr.r/lt~oli\f',u I ~ I L I I I I O I Iof rcfcvir~~l \cqlrcncc\ In C;rnlEnnkl' rrastlurcr\, w!71(*hI J ~ I ' IIOW (IVIIII(I/>IL' W T hE ~ O W L ' I / ~inI ~l u ~ ~mejRhbn"rjllRt lt oaalyds rolrtrnr7\ (for (Ic\r.rrpt ;on. \rrp !hr II(i!rr/?n\t~I ~ i t l r ~Iir,Jrl x ~ In tllc / I ? ! rr)(Iuct 1011 CJ I i i i ~ i r t o/ ( T r ~ ! ~ i ~ Jm\ , t rt7 02) For ( T ( I ~ T I ~\ ()l!( ~ / t ,~,( R ! ~ i *of~ J v .r rtn %-\j,rrit,\ vrrrrrrrl!i or rr~lr~tc,d grxnr, /onlllrlt n~r,lnhrbr\ crm rcnrlllv r:r-c*c'\w7rlh v onr. ox two T O ~ I ~ I01~ \~ ~ r ' i ~ h ! y r I)I R~( ~I / Vf \,I n, (which ~~ rlrr hriwd o r ] prr,-r.o~npt~t or1 nlrenmrw! r pcrlorlnrd 11wn.y 1 F ~ r u I I I ~ A S T(ilgr~rr ~ i hrn I,v thr, N C I ~ ~ TIFI$ ' ) flbcltt!rr 1 9 1710\t ~:\cfl:I kw rctr~r~vrrl of zcprprnt.r, runrrlrfs d~,po\i!ed rrT d ( i l ~ ~ ! > rlt~/t,r ~ w \ t l x ~ tihoc(, ~ 11\fr(!/w~lowTh11+,,rLJj~r(,\cntr1trvr ~ n r m / r r , rol ~ known uxr/lrr,nrra ~ ( I I I I O I O S ~gI r o l ~ p ~ n ,itrt7 y ~ II\IC(I to pcrrr~lt lnrtro/ drrcct rt.!rltvrnE~ h r r nuururc~on ~?trml?r'r,rI?~thor/rcf~~rerm or ~~c~rnrncfr~rrrre Follow~ng -rI~rcc! rnut.ras5~r~r~, howr,vrr, - -- n - c ~ ~ h h o ~ r rc~rlr~ly\~s r r ~ ~- -t .. i\ r!ror?~ly - ~ cc t ~ ~ ~ ~ r n c- nrrrFcnll/y f.r-, r- l ! o--n-m--d yrr~l~orr -ran-s~nrE - rrlr~!r~~~~cqt~criucr. I
Nnmcnclatiirc 5-HTI rcccptor prccurcnr: 5HTIR-AI ('long form'l
Specicc;, DNA
Acccwon
sourcc
Origrnnl ~solatc
Mousc ncurohlnstoma ccll cxprcssron tthrary
487 ;la; ~ g : gh M74425 23 nn ( 4 h i , plr: PLqYJ9 inatlire procltc: protctn) crr7 I'S0023h
FI"jTM1 55966 13.1
(from cl3Nh)
~
r
p
ScqucnccJ discussinn
Mnr~cq, Sclrmcc [ 19Y 1 1 254. 4.32-7.
cntry 05
Nomcnclaturc S-MTAreceptor precursor: 5HTxR-A? (’short form‘)
Spccics, DNA so~irccu
Original
Rat superinr cervical gang1inn library
461 aa
Acccssion
Scqucnccl
discussion
1solatc
not found
Likely rat variant of mousc S-
Fohnson, Soc Neurosci Rhs (l9UZ) 18: 11815.
I-ITqR-A,
5-HT,?rcccptor precursor: 5 HT.1R - As [‘short form’)
Rat superior
partiat cDNA
cervical gang1ion library
not fnund
IsenhcrR, NeuroR epor z [199,715: 1214.
rcccptor Mousc ncuroprccursor: 5hlastoma ccll MT3R-Aq lint NlE-115 (‘short form’]
460 aa
sig: 23 aa 53 17R Da [from eDNA)
gb: X7L395
Hope, E m 1 1%o rm o cnl (1993) 245: 187-92.
Related sources & reviews 05-56-01: Maior sources t h a t includc suhtypt dcfinitians”.“ ’.*’**,’; nthcr snurccs with a rcvicw clcmcnt includc - thc cstahlished and potential
therapeutic uses for S-HTa reccptor antagonists; hchavioural pharmacnlogy of S-HT\%rcccptor antagonists7’ thc ncumcndocrinc pharmacohm of scrw toncrgic (5-HT) ncuronmAd;molcculnr cloning and functinnal cxprcssion’; advances in clectmphysiological characterization of 5 H T 1 receptnrs’. ?.’; soluhi 1ization and physico-chcm ical cl~amctcrizatinnof S-FIT,?rcccptor-hind ing sitcs (Miqucl el at., lYR3, ~ e ~ I Rh Nenmsczcnccs, ~ s Vol. 1 1 - see Xesourcc E - Ion channel hook refcrenucs. m i r y 601.
’‘;
Rook references 05-56-02:
Andrews, P.L.R.and Sanger, G.J. (cds) (19921Erne in Anti-Cancer Therapy. Chapman and Mall, London. Fozard, J.R. [cd.j I19893 The Penpl~ernlActions nf 5-Hydroxytryprarnine. Oxford Wnivcrsity Press, Oxford. Frrzard, J.R. and Saxena, P.R. (cds) (1941) Serotonin: Molecuhr Biology, Receprors nnd Ftinctional Effects. Rirkhauscr, Bawl. Hamon, M. [cd.)(19923 Cenrrrzl and I’crrpheml S-HT,?Ileceprars. Acadcmic Press, London. Joncs, R.J., King, E. and Sanger, G.J. ( e d s ) 1994). 5-HT.7 Receptor dnfa,enniqts. CRC Press, Roca Raton. Saxcna, P.R., Kluwcr D., Wallis, D.I.,W(iuters, W.and Bcvan P., [cds] [I9901 Cnrdinvmscu /or P l ~ i ~ ~ m i ~ o ?of o g y5-Hydroxyt ryptnmincr Prospective TherrrpuuIic Applicntrons. Kluwcr Acadcmic, Dordrecht. Stonc, T.W. (utl.) [ 199 13 Aspccls of Synapric TmnSI??iSSz#n. LTI’, Gokanin, I1piord.s. Aritonomic and 5-HT. T ~ y l o and r Francis, London, New Ynrk.
cntry 05
-
Feedback Error-correct ions, cnhr~rrccmentond ex!ensjons 05-57-01: I'lcnsc notity spcc~flccsrnrs, c i m ~ ~ s i o nupdatcs s, ant1 commcnts on thrs entry hy c o n t r ~ h u t ~to n ~~ t se-mail feedhack file (for dr~trrzls,sce RCFOIFTCL' I, SP(IT(-JII;1rj!(,r111 ~4) CSN I l c v ~ l ( y ~ n ~ r lFor l t ) .this entry' scnd cmail rnrssagcs TI): [email protected], inrlicat~ngthc nppropriatc paragraph hy cntcring its six-figure index number (xx-yy-zz or other idcnt~flcr]intn thc Subject: flcld nf thc mcssagc ( c . ~ Suhjcct: . OK-SO-O?]. I'lcasc fcctlhack m only one specified paragraph or f i g ~ ~ rper c message, n o r ~ n ~ l hy l y s c n d i n ~a corrected-rtplwcrnent ~ c c o r r l ~ton ~zhu gui~Eclincsin Frpdl>ack c+) CCSN ~ c r c 5 r. E n l ~ ~ ~ n c c n ~ant! c n t oxtcnslcins s can also hc sumcstcrl hy thlr rovtc ( i h r d ) . Not1f1t.d changcs wlll hc. rntlcxcd via ' I ~ o t l ~ n k sfrom ' the CSN 'Homc' page (http://www.lc.ac.uk/~xnJ) frcm mid-1996.
Entry suppnrt
group^
nnd e-mrril newsluttcrs
05-57-02: Authors who have cxpcrtise in nnc or marc ficlds of t h ~ cntry s [and arc willing tn P ~ ~ F V Iuditonnl ~ C or othcr support for developing its cantcnts) can join its suppnrt group: In thiy cnsu, sand a rncssagc Tn: CSNOSkWe.ac.uk, ( c n t e r i n ~thc worrls "suppnrt grr~up"in thc Suhlcct: ficldl. Fn thc mcssagc, plcesc ~ndicntcprtnc~pal inturcsts (sut. ficldnr~rnecritrrm In the Intrl)dl1c'tlon for i.ov('r~;p,) t o ~ c t l l ~with r any rclcvnnt http://www site links (ustahlishcd or proposctl) ant! d c t a ~ l cs ~ any f othcr pclrsihlc cnntrthut~nns. In tluu coursc, support group n~crnhcrs w ~ l l (optionally] rccclvc e-mail new~lettrrs1ntundc.J to ca-ordinate and devclnp thc prcscnt (tcxt-hasurll cntry/f~cldnanic framuwork.; into :I 'Irhrsry' of intcrllnkctl rustiurccs covcrlng Ion clrsnnul s i K n n l l i n ~ Othcr (morc gcncral) ~nformation of intcrcqt t o cntry contr~hutrlrsmay alst~hc sent to thc ;ihovc ;~CIIJTCSS.for group distri2~utionant! fccrlh~ck.
Ilr / l)hnrn~ot.o/ ( 1957) T 2: 32.2-8. M:~r~cq, Sclr5nt*r, (199 1 ) 254: 4.12-7. " Hopc, Fur I I'l~rirn~r~cnl (19931 245: I X7-92 Ht~mphrtsy,Tr,,r~dcI'hnrnmnrol Sri [lYY.7\ 14: 23.1-6. ' IuIiub, Annt~Sicv NL'IITOCL-I II0F)IF 14: .135--00. "" I'croutka, Nt,rrror.hcn~(19BRl hR 408-1 h. R~chardson,Trr.nrl~ Nc~iro';cl(1WhJ9 : 424-6. 19crkacl1, Noflrri>1 1989) 339: ?Oh-'3 I)C~CTF, Trerdc I'hr~rr?rrrr.olS ~ ( II YXY) 10: 172-5 '" c ~ l t , I%vvcrol Lorlrl [ 19Xq 41 I : 2.57-69 *' NLamhcrt, Hr / I)h(lrnlncol {IYH9l 97: 2 7 4 0 . l2 YakcI, Ijroln H r j r (lY901 533 46-52, II Ysng, l'h~.c;olS.oncl'ot~1 l W 2 ] 448 237-56. " F i ~ r ~ t k a w[;Nr~,rophycrt>l ~, (19921 67: X I 2-19. S h ~ n , N ~ ~ , ~ o p h ~( I' Vc Y~I o) (15: l 630-X. ''l 7 T~cota,I'rr~t.Nrrtl Autrrl Sr, I I S A (19'9.3) 90 1430-4. Wcrncr, .lot. N i ~ ~ ~A!)< r i ( ~I V9<31 ~ ~ 19. ~ iI T (34 *'I V S u ~ t t a ,Nrwrott 1 I LIB21 R IY0-20,3. ~ [ I BHYI 251. 811.3-'9. Ill:an~lina, I'l~arrnirr.olF Y Thrv ~G;(llciuln,
"
I
entry 05
"' 22 2.1
Fiang, Rrnm Re< ( 1990) 513: 156-60. Rlandina, Eur [ l~horrnncr~l (1988) 155: 349-501. l ~ a ~ ~ t lHr ~ c[cI~horrnoccd , ( 1 991 ) 103: 17904.
Fcucrstcin, Nrrt~nyn-Scl~rnr(~drht~r~~ s Arrhh l'hnrrnncnl ( I YHhI 333: 19 1-7. Knpert, 1 l'hycml [ I Y Y 1 ) 441: 121-6. Martin, Rr J I'hnrrnncol(11V92) EOG: 139-42. 2n 3llicr, Hr I Phormocol ( I YY.3) 108: 13-22. 2' 13landin;1, I'hnrn~ncof Ex!) TIlcr ( 1 991 ] 256: 341-7. 2R Goldfarh, PI~nrrnncoiExp T l ~ r {r I 993) 267:45-50, 29 Harnes, N(~rure(IY89)338: 762-3. 30 H~ancht,Itr I)hnrrnocrll ( I Y Y O ) 101. 448-52 " Maura, N r ~ r r o r h ~ '/ m 1992) 58: 2-3.14-7. " Johnson, Snc Ncurnqc: A hc ( 19YZ) 18: 11.7-1 5. " Fozarrl, Il Sci ( l YY I 1 12: .E' 10-1 5. Fozarcl, N~n~~nyn-Schn~ied~her,y'.r; Arch I'hcrrmrrcoI (I9X4)326.3 U 4 . 7n I3utlcr, Hr I I'l~armncol1 PYRH) 94: 3YJ412. 7' Sangcr, Eur I I'horrnacol [ I'IXYI 159: 1 13-24, " Kilpatrick, Nnunyn Schrniedcl~rr.y'.r:Arch IJhormncol (1990)342: 2 2 4 . 77 Grccnshaw, Trcnds Phr;rn~(lcrllSr*r( I W , ? ) 14: 265-70. 74 RI~SSC! I, Rr 1 dnncqrh 11992)h9: Suppt. 1, 113-8s. " CostaII, Ilr Cunr-er ( IYY21 6h: Suppl. I Y, 52-SK 24 25
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"' *'
'' ''
" " " '' "
" '' " " "
.o-zo~'. h r 11-66 ~ I 1 I 177 S113rI ' Z I ~ '(JrC-hHZ : I f / [hh 11 /07WO.I, ~ 0 . 7 1 1 1 i U ~ l21r1)/ / t ~ f71Zlfv'N'Y 3P UPA 't.Ie-f61 . I 1 ( l h h 1 ) ' 4 1 1 t ( l J 7 . 7 ~ ' J ~ L ~ H .h-P<) .fll {h*fj\\ 12s l l ~ I L 1 J l ~ t / ( [\ [ ? l I t l l ~ ' % l l > c ~ l . L Z - ~ I E [Oh(;lI ~ L E . " I ~ ~ ) ~I I( I~ ~ $ . ~I J III :I) , E'.~PPOIR ' 5 1-01 P :C Iv6R I I [OiLfOI!l.7N l l ( l ~ 1 1 ! ( f ( ]1 1 / 1 3 ' 1 ~ 0 3 . 7 L hot.-667 'LEZ (I'66II /(k"il"J"Yltl l f i t 3 ' J ~ ' { ~ P I ! .x- I 6s :IOI (ohh I 1 I ~ ~ ~ ~ I ~I JI HJ '~31rnti I ~ Z ~ ~ % - I 65 101 { L S ~/()I ~J r j ~ ~ ~ J r ' ~/ ! ( i ' ~ ~ 1 ~ ' [ 3 1 1 p ) . l r ~ r r l J r ~ r ('11f'l~o3 ,l
I
'TO?-Ix I 'LD { o h h ! )J,?rf.r
Edward C. Conley
Entry Oh
Abstract /genera I description 06-01-01: In cxcitable cells, extracellular adenosine-5’-triphosphate (ATP)can act as a truc ncurotransmittcr by dircctly activating P2x subtype receptors which possess intrinsic cation channels ldcsignated FTxR-channels for the purposes of this entry). ATF-activatcd channels Rencrally confer an excitatory, dcpolarizing cffcct nn cells through their pcrmcahility to Na’ and K’ ions and En many cases thcy mediatc agonist-induccd CaZ’-cntryy.
06-01-02: Prior to expression-cloning1 of cDNAs encoding two distinct ismformst of PzxR, firnctionnl similarities with S-HT.3 and acetylcholincRated receptor-channcls Icd ta a general expectation that FzxR w ~ ~ u sharc ld structural charactcristics with these proteins. Surprisingly, the genes cncoding PlXreceptor channels predict a distinct subunit structure atypical of othcr cxtracellular ligand-gated reccptor channcls, consisting of [ i ) two transmemhrane domains, [ i i ) a largc cxtracellular portion [typically 5 6 6 8 % of total protcin length, rohahly comprising thc ATP receptor domain), (iii] a pore-forming matif reminiwent of potassium channcls, and [ i v ] rclativcly short mtraccllular N- and C-terminal domains.
r
”
06-01-03: The physicilogical rtllcs of PIX rcccptor-channels tncludc fast signalling across synapscs ot thc ccntral and peripheral nervous systems. In thc periphery, PzxRxhanncls arc involved in control nf effector structures such as cardiac or smooth muscle. For example, coeliac newrones that innervate thc gastrointestinal blood vcsscls relcasc ATP on to Pzx receptorchanncls cxpresscd in vascular smooth muscle to constrict the blood vessel. Ca” influx through ATP-activatcd channcls is also Iikcly to play important rnlcs in nevmqecretory processes, whcrc the cffcctor may hc (for cxampEc) an cxocrinc ~ l n n d ccll. 06-01-04: P,, purinoccptflr-channuls arc prcscnt both a t nerve tcrminals and ccll hodics of pcriphcral and ccntral ncuroncs. In thc CNS, ATP-gatcd channel4 are likely to mcdiatc rapid sensory, motor and cognitive functions. ATP has hccn dircctly demonstrated to act as a fast excitatory synaptic transmitter at nerve-nerve synapses hy activating 712x receptor-channcls.
06-01-05: ATP scwcs as a co-transmitter of acetylcholine (in postganglionic parasympathctic neuroncs], substance P (in sensory neuroncs] and noradrenaline (in postganglionic svrnpathctic neuroncs]. In kccpinp with rolcs in ncunitmnsmission, P?xR-channels activate with shnrt latcncyt ( i x . in thc ordcr of tens of milliseconds). Shortening of hnth activation and inactivation tirnc cnnstantst with increasing agonist conccntrntims havc
bcen dcmonstratd in somc prcparations. 06-01-06: In musclc and othcr cells, P,,R-channels display r h a l cxcitatnry functions by bath dircct cntry of Ca” through thc channcl and, via the depolanzrng effects of Na’ entry, activation of voltage-gated Gal’ channcls. In
entry
Oh
I
L
general, activation of P2xR-channels does not affcct phosphninositidc hydrolysis and suhscqricntly rclcascs only minor amounts of Ca2‘ from intracellular stores. 06-01-0J: Molccular cloning nf thu PzxR cDNAs has also rcvcaled a p o w i b l ~ link with apoptosist. Thc dcduccd amino acid sequence of one P2xR isoform is identical to that originally identified (3 years earlicrl as the RP-2 gene, activated in thyrnncytes induccd to undcrgo programmed cell death1 . Thus in addition to I t 5 role in fast signalling, it mtiy hc prw,ihlc that thc PTxR/ RP-2 protcin may also function as an ‘induccd rcccptor’ for ATP or another metaholitc rclcascd during apoptosis (,Fee Developmcntd reguhtion, Oh- 1 1). 06-01-08: There IS heterogeneity of PzxR suhtypcs, as indicated from distinct cDNA sequences, mRNA transcript sizcs, markcd diffcrcnces in currentvoltage relationships, single-channel propertics, aggcinist sclcctivityf, desensitizatiant hchaviour and variahlc pcrmcahilityt ratms.
Category (sertcode) 06-02-01: ELG CAT ATP, i.e. extracellular ligand-gated cation channels activated by extracellular adcnnsinc-S’-tripbosphate. The sugqestcd electronic retrieval code [uniquc cmhcddcd idcntrficr or UEIJ for ‘tawing’ of ncw articlcs of rclevancc to thu contents of this cntry is UEI: P2X-NAT (for rcports or revicws on nativct channel properties] and UEl: P2X-AET (for rcports nr rcvicws on channcl properties applicable to heternlog:nuhlyt expressed recomhinantt suhunits cncodcd by C D N A S ~or RcncsT]. For (I diwrssion ol the odvontsigec of IJEIs crnrl gujrlclines o n thcir implcrnen!afJnn,sre thr s r ~ : r o non Resoiirce I under intrdiiction d loyoul, entry 02, und for hrrther dctciils. scc Restiriruc I - Scorch urrferin CSN developmmr. entry h5.
Channel designation 06-03-01: Pzx-likc purinoccptars; P2xR-channels; the P?,x purinorcccptorchanncl; thc purincrEict channel; the ATP-Rated channel. Nate: For the purpciscr. of t h i ~entry, characteristics of the twn prototype PzxR-channels cxprusscd froin cDNA’.’ arc disttnguished by undcrlined prcfixcs dcnoting their rcspcctive opcn reading framc lcnnth, i.c. P2xR (ORF 399aa) and PzxRl - -(ORF 472 aa] (we Grnp Irimily. Oh-0.5). In official nomcnclaturcs, it has h e m rccornrncndcd that thc ’R’ suffix in ’PIxR’ dcsignations should be avoided. Fts use in thiq cntry is thcrcforc only for convcnicncc.
n 1
Current designation
06-04-01: Curicnts conducted by PI, receptnr-channels havc heen referred to as L C , A r P .
Gene family
P2xR-chnnncl primnry structure is distinct from other intexral ELG recPplfr-chnnnels characterized I n dote 06-05-01: Prototypc cDNA sequences encoding two distinct isofnrmst of Pzx-
Table 1. Genes encoding PPxpurinoceptor-channels (extracellular ATP-activated channels) (From 06-05-01) Subunit"
Description (for sequence discussion, see Database listings, 06-53)
Other distinguishing features
Encoding (OW
Molecular weight
P2xR (ORE 399 aa) gb: X80477l Equivalent to isolate RP-2 (see below)
Cation-selective channel; relatively high Ca2+-permeability; functional properties resemble those of native channels expressed in smooth muscle
Cation-selective channel; relatively high Ca2+-permeability
399 aa Sigh Transcript sizes: 1.8, 2.6, 3.6, 4.2 kb (see m R N A distribution, 06-13) 472 aa Transcript -2 kb (partial, non-coding)
-45 kDa [calc.)" -62 kDa [native, by
p2x R1 (ORF 472 aa)
Two putative transmembrane domains plus pore-forming motif; single subunit appears sufficient to form 'fully functional' channels. cDNA isolated from a rat vas deferens cDNA library by expressioncloning+ As above; cDNA isolated from a rat phaeochromocytoma (PC12)cell cDNA library by expression-cloningt The partial amino acid sequence of RP-2 appears to be the same as the PzxR isolate [ORF 399 aa) listed above. Now considered to be representative of an 'incompletely processed' primary transcriptt
gb: U14444' Isolate RP-2 [partial sequence)'
Initially described3 as 'apoptosisinduced protein' in thymocytes (for details, see Developmental regulation, 06-1I )
SDS- PAGE^ 1
-52.5 kDa (calc.) -
"In the absence of s stematic nomenclature (as going to press), the predicted (non-glycosylated) mol. wt based on [i)the cDNA open reading frame'( (ii)GenBank"' (gb:)accession numbers and (iii)trivial names are used here to distinguish isolates reported. 'Charged residues in the first 28 aa suggest the absence of a secretion leader peptide in this isoform. "This [calculated)mol. wt agrees with that of an in vitro translation product labelled with [35S]-methionine'. The higher molecular weight of the PzxR solubilized from rat vas deferens (62 kDaJ4is likely to be due to glycosylation of the mature protein.
1 Suhtype classificatinns Relo i iomh iy of P,, r m q or-ch ~ nnnels to oi her purjtwccp t ors 06-06-01: Rnscd o n pharmacological and functional propcrtics, purinoceptors wcrc classified as PI or lJLdcpcndin): on thcir prcfurcncc for ndcnosinc o r arlcninc nuclwtidus, rcspcctivuly. Extraccllular ATP oxurts its cullular cffccts vin Pz r c c u p t d I”? rccuptcirs arc hirthcr suhdividcd anto t h u Pzx subtype [refs'.' a n d this cntry) posscs\inK an integral ~ ( i n i cchanncl, while the purinoccptar svhtypcs P,y and P71Jarc G protein-linked receptorsH(see K P W W ~ G PA , rntry 56).Two furthcr whtypcs, drc PZT receptor a n d the P2z receptor may also possess intrinsic catinn channels. P,, suhtypcs have rcccivcd most attcntron, ant1 in suvcrd sturlics no whtypcs arc dcfinud hcyond thc 1’2 c a t c g n v . 06-06-02: Note. Thu clawhcstion of I’, scrics suhtypus has not hucn finalizcd sntl In due c(iiIrsc will h c d r t from more spc‘cific pharmacrilogical hlockurs/ activator< and primary st.qucncc Information provided hy molecular cloning. For rluvclopnicnts in piirinoceptar nomcnclaturc, rcfcr t o the latcst WPHAR N(iinciicl;lturc Comrnittcc rcciiiiimcndntionh V M thc CSM (sec F c ~ d h a c kc47 C S N m.i.i,$$, cntry 12).
Frnr urcs of P ~ rind L P p 1 purinergic r e c q i t o r s i i h t y p s 06-06-03: A P 2 ,rrceptnr siihtypv spccific for ATI’ is found prcdciminantly on inast c c l l ~and othcr iinmunc cclls. Activntion of thc PI, rcccptor by high ATP conccntratrons ,appears to hc linkcd to opening of an integral channcl. T h c P2T receptor suhtypc (if plattlctr has hcun rcpcirtud to inhibit adcnylyl cyclasc and to stiiiiulatc intraculluhr Ca” rcluasc. Notably, ATI’ and AMP arc antagmists fnr this rccuptor type whilu MI!’ is n potciit ngmr Functionnl e v ~ d c n c eTor m u l l iple ATP receptor-chonnel subtypes Oh-06-04: Differing hnctirinal propcrtics in ;Erangu of pruparazions predict tho cxistuncr uf piirinetgid channel siihtypes‘. ~uhtypusmay hr diffcrcntiated hy c h a r a c t c r i ~ t i ~ajynixt Sclcctivity, dcst‘nsitizattonl hehaviour and pcrmuahilityi r a t ~ o s In particular, distinct distrihuticins of chnnnel opcn times in single-cell prcpar~tir)nssumcst the uxistcncc of rnultiplu ATPg:3t~11 chnnfitl s~ihtypcs( T C ~I n r ~ivrlt~on, t 06-37)
Dc.cpitc thrir s t riictriral dzfferences, the functions of PpxR-chonnels rcsemhle thorc of other ELC: chnnncl types 06-06-05: Fmctionnlly, ciirrcnt? through P,,-likc pttnnticcptnrs show c h c r rcsctnhlancc to nicotinic cation channcls ( W P El.(; CAT nAChR, cantry 09) and AMPA-sclcctivc Rlutamntc rcccptor channcls ( S P P F I X CAT I : I . I I AMPAIKAIN. cnlry 117) than they do to NMnA-scluctivc glutatnatc rcccptnrs.
Trivial names Unsystemotic names for several 'ATP-activated' chnnnels should not he confused with Pzx receptors 06-07-01:ATP-gated catinn channels of the Plx-likc purinorcccptor suhclass (thi? entry) should nnt he cnnfuscd with similarly named channel currents which may hc jmijrectly activntcd by scvcrnl suhtypcs of G protcrn-linked purinoccptors (see clnrrficnt i ~ m IR S u hr ype ulas d r c a t ions, otl-rjtl). Similarly, ATP-gatcd PIxR channcls (this entry) should not he confused with novel inrmcelhlnr ATP-activatcd K' channels, such as thosc dcscrihcd in pancrcatic 0-cclls isolatcd from ;1 typc-2 diahctic humanY or with thc ATP-inhihitcd K' channcl family (ser! I N R K ATP-i, entry ,TO). Putative 'ATP-activated Na" channels' recorded in dc-fdliculatcd Xenopus oocytes'" are also likely to hc activatcrl by a scparatc (non-integral] G protcin-linktd ATP rcccptor. 'ATP-activated' inwardly rectifying K' channels of calf atrial cellsFF(see I N R K GIACh, c n t r y . 3 1 ) are also unrelated to P2xR-channcls.
Trivial nnrnes presently in use 06-07-02: ExtracclIuIar ATP-Rated cation channels; fast-activated ATP-gated channels; directly activated ATP channels; ATP reeceptor-channels. Note: Fast channcl activation (in the millisccond rangc] is thc most immcdiatc distinguishing charactcristic of thc Pzx-likc P13rjnorcccpItar-channeIs.
Cell-type expression index M-08-02:Consistent with their role in mumtransmission, neurosecretion and effector+ cnupling, PlxR-channcFs arc exprcsscd at scvcral synapses of thc C N S and PNS, togcthcr with a number of charaetcrizcd sccrctory and musclc ccll types. Some of the important prcparations that havc hccn uscd for thc study nf PzxR cwrrcnts are as follows.
acinar cells, c.g lacrimal and parotid types''. blood vcssels cardiac atrial cclls, hullfrog" liver, hepatoma cells2' cardiac parasympathetic ncurones2' medial hahenula ncurones, CNS" cochlea hair ccll neuroncs"-"' cultured h ippncampal n ~ u r o n e s " ~ dorsal horn neumncs" nucleus-solitarii ncuroncs" coeliac ganglion ncaroncs, PNS'"
sensory ganglia ncuroncs'R**' phacnchromt~cytoma-dcrivcdPC 12 ccll lines (rcscmhlcs scnsory ncumne P~XR)"-~'
entry 06
Iocus cocrulcus ncurrmcs, ratz7 skcktal muscle films, adurt rat2’
vascular sm4)nth muscIcT2-.’“ vas defcrcns smooth rnusclc7”.”’
hladdua smooth musclu”
M a f c h ~ n ~nntiw y !hsiw-speciiic pruperties of PPxR Mrlth properties of cloned J’*xK 06-08-02:PAxR 1 [C)RF472 aa]: Native extracellular ATP-gated cation channels havc hccn-inrgcly charactcrizcd on smooth rnusc~ccells and autonomic sunsory nuuroncs. The PzxR type clrincd horn PC12 cclls (472 an’) rcscmhlcs nativc PLxR on PC12 culls and some scnsory and autonomic neuroncs24.z~. an. . T h c prnpcrties nf the 472 aa isoformr differ from those of P?xR in vascular smnnth musclc, vas defercns and somc CNS neurnncs, .PzxR-.(ORF 390 aa): The prntotype receptor expression-clnncdt frnm vas
dcfcrens has properties ctinsistcnt with the native PIxR in this preparation (C.R.whcrc 2 , P-mcthylonu-ATP acts as a potcnt agon1st”], vascular {artmall smooth muscle*”, and nthcr smooth muscle preparations‘, 7. [For further propertics of thc cloncd/nativc PTxR isoforms, see the respectiw jieldq wifhin the FLE17TROI’HYSiC>i,OC:Y und P H A I1MACOLOGY sectinns.]
Cloning resource Pro t n t ype Ppx R -c h on n I* I c fl NAs isn 1r1 t L‘ d 17 y c x pw s s j un - c 10 R in,qt prntrnco?.~ 06-10-01: Oocytes inicctcd with pnPy(Al‘ mRNA from rat vas dcfcrcns and urinary hlsddcr support ATP-cvokcd mcmhranc catlcinic currunts which do
not :ippe:w In unmicctcd rmytcs‘. Undircctional cDNA Iibrarm constructed with poly[hl’ R N A from VRS defcrans and rat phacochromocytoim (PCI21 cclls wcro Irscd a s stiurccs tn isolate thc prototype cl3NAs cnctdmg thu rcccptor-chnnncls by cxyrcssion-clonin~tprntncols’.’. (Fnr further potential soiirccs nf mRNA cncnding P ,xR-channcls, .WP m R N A c i r c t r i h f w n , 0 6 - 13.)
Developmental regulation Possible role of PPxR in npoptosis Oh-11-01:In addition to a welt-cstahlishcd role in synaptic transmission, thc P,, receptor may h a w a role in the Ca”-influx assncratcd with induced apoptosisT. Fdlowing subtractive hybridization1 prrxcdurcs rlcsffincd to cnrich mRNAs cxprcssctl in thymocytus indiicctl to dic (by culturc for 8 h in thc prcsencc of dexamethasnne and cyclnhexirnide), an carlicr study3 iwlatcd n pnrtint c D N A suqiicncc (RP-2)which was 1att.r shown’ ti, hc idtntical tri part of the ,399 aa PIxR isnform. Thc first 3 7 nuclcntides of the rcpnrtcd RP-2 s q u c n c c (whrch did not match thc PzxR uxprcsscd In YBS dcforcns cDNA) caii hc attrihutcd to an intronzct scqucncc not prcscnt within spliced PPxR mKNA [ t h u s thc orginnl RP-2 .;ctluciicc was likely to hc a traEmcnt of an incompktcly proccs~cdPTxR Ecncl. Intcrcstingly, ATP (variably)induces cell
death in thymocytcs", hupatacytcs" and scvcral lymphocytic cell l i n c ~ " ~ (rcporacdlyby action at P1z or P?y suhtypc purinoccptorsl with concomitant incrcascs in thc intraccllrrlar calcium conccntratian. Thc idcntity of RP-2 and P2xR may indicate a rolc for dircct activation of C ~ ~ ' - i n f l t ifollow x in^ aE:onism nf P2xR during some forms of apciptnsis.
ATP-gated conductances nre not nctivoted h y nerve growth factor in
PC72 ceJIs
06-11-02:Ncrvc growth factor 1NC.F)-zrcatcd rat phaeochromncytama PC 12
cclls cxprcss an ATP-gatcd channcl (scu Rlockers, Oh-43). NGF stimulates the wptakc nf radicmtivc calcivm intn PC12 cclls, hut i t has hccn cnncludcd that thc NGF-activatcd influx pathway is indupcndcnt of hnth ATP-activatcd and L-typc calcium channcls".
Cationic- and anionic-permenhle chnnnelx in developing rnirscle Oh-E1-03 Micromolar cnnccntrations of cxtraccllular ATP havc hccn shown to clicit rapid excitatnry responses in developing chick skclctal muscle. Unusually, these studies concluded that a singlc class of ATP-gated channels wcrc able to conduct hot11 cations and small anions4' (scc SeJc L' t ivi t y, Oh-40).
mRNA distribution Strong expression of P2,$ mRNA in smooth muscle 06-13-01:~P2xR (ORF 399 aa): Nnrthcrnt hlot analysis shows radiolahellcd hands with cstimatcd sizcs nf 1.8, 2.6, 3.6 and 4.2 khF. The representatinn and intcnsity of hyhridization of cnch 01 thcsc transcripts acccirdinfi tci prcpamtinn, with thc strongcst signals in vas dcfcruns and urinary hladdcr (0s srrrnrnorrzod 1R TdMc 21. mRNA distnhutions judged hy in sitii hyhndization show prominent signals in the smooth muscle layer of thc urinary hladdcr and thc smonth musclc layers of smal I artcrlcs and atturiolcs'. 06-13-02: -P7,RI-(ORF - 472 an): Thc rcprcsuntaticm and intcnsity of hyhridimtion of thc 2 kh transcript in various prcparations are shown in TAMe 3 . I _ -
Phenotypic expression ATP acts
0 s cf
transmitter in the CNS and the periphery
06-14-01:A wide range of central and pcriphcral functions are mediatcd hy cxtraccl lular ATP acting a t Pzx-type rcceptor-channcls. Physiological roles of Plx&channels frequently involvc 'fast' signalling, e.g. synaptic transmission hctwccn central ncurnnesth*2',4Rundcrlying rapid sensory, motor and cognitive functions and fast responses af smooth muscle5 following syrnpnthctic ncrvc stimulation. ATP has heen conhrmcd tci act as a cn-transmitter with nosadrenaline in thc sympathctic ncrvous system'5*.36+41'.In kceping with Its rnlc as a truc ncumtransmittcr, thc stimulus-cvokcd rclcasc of ATP has hccn dcrnonstratcd. Ca"-intlux through ATP-activated channels is also likcly to play important rolcs in neurosecretary processes. T ~ h l c4 ~ u r n r n ~ rfunctional i~c~ rolcs in n numhcr of tliffcrcnt preparations which arc likely tn invdvc Pzx-typc sign all in^. Furthur cxarnplcs arc dcscrihcd clscwhcrc in this cntqv.
entry 06
: of dofn d m v e d Tahle 2. ,I (ClRF 399 m) i n l l N A d i \ t r ~ h t l t r o ~ lsunimrzry /rrtim Norlhern iinoJy.cr~*‘ (Frtirn 110- 1.7-011
Prcpnration
- I . # kh transcript”
Ihin
ItI
Cocl1nc ganglia
[t
Lung rc12 E
4 t ~ J N
Rctina Spinal cord Splccrl Thymus Urinary hladtlcS: Vss dcfcrcns‘
I‘I
I l l
(+I
($1
+ t -t -t
-.7.h kh
transcript‘ transcript
+
1
+ (handshiftl’
++
kh
-2.6 kh
transcript
+ + +[I t (+ 1 t t‘l
t (handshiftl’ I I t-t+ i i
+++
-4.2
[+
k
Ill
1 1
It1
I
If ! (3F I+) I+)
t + t t a tt
[+I
i t 1 ++-I t +
(+I 1-
t
+
‘rThc’rclativt. abundance’ of thc various transcripts Arc shown on an arhitrary walc (+ to 1 -I t Y -t and (-t]nr 1-1 for law o r zcro dctoctahlc cxprcssion In singlc trials1 as lurljiurl from the puhlishoti data”’. ‘ T h e 1.8 kh tranwrrpt corrcsponik in sizc to that cncnding thc full-lcn~th (post spliced) O R F ~ . ‘Thc ;l.h kh transcript may scrvc 3s ;1 prccursor f o r P?x mRNA a s i t can hc dctcctcd using 3 lTLX tntront-spccific prohc (crlrd in rrf.’). rl Thcsc wgnals m l i y rcsult from smooth muscle In thcsc organs. “l’hacr,chrnmocvtr,mn cclls diffcrcntiatcd with NGF (ncrvc growth factorl. ’A handshift t o ’slightly larger’ transcript sizos arc nppawnt f o r thcsc hands, although this docs not sppt‘nr to ;iffcct thc 4.2 kh tmnscript. '!The ’prcdominant‘ 2 h kh m R N h h:is a 3‘ untr;inslatccl cxtcnsion a s dc t u m lncd h y scq w n c i ng .
Protein distrihution
1
Co-disiribtltion of metylcholine c~ndA Tl’-rcleasc sires 06-15-01: Distrihutions of ATP-rulcasc sitcs in the CNS arc chnscly associated with thnsc of acetylcholine, suggcstinl: ATP may hc a to-transmitter a t chnlinergict synapws as shnwn to he the c a w in thc periphery (scc R w r p t or o p m i v t s. Clh-50)
1
Subcellular locations Sires of ATP-induced cntron influx In PC12 cc1J.q
Oh-16-01: Activation d I’7xK chnnnels in rat phamchmmocytnma (PC12) culls rnduccs A ’rnixtxl’ Na’/Ca’‘ inward current, hut docs nof rclcnsc Ca” from inturnnl stnrcs (WY Srlrctivity, Oh-40) This dcpnlarizing current raiws vr>ltagc-g,atucl calciuin channcls on thc c c l l wrfncc to thcir firing thrcsholdt . I’C12 ccll ATP-Ratcd influx sites arc v:iriahlc in ccll hociics hut niort. hrmmgcncous in Krowth cont‘s5’.
entry 06
Local depolarization affecring ATP ngonist release synaptic contact
~t
regions of
06-16-02: P2, purinoccptnrs are present at both nerve terminals and cell bodies of peripheral and central ncurones. Local K ' depolarrzation of thc cntls of coeliac ncuritcs of the guinea-pig evoke single-channcl currcnts charactcristic of PzxR in outsidc-out patchcs whcn patchcs arc positioncd near thc rcgion of apparcnt synaptic contact. This cffcct is not nhscrvcd when patches arc positioncd at remotc regions5".
Transcript size 06-17-01: See mRNA distribution. 06-13.
Table 3. PZxR1 (QRF 472 nu) mRNA distrfhntinn:sornrnary of data derived from Northern andyses" [From 06-23-02)
Prcpara t ion
-2 kb transcript
Adrenal
+ +
Brain Heart Intestine, large Intestine, small Kidney
Liver Lung
Ncuroncs, supcnor ccrvical ganglia Ovary
PC 12 cclls Pituitary Skclctal mwsclc Spinal cord Spl ccn Testis Urinary hladdcr Vas deferens
{-I)'
+ + If1 Efl
I4
+ +" +++++ + t + + +"
I*!
I-?
++
(%I
4-
+
f+++
"The'relative abundance' of the various transcripts are shown on an arbitrary scalc I+to + + + + + and (&I or (-3 for low or zero detectable expression in singlc trials) as judged from the published data'"'. ?he absence of transcripts in these tissues {where oativc PIX rcccptars have bccn chnractcri~cd"-~~) plrohnhly indicates the 472 aa P2xRl isofnrm docs not underlie responses in these preparations. 'ATP-gated inn channels have heen characterized in sensorJ ganglia". "Although 'striing cxprcssion' of mRNA cncoding this isofmm has hccn demonstrated in both the intermediate and antcrior lobes of thc pituitary a [hnth ncumsecretory cclls and stellate support CCIIS, cited in physiological role fnr cxtracelrular ATP has not been described in thcsc ccll 'types to date.
Table 4.
Functional roles of PPxR-channeh in varinus prcpurations (From Oh-14-01)
Prcpara tion
Fcaturcs/functionaI roles of punnoccptor-c hanncls
Refs
Hcart, neurotransmission
Extracellular ATP-activated cation channels in smooth musclc gcncrally producc contractile [cxciltatciry]rcsponscs by dircct admission of Ca7,+.In cardiac neurnnes these channels may contrihute to nnn-adreneqic non-chnlinergic (NANC1 neurotran sm issi on and mediate, in part, the vagal innervation of the mammalian heart
."
Caz'-influx thmugh ATP-activated channels in ~ ~ cells 1 (hut 2 not voltage-gated Ca2* channels) cnntrrhute to ATP-evoked noradrenaline release and at the samc timc inactivatc thc Ca2'-selcctive channels in thew cclls. Thc PC12 lATPnr rcscmhlcs the PlxR expressed in scnsory ncurnnes. A number of ATP analogues arc effective in stimulating catechohminc rclcasc, and thc receptor antagnnists suramin and Reactive blue 2 inhihit the nuclcotidc-induceti naradrcnalinc release [see Rrceptor r z n t ~ ~ ~ o n r s06-5 r s , I , nnd Receptor ngnnistq,
"
Phacochrnmoc ytoma PC12 cells
26
''
06 - 50)
Coeliac ganglinn ncuroncs
ATP acts as a ncurntransmittcr at scvcral junctions between autonomic nerves and visceral musclc. Feature? of excitatrrry lunctiont currents in the coeliac ganglion, c.g. rcvcrsal potential+, time cnursc and I-V rclationt Ins dmm 3n Current-voltage relotion, 00-.75)can he mimicked by gpplicatinn nf cxo~cnousATP Synaptic currents measured i n s 3 t pr~sscss ~ similar current-voltagc rclatianships ta currcnts produccd by ATP, arc incrcased tn frequency hy K ' depnlarizatinn (ina '0"-dependent' manncr), ant! arc rcdWCd hy ATP antagonists (see Reccptor nntngoni7ts, Oh-s1 )
Hippocam pal nuiironcs
In cultiircd hippocnmpal ncuroncs, ATP directly activates small sustained currents, and indirectly induces the transient currents by cvnking glutamatc rclcasc
35
Table 4.
Continued
Preparation
Fcatures/~unctionalroles of purinoceptor-channels
Refs
Smooth musclc
ATP acts as a (co-)transmitterat several junctions hctwecn autonomic ncrvcs and vascular smooth muscle. The ATP-activatcd channcls provide a distinct mechanism for excitatory synaptic current and Caz+-entryin smooth muscle. P2x receptors mediate
32
sympathetic vasoconstriction in small arteiics and a r t c r i n h . ATP may also initiate smooth muscle relaxation (vasodilation]hy
5tn52
indirect agonism at endothclial cell ATP receptors coupled to second messenger systems (see Appendix A, entry 56) Smooth muscle, hladder, non-human
The bladder of most non-human species receives dual purinergic and cholinergic , excitatory inncrvat ion. Activation of P purinoccptors dcpolarizcs thc cclls, incrcascs thc spike frcqucncy and causcs contraction. Addition of agnnists rapidly activatcs nonsclcctivc catirm channcls, which underlie the excitatory lunction potentials seen on stimulation of the intrinsic ncrves
.39
53
’’
Skeletal muscle fihres, adult rat
Extracellular ATP ( 5 0 - 1 O o p M ) has heen shown to activate junctional and cxtralunctional currents similar to those of acetylcholine receptor-channels in isolated adult rat skeletal muscle fibres, hut exhihit a shorter open time
Liver, hepatoma cells
Calcium-permeable channels expressed in rat hepatoma cells are activated by extracellular nucleotides
Parotid acinar cells lPzzR channels1
In rat parotid acinar cells, cxtracellular ATP increases influx of Ca2’ across the plasma rncmhranc, in contrast to rcccptor-mcdiatcd rcsponscs to carbachol (which also clcvatcs ICa’+],-d-fi-fnld, hut primarily by release of Ca” from intraccllular storcs).Within 10 s, ATP [ 1 m M ) and carhachol ( 2 0 p ~reduce ) the cellular CI m n t c n t by 39-50% and cell volvme hy 1525%. Both stimuli significantly reducc (hy -57(15%) the cytosolic K ’ content of the parotid acinar cell through multiple types of K’pcrmcablc channols. ATP anti carbachol also stimulatc thc rapid entry of Na’ into the parotid cell, and elevate the intracellular Na’ content to -4.4 and 2.6 times the normal level, respectively - part of this flux is due to PZZR-channek (we alco Receptor anfngonists, 06-5 I )
entry 06
Encoding I'redrcred sizcs of pmlrfnx unundud hy P7xR genes 06-19-01: Thu prcdicturl prntcin encoded by thc P l x R cDNA svnthcs~zcdfrom vas dcfcrcns ~ R N A hns ' nn opcn r c a d ~ frarnct n~ of 399 a ~ n ~ acids n o (-45 kDa withnut ~lycosylationi1; thu opcn rcading framc of thc P7xR cDNA dcrivurl from mRNA fmm phac(~chrr~rnocytc~mn cclls2 prctlicts a protein of 472 arnlnr) acads (-52.5 kDa withnut glycnsylnticlnt\.
Gene organization Evidencc for K N A splicing within thc protein c ~ d i region n~ of PpxR gene? 06-20-01: P2,R [QRF 399 a,]: T h u cxistuncu of scvcral 'high molecular w u ~ g h t hantls' on ~ r ~ r ~ h c r n t l ~ v h r l d l z a t( ~~ocnrnllNd cs dlctril,trrlon, 00-1,7) and thc ohwrvation of ~rnproc~ssed Fnrrns nf thc PlxR gcnc ropruscntctl by isnlatc
RP-2" ncvuloprr~cntrrl ~ i ~ ~ ~ l lon, l r n !Oh-1 1 ) arc intlicativc of sn R N A splicing mechanism w ~ t h ~t nh t protein coding rcgion of thc sctlucncc ciicotI\ng thc ,399 an PJXRisoform. (z.cpr,
HomoIngnus isoforms
U
00-21-01: Scc thc scction ~ n a t c h ~ nn;ltlvc r: tissuu-spccific prnpcrtlcs of P?xR wit I1 prt ~pcrticsof 'clonctl' 1'1~11 untlur C c l l - ~ v l cxl>rcj.rcror~ ~r ~ncJr.u,O()-OX.
Protein molecular weight (purified) 06-22-01: Scc 'I'tr l ? l r h I ~lni!r,rC;i,llru ir~znrIy..Ill,-05.
Protein mo1ecular weight (calc.) Oh-23-01: Srr Trrhlc I urlrler I:i?nr Tcrmtly,Oh-0.5.
Sequence motifs ATI1-hindin'q site m o t i f r 06-24-01: A mot16 ~ i m r l r ~ tro thr Walker type A phosphate-hinding site c ; ( x ~ ~ G K ( x ~ ) ( Iis/ vfound ) " ~ hctwccn rcqiducs I,?] and 144 of thc I)2x pi~xinoccPtor-channrI1. Wnlkcr typc A motifs5' have also hccn indicatcd rln thc rcportctl pr1m:iry scqilcncc clf thu cI3NA cncoding thc P,xRI ~ ~ r r f o n n ~ s o l a t t dfrom rat PC12 cclls2 (setu I?rIc)w). Thc cxtmcclluEnr location.: c ~ f thcsc consensus clcmcnt.; a r t ~~rc.;urncrl to form p;lrt of the AT]'-hindinp, s1tr ( ~ ( /lvl'rA4/, ~ 0 Fi.y
f).
Typlcal form of "Cys-Cya bop" motif
Walker Type-A ATP-blndlng rnotrt (aa 131.144)
Consensus H-glycosyrrtlon sltes (actual podtlon8: aa 153, 194, 210, 284, 300))
(diagram rnatlc)
Proposmd P-Ilk.
'pon-formlngn
or HHS-likO domaln forming the Inner linlng (narrowest part) of the artracelrular hall of the h I c pow
Extracellular Not.: Tho multimerlc topography (I.& subunlt numbem per homo-multlmerle channel cornprmx and the exlatenee and/or stolchlornetry of hetero-rnultlmer8) 'have not yet been determined l o r thls class of channel.
- .
Monomeric domains _ _
Channel 8ymbOl
Na,K,Cs N H 2 (na 1)
(aa 599)
ATP
7 ~.
-I
V
Figure 1. Monomeric protein domain topography [PDTM] model for the rat extracellular ATP-gated receptor-channe] (Pp&) exemplified for the 399 amino acid isoform rsolated from vas deferens. Note: All relative positrons of m o t h , domain shapes and sizes are diagrommatic ond are subject to re-interpretorion.(From 06-2441)
entry 06
N-Glycosylation sites cind putative 'Cys-Cvs loop' motifs 06-24-02: PzxR .-- [ORF -399 __ aal': The N287 aa hydrophrlr (extracellular) region hctwcen the two hydrophobic (putativc memhranc-spnnning:)domains in this isnform shows five potential sitcs for N-linked glycosylationt (10 cysteine residucsl. PzxRl (ORF 472 sal': The -270 aa cxtraccllular region of this isoforrn dlsplays thrce potential N-linked glycosylation motifs and scvural rcgularly spaced cystcinc residues which resernhle 'Cys-Cys loop' motifst fnund in the nAChR and other members of the extraccllular ligand-gated channcl family. Note: Cys-Cys loop motifs are proposed to he important in stabilizing the stnrcture nf extracellular ligand-binding pockets (see o h [I'DTMj. F i g . I ond Protein phnsphorylfltron. 06-32), I
Apparent lnck of secretory leader (signal) peptrde Oh-24-03:PlxR (ORF,799 aa)': Thc prcsence of chargcd rcaduw in the first 28 ~
amino acids of thc open reading framef of thc prntntypc P2,R suggest the ahscncc of a sccrction leader. This supports a model In which both thu Na n d C-tcrmini arc in the cytnplasm of thc ccll (see [PDTM], F i g 1).
Amino acid composition I'rcdiction of L] novel strucriire for P2xR-chonnel subunits 06-2(3-01: R y hydrnpathicityt analysis, PlX receptors cxhihit only two
hydrophohict segments 'sufficiently long' to cxist as transmcmbrane domains (see /PDTA4/. F A R 1). Thusc hydrophobic scgmcnts are separated by n largc hydrtlphilicf Scgtncnt nf -270 aa [for thc 4J2 aa PlxRI') or -287 3.1 [for the .39<) aa P2xR'l which IS cystcinc-rich (10 cystcincs for rhc 472 aa P2XRIZand Y for t h c ,199 aa P ~ x R ' J Thc . structure and topography of thu rcccptor therefore diffcrs markedly from thosc of uther cxtraccllular ligand-gstcd channcls Icornporc rh hrr ELC: cntnes), which is contrary tn what was prcdictcd from clcctmphysiological and pharinacologrcal propcrzits alonc. (Scc n l w Lhrnain nmmncyeriimt, Oh-27. Domain consrrvii tion, 0h-2HI Doma r n fu nci inns, Oh-29, (ind i'rcdicrcd protein iopogmphv. O(I-.3R.J
Domain arrangement Expression of cloned PPxR subiinits displny properties resernhlinl:
native channels 06-27-01: The elcctrophysiolnRica1 propcrtics nt hctcrologouslyt exprcssed recombinant reccptors cloned 'to date' clnscly rcscmhlc nativc channcls, sumcsting that assernhlics of singlc suhunits form fully functional channcls (see Cell-type exprcssinn index, O6-0R). The stoichiclmctry and thc arrangement of the prntcin suhiinits cnrnprisin native or recombinant P2x rcceptors is presently unclcar, and hctcromcric asscmhlies have not hccn ruled out (see n?so Predicted profcin tnprqrrrphg, 06-30). Variability nf results in Hill plot1 analyses for native PT.x rcccptors suggcst the ATP-gatcd channels arc composcd (if rnultiplc subunits in common with othcr uxtracellalar ligand-gatcd channels (see Dosc-response. Oh-36)
4
Domain conservation Putative pore-forming domain 06-28-01: Amino acid scqucncc alignmcnts of portions of thc prototype PIxRchanncls in the region immcdiatcly prcccding thc M2 transmembrane domain indicatc same similarity with thc HS rCgjQn typical of 'poreforming domains' of voltage-gated and inward rectifier-typc K' channels (see the entries heginning VLG K nnd I N R K). For cxamplc, it is possihk to align residues .1,71-t3,7f4 of thc ,199 aa PzxR isoform' (TMTTIGSG) against thc 'pore r q i o n ' sequences of ;1 rangc of ion channcls (including voltagcgatcd K' channels (we VLG K cnrricr), Ca2'-activatcd K' channcls (see ILG K Cn, entry 271, inward rcctificr K' channels (see 1NR K entries), K ~ a p chmncls (see I N R K ATP-i, entry ,301 and cyclic-nuclcatidc-Katcd cation channels (see ILG' CAT C A M P , entry 21, nnd ILG CAT cUMIJ, entry 22). This structural fcaturc is not found in othcr 'cxtraccllular ligand-gated' channels [i.c. those described in nthca ELC, cntncsl.
Potentid ornphipnthic sc-helical regions nssocinted with the P2xR M2 domain 06-28-02: The 'topographical sirnilaritics' between thc PzxR and other ion channcls (see Predrcted prntcin topugmphy. 06-30) has led to predictions that the P2xR M2 domain is ahlc to form an imphipathkt 7-helixt whasc polar rcsiducs project intn thc inn pore. Thus comparative 'helical wheel't plots for the two protntypc PTxR i d o r r n s clcarly illustratc the potentia1 of this domain ta form such a structural motif (see F s ~ .2). Such a rnodcl is consistent with a multimcric channel complex in which thc lumcn of the channel is siirrnwndcd hy M2 domains from diffcrcnt suhunits2.
1
Domain functions (predicted) The larxe extracelhlar prrtntwe ATP-hinding domain
06-23-01: A major portion of both PIxR isoform proteins cloned to
dater.'
appears to hc a large hydrnphilic extraccllular domain which is likely to comprise thc ATP-binding site (sue Sequence motifs. 06-24.[FDTMI. Fig. 1). hlotc: For cviduncu supporting thc cxistcncc of a 'pow-forming' domain, see Domain cnnwrvntron, 06-28,
345
346 the nmphipathic M2 tranmembrane domuin of cloned Pzx receptor-
Figure 2. Cnrnpmntzvc ‘hclrcd wheel’ reprecentntjonq of chunnetc shnwng nrnino muid residues 325-346 of the 472 nu isnfnrm in comparisnn wrth the corresponding region of the 399 cia rcoforrn (equwnleni to icolate RP-21 The cimiIar chrirrictcr nf homologous amino acids in the two proteins SF ernphnsized by hvdrophohic rind non-pnlar recidrrec oppcarmg in outline font. M a r Oivdrophilic) residues nre predicted to proiect i R t 0 the pore. (Rrrced on representotion? rn Rruke et al. (1994) Nature 371: 519-23.) (From 06-28-1921
entry 06
Predicted protein topography P,,R topography is otyptcal of Other 'ELG channel' fnrnily members 06-30-0 1 : The majority of cxtracel lul ar 1gand-gatcd receptor-channels share the same overall topography, i.e. four prcdictcd hydrophobic {presumably transmemhrane) domains (see ECG Key f n ~ t s ,entry 04).PzxR rnonomcrs, howcvcr, appear ta ctmsist of a two-transmemhrane-domain motif with ;1 largc hydrophilict cxtraccllular (receptor1 dnmain and a region apparently contributing to the ion-selective pore.
Resemblonce of predicted 112,R prmein topography to other ion channels 06-30-02 The overall protein PzxR monomcric protein t q q y m p h y as prcdictcd from primary sequence has some sirnrlarlty to that proposed for the mechanosensitive channels of Caenorhnbditrs eleRnnsfM2 (see protein domain topography models fnr MEC (mcchnnosensitive), entry 36, and the rat amiloride-sensitive epithelial sodium channel"']. There is also snme topographical aesemhlance 'to cloned inward rectifierM and pH-sensitive K" channelsh5. Note therc is no primary sequence homolngy hetween the FzxR and these channcl types. A two-transmemhrane-domaln topography is also typical of the inward rcctifier potassium channel family (see the entrm heRinI7iRR IN!?K),
Protein interactions Functional interactions of PPx receptors with volrage-gnted Cn"' and colcium-nctivated K' channels 06-31-01: In single cells isolated from guinca-pig urinary bladder, rapid application of ATP (threshnki -10011~) depolarizes the cell rnernhranc with
supcrimpasit ion of action potentials, tollnwed hy transwnt hypcrpcilarizntian". Following addition oh the voltage-gated calcium channel hlocker DhOO, the amplitude of thc ATP-induced &polarization hecomes a function nf the ATP cnncentration [ECSo-0.5-1 pu)"'.
Coupling of PzxR-medinred Co"-infhx to activation of potnssium channels
n
06-3142:The interaction of ATP-Rated Ca"-influx prcccding thc activation of Kca channeh in rat phacochromocytoma {PC12)cells has also hecn dcscrihcd"'. Similarly, the action of ATP upon Kca channels in hovinc aortic endothclial cclls is related to secnnd messenger-mediated release of Ca2+ from internal stores [coupled to ATP-dependent calcium influx which i s also abolished a t dcpolarizing voltagesh81. In cultured aortic smooth muscle cells from rat, ATP-gatcd channels elevate internnl frec Ca2' lcvcls to subsequently activate hoth Ca2'-dcpcndent K' and C1 currents" (see ILC: CI Ca, entry 25 and ILC: K Co, entry 27.
Protein phosphorylation Pnten L in tion of nn tive F,R-chonnel responses h y cA MP-dependen t
protein kinase
06-32-Of: An ATP-gated channcl activation in ~ O U S Llacrimal ' acinar c c k is potcntiatcd hy stimulation of CAMP-dcpcndent prntcin kinasc activity
entry 06
rcsrilttng In an incrcased rcsponsivcness to cxtcmal ATP. CAMP-dependent potcntiatmn can hc Induccd hy Ii-aganists such as isaprenaline". In this prcparatmn, cxtraccllular ATP 11 !TIM\ promotcs Ca' '-influx (possibly thrtiuRh a PIz channcl suhtypc - see Rcceptnr lrn!si.yoniqtf, Ot,-51), which in turn activatcs K ' channcls rcwlting in a dclaycd, outward current componcntr2 (see ILC; K Cn, entrv 271. Thew ATP-induced rcqxinws, and similar oncs rn rat parotid .cc!!s'', occiir in the rihsence of phosphoinoqitidet hydrolysis (unlikc thrisc follnwing ACh applicaticinl. Supplementary note. CAMP cluvation following stimulation of {f-adrenergic ruccptars is a kcy scctind mcsscngca promoting the synthcsis anti sccrction by exacytosis of protcins stored in secretory grantrles. Potentiating cffccts of GTP and GTPy-S on ATP-activated currcnts have a I w been charactcrrzed in rnousc lacrimal ccIh".
Regions of PPxR susceptihle t o modulntion h y protein phmsphorvla tion 06-32-02: P2xRI (ORF 472 aal: T h c - 1 1X rcsiduc C-terminal segment nf t h e P2xRl isofrlrm has 20 prolinc and 13 scrinc rcsiducs2 and cnntarns putative sites at which channel activity cr,uld he modulated.
Activation A ifivotiot? k irictics f o r n i i t I V P P?,y rcccptor-chctnrirls 06-33-01: As is typical for iriirgrril cxtraccllular ligand-gated reccptorch:inncls, ATP-gated rcccptor-channcls.~-' iictivntc' in the millisecond time range tollt)wing ;igonist applicstion. For example, in singlc cclls Isolatctl trom xuinca-pig iirinan, hlsddcr, A T P x t i v a t c s a 'dose-dcpcndcnt' inward ciirrcnt with ;I short latency , I 8 111swith 10 I ~ MATP, rncasurcd as t h c timc hctwccn the s t a r t or applicntion rind l O % ot t h e pcakl'". Similarly, rhc ATI'-.;tiinulatctl C;I" condtict;iiicc in r;it hcpntoliin cc115 is trmcivnt in natiirc, commencing immcclintcly nftcr A T P ;itltlition nntl rc;iching ;I pciik ;it I niin or Ic.;?'. 06-33-02: In kccping with n role in nciirotr;~nsmis~ion, ATI'-E;itcd currents in ilorsal r o o t p n f i l i o n ncuroncs froni r a t s and hullirogs iictivatc rapidly upon :ipl~Iiciitionoi A T P ;inti quickly tlcc;iy whcn ATP IS rcmovcd. Short Istcncics [ < 10 msl hctwccn A T P applicntion and activation ot m c m h r m c currcnts arc n l q o typical f o r PIX rcccptors in r;it pnri~symp;>thctic cnrtliar gangli;i.
lkpctidcnce (J/r h n n c l Rot iriR kinct icc mi (7,yoni.st conccnfrtition 06-33-03: T h c ATI'-gntc.tl currcnt in dissocintcd r n t nuclcus solitarii nctirone5 show rimc constaiitc' (if ;Ictiv;ition (;inti inactivation! that arc dcpcndcnt on thc. cxtr.iccllul:ir ATP conccntr.ition, with both parsmctcrs hecoming fastcr .it highcr A T P conccntrntions" In rat scnsorv ncuronw, P?YR x t i v a t i o n kinctics iirc nlso t;irtcr at highcr ATP conccntr;itions, with t i m e constants ATI'tc)- IOitisat I O O ~ MATP. D c x t i v a t i o n tlccic.a.;in~from- - ? ( ) f l n i s ~O..%ithi t 100-LOO rnsl :ire i n ( ~ t ~ p t ~ r i d ol ( ~ ithc i t A T P ccmccntr.itioniR. kinctic5 -*
entry 06
Endogenous modulation of activatjon time constnnts 06-33-04: In rat phaeochromocytoma PC 12 cells, dopamine has hcun shown to shift dcpcndcncc of activation rate constantst c ~ nthc concentration of ATP toward R lriwcr cnnccntration range by approximately two-fold” (for details. see Channel moduln tron, 06-44].
Poten rial ‘stretch-sens.itivit y’ of A TP-goted currents 06-33-05:The current-voltage relationship of thc ATP-activatcd channcl in rat hcpatoma cellsZ” is ’identical’ to that of a previously charactcrizcd stretchactivated channel in the same preparation”. Note: Thc P2xR topgraphical structure resembles that dcscrihcd for ‘rnechanosensitive’channels of Cocnorhnhditrs e?c.ymsf(see MEC (mechanosensitrvc),entry 3h).
Current-vol tage relation Ruth native and cloned PPx receptors incorporate an i n w o r d y rectifying non-selective cation chnnnel 06-35-01: PTxR (ORF 399 aaJ472 aa): The currcnt-vdtage relationships of thc prototype PLxR d o r m s cxprcsscd in oocytes and mammalian cells shows a marked inward rectificationt reminiscent nf native Pzx receptors as dcscrihcd holow (see nlso ,WectivI!y, 06-40),
Compnrison of I-V relations for autonomic excitatory synaptic potentials and ATP agonism 06-35-02 Ovcr 95% of coeliac neumnes display fast inward currents in
rcsponse to oxogcnously applied ATP (in comparison tn -45% rcspanding tn acetylcholine and 4 0 % which respond to 5-hydroxyzryptaminel~~’. Current-voltage rclationships for cxcitatoqv post-synaptic cvrrcntst (EPSC) rccordcd in cultured cneliac ganglion ncuroncs show high similarity to currcnlts evoked hy exogenously applied ATP (Fig. 3). Tht EPSC at this synapsc arc ‘hlockcd’ by suramin and ‘dcsensftizcd’ h y ~,/h~wthylene-ATP hut arc unaffcctcd hy thc mngc of antagonists for other candidatc reccptnr channels (c.p,.nAChR, NMDAR, nnn-NMDAR, .S-HTRR,GARAAR). T h e w and additional data Icd to thc identification of ATP acting nn the P2,R as the prcdominant ncurotmnsmittc~/ruccptorcombination at this synapscXh.
Functional rmplications of ‘jnstnntaneous’ inward rectification in sensory neurones 06-35-03: Thc current-valtagc rclationsh ipt for single ATP-activatcd channcls in rat sensory neurnncs and other preparations is highly nonlinear and dernonstratcs inwardl y-directed rectification”. Functinnall y, inward rectification allows Ca”-intlux to hc maximal at hypcrpolarized potcntials without trimming post-synaptic action potentials. In rat and hull frog sensory neurnnes, strong inward rcctification 1s maintained cvun in symmetric solutions of divalant-frac caesiurn glutamatc. Examined a t microsccond resolution, thc inward rcctificatmn occurs within -1040 p (i.e. is instantanenus)”. Note: I-V curvcs obtained for singlc ATP-patcd channels in a number of prcparatians arc identical to thosc for rnncmscnpict current, hut cxcepzions cxist (see helowl.
entry 06
a
ATP(3uM)
C
I r 0.2
30
4
-1
100 -I
Figure 3. (a) Amplitude of the coeliac ganglion EPSP recorded from a single neurone at the membrane potentials shown (caesium gluconate in the pipette). The point of nerve stimulation is marked ns (0.1 Hz, 0.5 m s per 20 V). (b)ATPevoked current under the same conditions as (a). (c) Derived current-voltage relations for data in (a) and (b) (normalized so that current evoked in single cells at -110 m V was equivalent to -1 on the scale). (Reproduced with permission from Evans ( I 992) Nature 357: 503-5.) (From 06-35-02)
Differences rn I-Y rc1nrron.s: f o r unrtnry ond macroscopic recordings nf ATP-gntud chnnndc 06-35-04: The currcnt-voltage rclationship for macmscoplc ATP-cvrikcd currcnts in rat paa;isvrnpathctic cardiac ganglia also shows nnn-ohmic1 [ inwardlv rcctifvingi 1 huhaviour in thc prcwncc nnd nliscnce of cxtcrnal divalcnt cations with n ruvurwl pr*tuntial of t 10 m V [NaCl outside, CsCl insidt)2'. I-lciwcvur, unitary ATP-actavatcd currcnts in cell-attachcd mcrnhranc patchc? in this prcparatmn cxhihit a lincar (nhmic) r-v rclatinnship with a slopc conductance of apprciximatcly 60 pS2"'.
Alierorion of I-V relntionsh~psh y a channel hlncker 06-35-05;ATP-activatcd (which rcsumhlc thc
in rat phaenchrnmncytoma PC 12 cells rccuptors cxprcsscd in scnsory ncurnncsl show ' ~ C S S ' inward rcctificaticm in the prcwncc of the non-sclectivc channel hhckcr [ +)-tuhocnrarine, whcrcas thc receptor antagmists suramin or Reactive blue 2 do not alfcct the vnltagc-dcpcndcncv (we r11.w Rcccptor rrntrl'Snn!sts, O h - f i 1 ) . currents
mtry 06
n
Current-voltnge relotions of cloned P2x receptors expressed in oncytes and HER-293 cells 06-35-06: PzxR - .-(QRE - -399 _ _aa): ATI'-cvr)kcd currents cxprcssing recornhinant P ~ in ~ oocytes R display currcnt-voltage rclaticmst land ot~icrpmpcrticsj which closcly rcscmhlc thosc of nntivc channels cxprcsscd in srnnoth
'.
rnuscluA. 72.
1
Dose-response Positive and negative ngonist co-operotivit y in Ppx chnntrel ,Toting 06-36-01: ATP-gated channels gcncrally require morc than one apmist molcculc to bind to the channel in order to open it. Hill plotst of the cnnccn~ration-current response for low conccntrations of cxtcrnal ATP agonist (0.3-1.2 IIM ATPJ applicd to a hullftrig dorsal root ganglion [see Ax. 4) show a Hill slnpc of 3, sugjicsting a stoichiomctryt of (at !cast) ?, ATP rnoleculcs/channcl. Negative co-operativity hetwcen thcse h i d i n g
1
0
-1
-2
-3 -1
0
1
2
log IATRI
Figure 4. Rcspanscs of n singk h u l l / r q dorsnl mot (scnsnry) ganglion voltageclrirnped ot -80 rnV w f t h inword current mtivntcd h y mpplicntion of O . ~ / I M , O . ( l p M rrnd 1.211~ ATP (I,,,,, nctivnted h y ~ ~ O O / ATP). IM A t high cnncentrotions the slop hecomes less t h m 1, indicatinl: ncgntivr qqoni.;! cn-operntivity. (Ilepmducerl w'!h pcrniission horn Henn ( l W 2 JTrends Phsrrnacol Sci 13: 87-90.) (FromO M h - 0 1 )
sites is s u ~ : c s t e dhy the Hill slopc 'tmx)rninK 'Iuss than 1 ' at high agonist conccntratrons ( .c w F1.c. rl). ATP-gated current in tlissnciatcd rat nuclcus solitarii nczironcs incrcasus in a cr,ncentratir)n-rlcptndcnt manncr ovcr thc conccntratinn ranRc hctwccn 10 I'M and 1 r n ~ " " .N o f e : Channcls cxprcssed in vas dcfcrvns smnoth rnzmlc"'. " show two 'positively cn-operative' hinding sites (reviewed in rc/.') ( W E ( 1 1 ~ 0 Ilornriin rrrrrrn.yernrnt, 06-27).
06-3h-02 In thc bullfrog dnrsal root ganglion ncwonc prcpnmtion, the ATPgntctl cnnductancc i s half-maximallv activatcd by -,3 /IM ATP'R. Ar low conccntrations, thc condiictancc incrcnscs 3- t o 7-fdd for a douhling in ATP conccntratinn, further sumcsting that scvcral ATP mnlcculcs rniist hind in order to activate thc currunt. A stccpcr cr~nccntratinn-rcsponsu relationship thnn cxpcctcd from I : 1 hinding IS s w n in rat drirsal mot gnnglinn n c urn n c s *'.
Dose-response characteri.rtics Q/ cloned P2xR 06-36-03:PIxR 1 (ORF -~ 472 aa):Thc half-maximal cffcctivu ATP cnnccntration for thir isofnrm cxprcsscd in nocytus has hcen dctcrmincd as -60 (see OISO Hrll corlCicient. Oh-40). ___I
Inactivation V(ridiiJity in c ~ h s ~ r v erecepinr d dccensitizntmr propertics
-
06-37-01:Typically, ATP-gated itin channel current4 dcscnsitizct with rnaintaincd spplicathon of AT!' I t , rcvcml scconrls\. Nozahly, desensitization' IS slriwrr In IwIIfrog scnsory ncurrmcs than in rat" and is nnt ohsorvctl ;it all in somu pxcparatinns (c.g. guinea-pig hair cclls7"~"~ and rat parasympathctic cardi;ic g a n ~ l i f i ~ '(crtu ] rilsci Ifnltrzp>scn~itzvilv, 06-42)
R ( I I PS O J PPxlidcsen F it tzn I ion crnd 're wns it IziI r ion' 06-37-02: In smooth inusclu cells {if rdt vas dcfcrcns, the ATP-induccd current rlisappcars within 2 min cvcn in thc continuous prcscncc of ATP7-'; ccllr of the same prcpnmtirm rcctiver frnm dcscnsitization in the nhscncc of A T P with a resensitization h n l ~ - t ~ mr icb 2 rnin.". ATP-activated currents In dcirsal root ganglion ncurrmcs from rat< and hullfrngs are similar, cxccpt that ciirrunts in rat ncuroncs dvscnsitizc ;It ;I faster wtc" In singIc ccIIs iwlatcd from guinea-pig iirinarv hladdcr, thc tiino course of rapid dcscnsitizntmn 1s a function of thc ATP conccntration and can hc frttcd hy t w o cxponcn t ials"".
M m o r effeciq nf P Z x mRnnism on relense of Cn2' from inttocelhlar st nrcs 06-37-03: In the rat nciirosccrttory phacochrnmocytoma (PC12)cell 1 ine, ATP evokes a risc in ICa"], which mpirlly inact~vates'~. The minority of thc total rcsponsc to ATP in thcsc cclls [<20%)is due to intraccllzllai Ca" rcdistrihzitFcm, cnnsistcnt with a small increase in innsitol 1,4,5-trisphosphatc Icvcl. Thc rnaloriiy [ > H0%) can he accountcd for hy A T P - x t i v a t c d cation channcls ant1 voltapyptcd Ca-'' channcla.
cntry Oh
SeIective modnlnrion nf inociivation properties 06-37-04: Thc inactivation of the ATF-activated currcnt in rat phacnchromocytoma (PCF 2) cells is accclcrntcd by thc non-selcctivc ATP channel hlncker (+I-tuhocararine hut not by the ATP rcceptnr antagonists suramin or Reactive blur? tsce Illocker.7, 06-43,and Receptor nntogonists, 00-5I). Cell-frcepatches of PC12 cclPs show channc! inactivatinn with a half-time of ahnut 5 s f 5 . Accclcration of mxtivation and thc deactivation nf ATPgatcd channtls hy rlopaminc has hccn rcpnrtcd in PC12 cclls (as dctcrmincd from thc currcnt dccay upon washnut of ATP) (for frrtthcr d e i n i l r . sec Chnnncl rntidoin tion, 06-44)
z2'
Functionnl evidence for A TIj-Rated channel subtypes 06-37-05:Opcn timc distributions for ATP-activatcd channels in rat sensory neurnnes can he dcscaihcd hy a sum of two exponentials, probably rcfkcting the cxistcncc of two channel subtypes within thc
Effectsof P2x-rnediated Cn"-infIux on innctivntion of other chnnnels 06-37-06: Ca"-influx through ATP-gated channels in guinca-piE urinary hlacldcr incrcmcnts thc intnccllulat Ca2' conccntration, and this has hcen propnscrl to inactivatc voltage-gated Ca2' channcls in thc samc cetI3". Thus, Ca"+-influx throvgh the PlxR non-sclectivc cation channels has hccn prnpriscd to hc thc main dctcrminant of intracellular Ca" concentratinn in thcsc cclls undcr physiolngical cnnditions~".
Kinetic model Thc complcx opening and closing kinetics for P>xR have nnt hccn analyscd in detail. A model for thc rapid activation and dcactivatinn kinetics for ATPactivstcd currcnts in dorsal root ganglion ncumncs from rats and bullfrogs rcquircs ATP hinding to threu identical, non-interxting sitcs fnr chnnncl activation ( . w r D n w -reqponsc. ~ 3 h )
Selectivity ATP-gated channels
0s a
pnthwny for calcium influx
06-40-01: PIX purinoccptors posscss integral non-sclcctivc cation channcls' possessing significant perrneahility tn Ca2+, Na', K ' and Cs' inns. Direct
cntry of Ca2' into thc ccll appcars to he relatively large in rahhit car artcrial smooth muscle (see Table 51, urinary hladdcr smonth musclebTqand cochlear hair cclls"'. Measurcmcnt of variable permeah~lity ratios in diffcrcnt mcrnbranc preparations may indicate an unclcrlying hetetngeneity of channel suhtypes (see rxnrnplcs i n Tahic 5).
Select ivjry chnractcristics of c l o n d P ~ x Rexpressed in nocytes 06-40-02 PzxR (ORF ,199 na]: Ion-suhstitutinn cxpcriments show thc ratio of Cn" t o Ns' pcrmcahility IS 4.8 f 0.4 for this isofom when hctcrolngously cxprcsscd In oocytcs'. Thc sclcctivity cxhihitud hy this isofom (and nthcr clcctrophysiolagical prnpcrticsj clriscly rcscmhlc thosc nf nativc PZx
rcccptnrs of smtioth muscle.
entry Oh
Table 5 . Sulcctivity chnrrictrriTtm of P2y ri~ueiilor-i-hrinnrls(Fmni06-40-01 ) Preparation
ScIectivity charactcristics
Cardiac parmympathct ic
The amplitudc of tlic ATP-cvokcd c w r c n t in cardiac parasyrnpathetfc neuroneq is dcpcndcnt (in thc uxtracclPular Na’ concentration. Thc dircctian of thc shift in rcvcrsal pntential whcn NaCl is replaced with mannitol indicates that the purincrgict rcccptor channel is cation-sclcctivc. In this prcparatinn, the cation pcrmcahilitv rdativc to Na’ follows thc i m i c sukctivity scqucncc Cn” [1.48) > N;l’ (1.01 > Cs’ (C).67’), with anions hcing ’not inncasurahly pcrmcant’
ncuroncs
Scnsory ncuroncs
ATP-gatctl inward currunt m rat and bullfrog sensory neumncs is grcatly rcduced whcn N-mcthyl-r,-glucaminc is suhstitutcd fnr exturnal Na’. ATP-activatcd inward currents can hc rccnrdcd with Cs” a s thc sok external cation. From rcvcrsal potcntials, thu ratio of Ca” to Na’ pcrrncahilitv i s -0.3 : I rn this prcparetion Fcf. rrzlilirt erir or!er>r,
Rcfs
’‘
’‘
h h Wj Cocliac nuuroncs
ATP-activatcd channels which mediatc cxcitatnry synaptic transmission bctwccn coeliac neurnnes of the guinea-pig show a Na’ t(i C s ’ pcr~ncal,ility ratio PN.JPI.+- 0.6
Nucleus sol i tsri i
Calculatcd rclativc purrncahility ratiosf for the ATP-RatcJ currcnt in rlissociatcd rat nucleus solitarii newones KC P ~ J P ~- , 1 .h4 [ [ N;l *II, .In- I 50 IIIM.~), I’~..,JP~-, - 2.1 7 [[C:n”I,, - 2 m ~ hnicins ] arc not rnuasumhly pcrmcahlc in this prcparation
ncuroncs
50
’‘
7
Vascular smooth inu sclc
Although the rnajnr cation cntcring thrnugh the ATP-gatctl channcls In vascular smaoth muscle cells is Na’, PIXpurinoccptor activation also guncrates suhtlc, I(ica1izcd incrcast‘s in calcium conccntmticm. In rahhit car artcria! smooth musclc, ATP activatcs channcls with appnixirnatcly .? : I sdectivrty for Ca” ovcr N a ’ at ’ncar-physiol~iR~cal’ conccntrations (uf. sr*nsory neurnnrq. n !?owl
31
’‘
Table 5. Continued Rcfs
Preparation
Sclectivity characteristics
Skeletal muscle
Permeation of both cations and small anions has been shown to occur through a class of ATP-activatcd ion channels in developing chick skeletal muscle. This conclusion is based on fluctwation analysist about the mean current induced by ATP. At both +40 and -SQmV, ATP elicits a clear increase in noise, ‘hut a t the reversal potcntial of the ATP current ( - 5 mV1, no increase in noise abovc background was observed, indicating that only a singlc class of non-selective excitatory ATP-activated channcls was prcscnt. Rased on analysis of noise spectral, the conductancc of individual channels is cstimatcd to be 0 . 2 4 4 p S in this preparation. Calcium is the most pcrmcant ion for this class of channels, with NO; and I- calculated to be of equal pcrmcancc to Na’
‘‘
Selectivity choracteristics of cloned PpxR 06-40-03: PzxRl (QRF 472 aa): Ion-substitution cxperiments indicate thc 472 aa isoform incorporates a non-selective cation channel equally permeahlc to Na’ and K’, allowing conduction of even large cations. The P2x reeceptnr expressed in oocytes displays inward rectification with a reversal potential of approx. -5 mV. Rcplaccment of all extracellular monovalent cations hy large organic cations [c.g TrisJshifts the reversal potential to -45 mV and rcduccs the currcnt amplitude significantly. Replaccmcnt of cxtraccllular Ca” with Ra2‘ has nn effect on thc ATP-induced currcnt2.
Single-channel data Heterogeneity of PPxR suhsypes is evident at the sin,qle-chonnel level 06-41-01: In general, singlc-channel currents arc significantly larger in neiiroms, smooth muscle and PC12 cells than thcy arc in cardiac and skeletal muscle, whew estimates of small single-channel conductanccs
(typically less than 1 pS) arc difficult to obtain by patch-clamp and require tcchnrqucs of noise analysist.
Flickering hehnvioirr of unit nry P2xR-channelx 06-41-02: Singlc-channcl npcn statcs of ATP-gated currents in a numher of
prcparatinns display prominent fltictuatinnst . For example, in sensory ncurones, fluctuation amplitudcs in the kHs fruquuncy hand display approxirnattly 30% of the mean currcnt arnplitudc [mcasurcd as a douhlc
cntry Oh
rrns). Autocorrclaticin functions for fluctuations in an open channcl can hc approximated hy a single exponential (timc constant -10.4 msl and do not depend on the presence of divalent cations in thc external r n ~ d i u r n ’ ~ . 06-41-03: Singlc ATP-gated channels in dorsal root ganglion ncuroncs also display Wickery‘ open-channel gatingt hehavinur whcn activated, prndzrcing a mean current of ahout 0.5 PA at -100 mVF8.High ATP agonist concen-
tration? lengthen the periods whcn the activated channcl flickers, while lowcr tonccntrations of ATP produce periods of flickering intctspcrscd with closurcs”. Wholc-ccll current fluctuations display the expected characteristics if such flickering channels underlie the macmscopic currents7z.
Mensured vrrlues of ATP-gated unitary conductances 06-41-04: ATP-gatcd unitary channcl activity can he rccnrded when agonists arc applied to outside-out1 patches u r in thc ccll-attachcdt mode. Although a nurnhei of different rucording cnnfiguratinns and condbtions have been
emplnycd to measuic unitary PzxR channcl activitv, variation? in the values ohtatncd prnvidc suppnrt fnr the cxistcncc (if channel. subtypes. Further examples of studies rcpnrting measurements of unitary mrrctlts are shown in Ta’hlu 6.
Voltage sensitivity Volrqe-dependence of ATP ngonist potcncy Oh-42-01: ATP-activatcd currvnts in dorsal root ganglion neurones from rats and bullfrogs display potency and kinetics of ATP action that are vnltage-
depcndcnt, with hyperpolarization slowing deactivation and increasing ATP‘s potency. Note: In this preparation, dcactivatinn kinetics are sensitive to the cnnccntratian nf CXtCmal Ca” (hccoming fastcr in higher Ca”). ATP-activated channels in rahhit car arterial smrmth musclc can hc opcned cvcn a t very ncgatrvc potcntials and arc characteristically ( 1 ) rcsistant to inhihitinn by cadmium nr nifedipino (uf. V K Crr, entr,v 41) and (iil arc relatively inscnqitivc tn cxtraccliular Mg” hlock in thc rangc 1-5 mM (d. F L G CAT GLIJ N M B A . cntry OX).
Differing vohnge-rlepcndcnce of externd vcmus internal Cn2’ hlock nf P p x R -chnnnels 06-42-02: Extcrnal Ca2‘ block of ATP-activated channels in mt PC12 cclls ( w e Rlockrrv, 06-4(71docs nor cxhihit valtagc dcpcndcncc hctwccn - 100 and 210 mV. Hnwever, inhibitory effects of internal Ca” arc voltagedcpcndcnt, with thc inhihition heinR relicved with hyperpalatizatian”.
Wenk voltagr-clependencc of desensitimtion pmpcrries 06-42-03: Tho ratu (if ATP rcccptor-channel desenqitizatinnt (set I n q trvmtron, 06-37)1s wcakly vriltaac-dcycndcnt in cardiac
Dcpcndencc on mnintnined rnemhrnne potcntinl 06-42-04: Thc ATP-stimulated Ca2’ cnnductancu In rat hcpatomn G C h is i n h i h t e d by 70% upon dissipation of thc memhranc potcniial using thc K’ ionophnrc i vaIinarnycin2”.
entry 06
Depolarizing effects of PzxlE-channel nctiva tion 06-42-05 ATP-activated channels in several preparations7'- '* serve a dual excitatory function involving ( i Jdirect entry of Ca*' through the integral ATP receptor-channcl and [ ii) indirect activatinn of Caz'-cntry through voltagc-gatcd channels [fdlowing Na'-entry and depolarization). Roth actions promote x t i n n potential discharge. The ATP-activated rise in Table 6. Single-channel charmteristrc.7 nf Ppx receptnr-chonnelx (From 06-41-04] Prcparation
Sensory neuroncs
Refs
Single-channcl characteristics
-
In rat sensory neurones, the mean conductance
'9
of single ATP-activated channels is 17 pS (in saline containing 3 mM Ca7+and 1 r n Mg2'; ~ holding potcntial -75 mV]. Suhconductancct levels havc heen detected in this preparation. These channels rcscmhle the PzxR cxpressed in phaenchmrnocytnrna
cclls (see hehw) Coeliac neurones
ATP-activatcd channels which mediate cxcitatory synaptic transmission between coeliac ncurones of thc piinca-pig display inward rcctificationt and a mean singlechannel conductancet of 22 pS at -50mV
5fl
Phaeochromncy toma
ATP-activatcd currcnts in thc phacochromocytoma PC12 ccll linc display a unitary conductance of -13 pS [outsidc-out, ccll-free patch with 140 mM Na' in the cxtcrnal solution)
75
Artcrial smooth musclc
ATP-activated channels in rahhit ear arterial smooth muscle cells havc a unitary innic cnnductance nf -5 p s in 1 10 I~IM Ca7+ na2+
Vas dcfcrcns smooth rnusclt
In smooth muscle cells of rat vas deferens, an clcrnentary current (mean conductancc -20 pS, zcro currcnt potcntial -0 mV)is nhscrvcd in cell-attached patch clamp when the intra-pipette solution is changcd to ATPcontaining solution
Cloned PzxR (899aa isoform) cxprcsscd in oncytcs
ATP (+10 nM] cvnlccs unitary currents in apprnximatcly 64% oh outsidc-out mcmhranc patches, while the effcct of ATP dcclincs with subscqucnt applications to the samc patch. Thc chord cuntluctancct hctwccn - 140 mV and -80 mV has been detcrmincd as 14
ps
.'
''
1
n
[Gn"I, within rahhit car arterial smunth musclc culls7" 1% voltagc-dcpcndcnt as out wrird currents rvokcd by AT]' [at pcjsitwc m c m l m n c potentials) arc nnt associated with n changc in [Ca"]," (undcr thcsc conditi(ms, approximatuly 10% of thc ATP-g:atccl currcnt is carricd hy Ca" ions).
Blockers (.%p u l w common i r s ~ n( I T p x recepror mnrmgoniTis ~n'IhukInR' ex~rrrcellrrlc~r R TIMrpendent rcsponsrv iindcr Ilroep!or ontmpniqts, 06-5I).
RInck of PTXR chnnnuls b y cxtcrnn? Co2' ions (it high mlllirnolar cmcen t m i mn.s
06-43-01: AtthouRh it is pcrrncant, rclativcly high concentratinns nf external calcium ions can rcducu inward cztrrents carried hy Na' ~ 0 n s ' ~ ~ ~ ~ * ~ 75. ~~~~'~'~ In PCI 2 cells, thc hlnck is conccnt~~ticm-dcpcndcnt with a Hit1 coefficicnt of I and a hall-maximal conccntration (if npproximatcly h TIM. A similar Hock is nhscrvcd with other divalcnt cations, with an d c r of pntcncy nf
MK''=Ca I . z Pa"'. Characteristically, high canccntraticins nf Ca", MR" and I h " do m r hlnck complctely, prnlxhly because thcsc ions can also carry currcnt in thc ohnnncl (rec rilsn Voirtigr srn.citivI!v, Oh-42). Cd" > Mn"
>
Internti1 Cn" ion hlock of
rcccptnrs
06-43-02: Thc amplitude of inward channel currcnts cihtaincd with 150 mM cxtcrnal Na' nsc rcducctl h y incrcasctl internal C d ' rn thc insidc-nut patchclamp configuratinn. This reduction is cihscrvctl at hwur concentrations than that hy cxtcrnal Ca". Internal h'' and Cdv+ ~nrliicc similar r d t t c t i m s tn ciirrcnt amplitiitk". A qimplc nnc-lmdin~-situmodcl with symmetric energy 'harriers I$ inwfficicnt to explain I~~drrcctic>nal Ca' ' hlock in 1'C 12 cL.lls22( w e r 1 l c o V O h I p ~cran.5it ivrtv, M-421.
Ionic hlockrrs rd A TI'-,qritud channels 06-43-03: An cffccttvc t h c k e r of thc ATP-stimrrlatcd Ca" cnnductance in rat hcpatoma cells is gadolinium inn2". Voltagc-gatcrl calcium channcl blockcrs such as nifcdipinc ;ind vcrapamil fail to inhibit "'Ca'' uptaku in thcsc cclls". High conccntrations of zinc ions rcducc and p i d o n g ATP-activated currcnts in rat syrnpathctic ncuronm (crmsistent with open-channel Mock)
whilc lnwrr (micrnmnlar] conccntratirms potcntiatc I,,, Chnnncl modiilntion. 06-44]
I,hr detoiE7,
we
P hn r m acnlocqicnlblockers
06-43-04: In thc prcscncc nf thc non-sclcctwc hlockcr (+-J-tuhocurarinc,
maximal rcsprinscs of rat phncochromocytarna (PCI1) cclls to ATP arc dccrcnscd, hut ATP cnnccntratirms pmducing half-maxima! rcsponses a f v unchsngcd25. Narc: Thc blocking action of (+j-tuhocurarine affccts influx through othcr c x t r a c c l l u h Iigmd-gntcd channcls and vdtagc-gated channcls powcssing dtstinct stmct~ircs.( F o r cxnrnple, wr' ELG CAT 5-HT,7, critry 0 5 . F I A ; CAT n A C h R . r n t r y 09, nnd V l X C(i, enfry 421.
Scpnrahle PPx purinergic and nicotinic receptor-channel responses
06-43-05: ATP-activated and nicotinc-activated influx currents in ncrvc gmwth factor (NGFJ-trcntcd rat phaeochromocytoma (PC12) cclls show many similar propcrtics, and tentativcly, the possihility that thc channels . inward underlying thc currents wcrc idenricnl was ~ f f e r e d ' ~Hnwcvcr, currcnts mediated through ATP-nctivatcd channcls in thcsc cclls can hc sc~cctivclyantaganizcd hy suramin7* (we Receptor nntrljymists, ~ t l - 5 1 ) . Furthcrmnre, ATP-ptcd currcnt IS not affected hy -100 I I M hirsutine (an alkaloid that praduccs a potcnt ganglian hlocking cffcct by potently Hocking nicotinic rcccptnr channels and partially inhihiting voltagc-Katd Ca" and K' ~ h a n n e l s ~ ~ ' .
Channel modulation Positive and negotive modulation of native PZxX channels h y Zn2' ions 06-44-01: Two distinct modulatory sites of action for Zn2+ions have bccn proposed for ATP-activated channels in rat sympathetic neurones4'. First, therc is cvidcncc tor a positively acting allosteric site that enhances current amplitude (-hc-fold with micromolar Zn''1". Ry modulation a t this site, Zn" ions can increase rncmbranc dcpnlarization and action potential firing elicited hy ATP a~onists"'. Thus low concentrations of extracellular Zn' rapidly and rcvcrsihly potcntiotc both I AI I,nr and the introccllular Ca'" rise. The potentiation hy 10 (IM Zn" is dependent on agonist concentration"'. Zn" ions increasc thc sensitivity nf activatmn without potentiatmg thc maximum rcsponsc (i.c. possihly by increasing thc affinity of PzxIi for a g m i ~ t " " ' ~ ' )Sccnndly, . there is evidence for a negatively acting modulatory site for Zn2' (pcissihly within the pow) that blocks cnntluctancc through the I'2xR-channc14".
Positive modulation
of cloned P,,R expressed in m c y t e s 06-44-02 PzxRl (ORF 472 aa): Addit'Lolon of lil pu Zn2+to thc bathing solution shifts the EC5{)fnr ATP from 60 p~to 15 p d .
Dopnminergic modulation 06-44-03: In pfraeochromocytnma [PC12]cells, ATP-activntcd channcl currcnts are enhanccd by dopaminergic mechanisms, although this motlulatinn has not hccn attributed tr) any single class of dopamine receptorsR'. in these cells, 10 JIM dopamine enhances a n inward currcnt nctivntcd by 100 pu ATP. Similar cnhancements are produced by 10 I ~ M apornorphine, a non-sclcctivc dnpaminc rcccptor agonist, 10 I ~ M(+J-SKF38393 4a sclective dopamine n, receptor agonist), and 10 JIM (-)guinpirnle (a sclectivc dopamine DI receptor agonist). Morcnvcr, 30 I ~ M/+)-SCH-23390 [a dopamine Dl reccptor antagonist, and 30 IIM (-)-sulpiride (a dopnminu LJ7. rcccptor antagonist] also cnhancc thc ATP-activatcd currcnt".
Mechanism of dopominergic modulatr'nn of P2xH-chmnels 06-44-04: In PC12 cclls, thc 'dopamine effect' (see rilrwel has hcen shown to shift the dupcnrlcncc of activation rate canstantst on thc conccntration of
ATP toward a Irwcr cnncentrattnn rangc hy approximately two-fddz'. Dopaminc also x c c l c r ~ t c s thc inactivation and thc deactivation [as dctermincd from thc currcnt dccay upon washout of ATP). T h u s dopaminc augments thc ATP-activatcd inward currvnt by facilitating association d ATP tn its binding site. This augmcntation may hc mcdiatcd thmugh some protein kinasc which is diffcrcnt from cyclic-nucleatidc-dependentprntein kinnsos o r protcin kinasc C2'.
Comparative note: ATP rrs
fl
multiple modulator of other celhlnr
pmtcins 06-44-05: In addition to dircct and indircct ( G protein-linked) gating of ion channels, ~nimcellolmrATP is n candiciatc for multiple modulation 4311 other ccll-signalling moTvculcs ( f mexornplc, see ILc; Ca Ca Ryi<-Cu/,entrv 17. ILL' Cn { r ~ s i entry ~ ~ , 19. mnr? Protein interactions. oh-.? 1).
Equilibrium dissociation constant 06-45-01:In singlc cclls isolatcd frrnrn hwinca-pig urinary bladder, the rclationship nf the pcnk current vcrsiis A T P concentration i s wcll-fitted hy a Michaclis-Mcntcn equation with a K,I of 2.3 ~ I M .For ATP-gated current in dissociated rat nucleus snlitarii ncuronc's, the half-mammum concentration IS 3 1 I'M ATP'4. Suhstitution nf ATP with z,/l-rncthylcnu-ATP shifts thc K,l to - I 0.4 p~ (sec r r I w innctivnrinn, 06-37. a n d Hilt cnefficjcnt, 01,-41rl". Dopaminergic mcdulation can incrcnsc association of A T P fnr its hindnng site (sec Chrtnnel mwiutrt~ion, 0644).
Hill coefficient 06-46-01: ATP rusponscs in singlc cells isolatcd from guinea-pig ur~nary hladdcr displny n Hill cocfficiunt of 1.7. Suhstitutinn of ATP for T,/L
-
mcthylcnu-ATP (FCP E q i d i I m i E r n dicwciritmn consiwi. Oh-45)& i t s not significantly affuct t h t Hi!! cocfficicnt (n I .6ltA.For ATP-gatcd current in dissnciatcd rat nucleus solitmi ncuroncs thc Hill cocfficrcnt was calculated as 1.2'4 ( w a l w I ) o s c - r e ~ p o n ~ c06-3h). , T l x R l [ORF 472 aa): The Hill cocfficunt for this i s o h m cxprcsscd in oncytcs has hccn dcrlvcd as 2.0 for ~
I
_
ATF.
Ligands Oh-47-01: ATP as a free aninn (ATP4 1 can activatt thc rcccptor 1i.c. ATP can activate the channcl In thu ahscncu (if drvalcnt catinns a s well as in their prcscnccl. CR'+ ins ducrcasc hoth macro- and microscopic ATP-activatcd currcnts with il cnncentra~inn-deycnJencc that can not hc fittcd with a
singlc site binding isothcrm'9.
I'hotrbm ffin iiv -I(! hellrn~s t tidips
06-47-02 In nn attumpt tci idcntify th c ATP receptor protein in phncochrr>mt>cytr>mn[PCI21 mcmhmncs, cclls wcrc phntnaffinity-1ahelIcd with
the radio! iRand [32P1-3'-O-( 4-henzoyl-hcnzoyl IATP ( [32P]-BzATP)24. SDSPAGE$ analysis revealed that labelling of a 53 kDa protcin was inhibitcd by ATP and its derivatives, as wet! as by the P2 antagonists sutamin and Reactive blue 2. Note: Thcsc antagonists also inhihit nucleotide-induced noradrenaline release in these cellsz4.
Receptorltransducer interactions Role of PZx receptors in non-adrenergic, non-cholinergic excitatory transmission
06-49-01: In vas deferens" and Madder smooth musclc", ATP-gated cation channels produce a component of fast, non-adrenergic, non-cholinergic (NANC) excitatory transmission by sympathetic ncuroncs. In rabhit car artcry smooth muscle cells, entry nf Ca" ions through thc channel is directly associated with contractile events32*
'.
Discrirninotim of direct versus G protein-linked Ca*+-infJuxb y ATP
ngonists 06-49-02: Indirect activation of catinnic currents hy cxtracellular ATP and the inndulation of calcium current through G protein transducers have also been described in rabbit portal winR.' (see n h o Resource A , entry 56). Notahly, the P>x subtype receptorxhnnncl of rabbit car artery smooth muscle cells is unaffected hy SKWF 96365 ( a novel inhibitor of reccptnr-mediated calcium entry7uvR").Ry contrast, this compound reduces 6 protein-mediated ATPstimulated currents by ahout 80% in human neutroyhils"".
Receptor agonists ATP evokes inward currents through PPx receptors with short latency and fnst rise time 06-5041: ATP, as well as the rclatcd molecules s,fl-rnethylene-ATP, 2rnethyltbioATP and ADP evoke inward cuncnts with shnrt latency+ (typically < 2 rns minimum) and fast rise timc (twically < 10 ms for 10-90% rise) following application. Thc PIXrcccptors display variable desensitizationt kinetics with ATP and ATP-dcrivcd agonists [with some voltage-depcndencel, dependent on thc preparation and (prcsumahly) molecular suhtypc (see Inactivntion, 06-37, Voltage sensitjvrty, 06-42, and Table 7).
Apnisrn nt clnned Ppx receptors expressed in oocytes and HEK-293 ceIIs - comparisons with native P2,K 06-50-02 PzxR (ORF 399 aa): 10 pu ATP, x,/Lmethylene-ATP, 2methylthioATP and ADP evoke 'typical' inward currcnts with latency+ of c 2 rns and a rise timc of -7 ms'. For this isofnrm the order of agonist potency is 2-mcthylthioATP 2 ATF > x,P-methylene-ATP >> ADP. F2xltl (ORF 472 aa]: ATP, ATP-7-S and 2-rnethylthioATP arc equipotcnt as aganists, whcrcas z,/bmcthylcnc-ATP and P,;.-methylene-ATP are inactive as sgonists or antagonists. Note: Thc 472 aa isofclrm displays agonist sensitivity that resemhlcs nativc PIxR a n PC12 and ccrtain sensory and
entry Oh
P2 receptor aRonism by 2-methylthioATP on cardiac sympathetic neurnnal P,,R
ATP rcceptnr4mm-wls cfcscrihcd in rat cardiac 21r syrnpathctlc ncumncs show an cirdcr of agcmist potcncy of 2-incthylthinATP = ATP > ADP 7 AMP I, adcnminc = z,/f-rncrhylcnc-ATP > /i,;.-methylcnc-ATP (a sequcncc alsn consistent with the C, prritcin-linked Pzv rcccptnr suhtypc). ATP and AMP are a n ! n ~ o n i s ! snf thc PIT rcccptnr channel suhtypc expressed nn platclcts {.we Sriluype cl~icsrfmtions.Oh-QhI.Note. ATPvvokctlcurrtnts in thispreparatirin Arc nrrcnrrnreclr by r,/i-rncthylcnc-ATP lICGcF10 p ~see , hchwl snrl rcvcrsihly inhihitecl in a dosc-dcpcndcnt manner hy Rcactivc h l w 2 (KLI= 1 MI R9 x,/l-Mcthylcnc-ATP is a mcta‘holically stahie ATI’ ~na111gw that charsctcristicallv activates then dewnsitizcs PgXrccceptors. Arylazidnarninopropumyl ATP(ANAAPP.3)covalcntlyhinds tp Pzx rvcvptors fo!lowin~irrdiaticin, and ISa l w capahlc ot inrlucing rcccptw activation then hlockadc Fast-synaptic currt‘nts within much1 hahcnula 2R ccntrAl neuroncs (part of A wcll-charactcrizcd chcilincrgic pathway which arc hlockcd hy suramin descmitize fnllowing application of .x,/~-int.tt~ylenc-ATP. Miniaturc post-synaptic currents ohscrvcd following spontancnus rclcasc of trmsrnittcr Tn this preparation arc alsti rlcsunsitizccl hv this ngcinist Lhcnsitization hy r,/l-mcthylcnu-ATP also hlr1ck.i the rcspnnsc to ATP in single cells isolated from guinea-pig urinary hladdcr. rJ-Mcthylcnc-ATP i s -50-100 tirncs marc pnttnt than ATP at u l i c i t i n ~a ctintaactilc rcsponsc of strips nf dctrtisor smooth musclc. 1 Similar ricscnsittzatson hchaviour t(i ATP [ 2 1 I I M ) is chstrvuti in cells cxprcssing rccornhinant PlxR ( O R F ,199 aal PlxRl _ _(ORF _ 472 aal: Arnnng scvcral nucleotide and nuclcosicfc dcrrvativcs cxarnincd, only CTP ant1 dATP clicit small hut detectable current responses from this isofnrm In cxcitatnry synaptic trammissinn hetwecn ccwliac neurtincs of thc guincn-pig, ATP cvrkcs rnward currents with greatcr pntcncyt and clficacyt than acetvlcholinc IACh)
-
.x,/l-Methylene-ATP and ANAPP3
Induction of duscnsitizntian by r,/l-methylene-ATP on ncurnnal P l x R
Inductinn of dcscnsitization by r,/l-methylene- ATP o n smooth musclc b X R
CTP and dATP
ATP [cxamplc of tissue-dcpcndcnt, cndogcnous agon25t rcspnnses
1
autonomic neurnnc~"*~'.~"~' (this pattern differs from that ohsewed fnr P2xR on vascular smooth muscle, vas defercns and some CNS ncuroncs, where r,/f-methylcnc-ATP acts as a potent agonist"'"').
Multiplicity of modulntory and ngonist roIcs of ATP 06-50-03: ATP has multiple actions on nthcr proteins, including ( I ) indirect gating of ion channels through G protein-linked receptors (see Kesorirce A C; prcjrein-linked receptors. entry 5h) and ( i i ] as a modulatory factor on sssociatcd signalling mmponcnts, including ion channcls x t i v a t c d hy othcr ncumtransmittcrs or second messengers (see, f o r exnmple, I L G C n Ca KyRCaf. entry 17, nnd ILG Cn I n ~ l ' \entry ~ , 191. Partly hccause of this multiplicity of targets, highly sclcctivc agonists for PZx rcccptors arc cuncntly not
svailahlc. Tahlc 7 lists m m c applications of thosc presently in
USC.
Receptor antagonists 06-51-01: The [non-suhtype-selective)ATP receptor antagonists Reactive blue 2 (RB2) and suramin reversibly block hinding of ATP to P2 receptors Id.[+)tubocuradne, a potent antagonist of acetylcholine- and serotonin-gated channcls acts as a non-sclcctivc hlockcr of ion permeability through the ATP-activated channel - sec Rlockcrs, 06-43),RA2 and mramin can distinguish PzxR responses from othcr extracellular lipnd-gated channels''. Comparative studies of clcctrophysiological effects of the ahovc compounds show all thrcc of these compounds inhthilt ATP-gated current in rat phactv chromocytoma (PC121 cells in a concentration-dcpcndcnt manncr [ordcr of potency RR2 > suramin > ~+]-tuhocurarinc]25. Unlikc for surainin or RR2, hlnckadc indiiccd hy (-t)-tuhocurarinc is not rcvcrscd after a 5-min washout Furthcr characteristics of thcsc antaRonists arc Iistcd in Tahlc 8. In gcncral, thcru is a nccd fnr morc sclcctivc antagonists actinK at PIXpurinoccptors.
Antagonism at cloned PPxR expressed in oocytes and HEK-293 ceJls 06-51-02: PTxR [ORF399 aa): Cutrcnts cvokcd by ATP, x,/bmcthylcnc-ATP, 2methylthinATP and ADP arc rcvcrsihly blocked by summin (3-100 J I M )and by
~ytidoxalphosphatc-T,-sxophcnyl-2',4'-di~ulpho~i~ acid (FPADS, 1oL30 puJ hut not hy arnilnride (100 p ~ ) T. h e w propertics scrved to idcntify thc cxpresscd rcccptors as of thc PIX purinoccptor subtype'*'. PzxRl (ORF 472 aa]: - Both suramin and Reactivc blue 2 reversihly antagnnize ATFcmkcd rcsponscs of this isoform by > 95%; [+j-tuhocvrarinc only partially blocks (-50%) ATP-evoked responses of this isoform'.
Relative potency for P2-rnedioted release of nnrndrendrne from IT12 cells 06-51-03: The relativc potcncy of ATP and a number of analogues for cliciting nnmrircnnlinc rclcasc from rat phavochnimocytnma [ PC 121 cells in thc presence d cxtraccllular Ca" has hcun shown to follow thc nrdcr adenosine 5"-0-(3-thint~iphnspRate)> ATP > ndenosinc S'-O-[1-thiotnphosphatcl = 2mcthylthioatfcnosi!ic S'-triphnsphatc [MUSATPI > 2'- nnd 3'-Q-(4-hcnzoylhcnzt1yl)ATP (RzATP) > hDP > ~-nd~nylylimidndiphosphatc2~.
Table 8. Chnmctmstics r d AT{’ nntngonists (From Oh-SI-OIJ Antagonist
Rcactivc hluc 2
Suram in
Application [cxamplcsl
Refs
Thc ATP-actwnrcd channel in rat hepatoma cells is inhihrtcd in thc prcscncc of Reactive Blue 2 (RR21, suggesting that channel activation is dcpcncicnt nn purincrgict receptor interaction, ~n coeliac neurnnes of the guinea-pig, the antagonists Reactkvc hlut 2 (and suramin, see helow! rcduce the effects of ATP [1Ci0- l - l O p ~ Fhut not acctylcholinc agcinists. RB2 is a slowly-acting antagnnist (if t h c ATP-Ratcd channel. In rat phaeochromocytoma (PC12) cells noradrenaline release is inhibited hv RR2 [ I C 5 0 - l - l Q ~ ~ ~ F
2o
5o
24
lri The ATP rcccptor antagonist surarnin lacks sclectivlty for 112 purinoreceptor suhtypes, hut can discriminatc hctwccn P*x responses and those of othcr fast ncurotransmitters. Suramin is a competitive hlrnckcr of hoth cndogcnous transmittcr and ATP-cvokcd cumcnts in coeliac ganglion preparations (see Cell-tvw exprmsmn 1 R d C X . Oh-OX). Fn rat phacochromocytoma (PCl21 cclls, noradrcnalinc release is also Inhibited hy suramin (IC5r! 30 prw)
’‘
-
Stilhcnc Scvcrnl stilbene isothiocyanate a n a l n p e s nf the calcium-activatcd chloride current inhihitor DIDS isothiocyanata analogues at Pzz [dihydm-DIDS, SITS hut not DNDS - we purinnccptors Appendix C. cntrv S8F can hlock hoth the binding (comprirrrt I ve m t c of I ”PI-ATP t o intact parotid cell? and thc only) , rurinaceytclr-channcl~~. activation nf thc P T h c pntency nf thc stilhene diwlphonates is rehtcd to the numher of i ~ o t h i o ~ v a n aErnups te on cach cnmpound ( c g . DIDS, Ic5(,35 p ~ S ,I T S ICGr, 125p ~ DNDS ; lacks isothincyanatc (SEN 1 groupS]. Eosin-5-isnthincyinate (ElTC) and fluoroscein-5-isothiocya~ate (FITC1, nanstilhcnc isothiocyanatc compounds wsth single SCN groups, also hlock thc response to ATP but arc less potcnt than DlDS. TrinitrophenylATP (TNP-ATPJ,an ATP clcrivativc that is not an cfftctivc agnnist of thc parotid acinar cell PIZR, hlocks thc covalcnt hinding of DIDS to the plasma mcrnhranu, suwestiny: t h a t ATP and UIDS hind to thc same sitc. Thc drstilhcnc DIDS and 2‘,?’-dinlduhyde-ATP irrever4ihlv inhibit the skclctal rnuwlc ATP-gatcd channcl. DJDS also irrevcrsihlv hlocks ATP-induccd Ca”-entry in parotid x i n a r cclls of rat
-
+
’’
’‘
’‘
entry 06
Table 8. continued An tagonis t
Application (examples)
Refs
Adenosine derivatives without aRonist activity
These types of compound usually act as weak competitive inhibitors of PzxR (c.g. adenosine 5'-1P,;.-dichloromethylene~~ziphosphanate, ICqo-21 pu at neuronal PlxR)
RR
rJ-Me thyleneATP
a,fl-Methylene-ATP possesses agonist activity in some preparations (see Receptor agonfsts, Qh-5QJ and may therefore act via a dcscnsitization mechanism. rx,/f-Methylcne-ATP inhibits agonism by ATP in parasympathetic neuronal and cardiac atrial preparations, hut not in vas deferens, skeletal muscle nr sensory ncumncs
26,
37B 77. Rh
Effect af ATP nnnlo,ques on P ~ X Rexpressed i n smooth muscle from rat VQS deferens 06-51-04: In smooth muscle cells isolated from the rat vas deferens, the analogues 9,P-mcthylene-ATP and AMP-FNP (lt,y-irnido ATPJeach produce a small, relatively sustained inward currcnt (not resembling the ATP current\. The analogue AMP-PCP (I{,;-mcthylcnc-ATP] has little or no effect in this preparation9'.
Agonism b y other adenosine derivatives and ATP-y-S 06-51-05: Ry definition, PI receptors arc sclcctivcly agonized by ATP over adenosine. ADP is a weak agonist (seF above) and CTP may elicit some currcnt in ncumncs", hut GTP and UTP are nnt effectiveas agonists. ATP-7-5 (adenosine 5'-0-&thiotriphosphate, see above) i s apparently equipotent with ATP in PC 12 cells", cardiac muscle", skeletal muscleR', and neurones2'.
inactive agonists nt cloned PZx receptors
06-51-06: PzxR (ORE 399 aa): UTP ( ~ O O ~ U ]GTP , (100pu3,acetylcholine ( 100p ~ and ) 5-hydmxytryptarnine (SOJIM) are ineffective as agonists'. PIxRI (ORF 4J2 aa): ADP, AMP, 5'-adenylylamido-diphosphate,adenosine, GTP, UTP, CAMP, cGMP, acctylcholine, glutamate, glycine, 7-aminohutyric acid (GARA] and 5-hydroxytryptamine (serotonin] do not activate this isoform when expressed in oocytes'.
~
1
~
~
~
~
@
l
~
~
~
Database listings/primary sequence discussion
06-53-01: The relevtint dntnlinsc is tndrcnted hy the lower cast' prclix ( e . g gh:,, which .sliould not 1 7 ~typed (.wc Introduction el 1ctvotJt of entries. e n t r y 02). D a t a h e 10ctr.5 ri~fniustinil ~ ~ C C P S Sntrnlhfrs ~ O ~ immetiioteh follow the colon. Note t h t a comprehensive listing of iill nvnilnhlc accession numhcrs is superfluous {or location o{ re!eviint s e q u r n c r in
~
~
cntry 06
GenRnnk" rrsnurcrc, whrch ore now ov(rrlnble wit11 pnwerlul in-/milt neighhflaringi onnly.;is routinrs (Tor d~vcriptron,FCC the Dotnhnse l i s t r n ~ s {irlrl In rhr Introduction nnd l o ~ o u ! oT cntrles, entry 02). For exnmple. sequcnccs of C~OFF-SPCCIC.C Iwrrc:nf\ or IL'J(JIL'II gene (om11 rn~rnherccon he rcr~dilvrrcccr..;sed t>y one o r two rn1:nd.s oi nrighbnuring +ana[vsiq (wl~rch art. /~oscrJ on prc-comp!rted o b ~ n m r n t cprrfnrmrd using the R L A S T ~ o l ~ o n r h r n h y tlac NGNITj. Thrc ienltlrr! mnqt slwiul Cor retrrevol of sequrpncc cntrrev dt.povit~'~j rn dlltr~l>(t~i~c Imt~rthmn thnce livted Irelnw. Thus, rr>prcr;rntutrv{> mrmhrrs ot Irnown wquunce homology grnupin~srrrc Jrsr cd to pcrrnlt ~ n lrr11 t d ~ r r c t rrvrrievr~lq hy at-cr w n n number, nurhorl reletcnr*i3 or nomrnclulrrrr Fnllomng rirrcct ncccssion. J~owevcr. neighhour_lngf --unnlj~eiq i.s qtrmgly rrcornrnrndtd t o ~rientrty newly Efyc~rtir/nnrfrclrrtcrl scsltlpnocq. --- -
Y
Nomcnclaturc
Speclcs, DNA sourcc
Orig~nal isol:~tc
Accession
Scquuncul discussion
P2xR
Rar, vas
ORF 399 aa
gh- XH0477
Valcm, Nnrure
bxR1
Isolatc RP-2 (partla1 cUNA scqucncc)
dcfcrcn~
(1994)371:
cDNA library
516-19.
Rat, phacochrnmocytoma (I'C 1 21 cDNA l i h r ~ r y Dtr~vurPfrom a suhtmct~vcf hyhndlzatlonf library
ORF 472 aa
gh: U 14444
Rrakc, Nflrure ( 19'141 371:
5 19-23. Originally
dcscribcd as an 'apoptosis~nduccd protein' - see
gh: MR0602
Owcns, Mnl CPII Rrnl (1991111: 41 77-88,
Ilcvclo/?mental rcgr11ntlnn. O(I-I I
1
Related sources and reviews 06-56-01: Pharmacalogical and clcctrophysiological charactcristics of ATPactivated inn channelrs'; ATP-activatcd channcls in cxcitahlc cc11s7 and vascular smooth musclc ~ o l l s " ~ATP ~ ~ ;as a co-transmatter with noradrenaline In thc sympathetic nervous systcm's~"P;ATP rtccpzor classif~catirjnsnntl: n ~ r n e n c l a t u r e ~ 'Ecatures ~~? nf clnncd PzxR ~sofnrms'~'.
Feedback
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Entry support groups and e-mail newsletters 06-57-02: Authors who havc expertisc in onc or more fields of this cntry {and arc willing to provide editorial or other support for dcvclnping its cnntcnts) can inin its support group: In this c a w , send a rncssagc To: [email protected], (cntcrmg the words ”support group” in thc Suhluct: ficld]. In thc rncssagc, please indicatc principal intcrcsts [ scc Jrcldnnrne criterio m the Introduciion for coverqy) togcthcr with any rclcvant http://www site links [established or pmposcd) and dctails af any other possihlc contributions. ln due cnursc, suppart group memhcrs wiIl (optionally) receive e-mail newsletters intendcd to cn-ordinate and deveInp the present (text-based] cntry/fieldnarne frameworks mto a ‘library’ of interlinked rcsources covcring ion channcl signalling. Other [more general) information of intcrcst to cntry contributors may also he scnt tci thc ahovc address for group distribution and fcecihack.
1
2
3 4 5
d
7 R 9 10 11 12 1.7
14 15 16
17 IR IY
2n 21
22 27 24
25
26
Valcra, Nnturc (1994) 371: 5 16-1 9.
Brake, Nntrire ( 1 994)371: 519-21. Clwcns, Mnl CPII R i d (19911 11: 4177-88. Rn, J N z d Chem (1942) 2h7: 1 JSKI-7. himstock, Ann NY Acnd Sci (1990)h03: 1-17, I
H ~ r n i Nrurocc;ur~cc ~, ( 1 YY2) 48. 94 1-52 Edwards, N r ~ t ~ i (r1992) r ~ 359: 144-7. Mozrzyrna.;, Nrl1rort.i I.(,/ 1 ( 1 YY2) 139: 2 17-20. N a k a ~ a w ; ~/ ,Nallrophv\rol ( 1 YOIIJ (r3. l OhK-74. Ashmorc, Phv\lol (1990) 428: 1 O9-,3 l Ilcnhnm, Nrriurr' ( 19H7) 32R 275-K Iknharn, A~lriNY Ari~riSr 1 ( 1990) h(13. 275-XS. Stl:~rnc, 7'ri,rlrf\ Nr1rrort.1 I YKhl 0.557-48. Inouc, Nr'urosr.r I,rbti 1 1992) 134. ?I 5-1 X Sncddon, Si~rr~nui~ 1 19821 21R: (1'1.1-5. Fricl, i'hrrlol tor~rf[IIIKKI 401: .?hl-HO. J ~ h rNrrirlrr , ( 1 YX.3) 304. 7.30-.1.7. Sclincidcr, I'hyrrol 11 99 1 1 440. 479-46. Clouuf, I)ffl~~ytprc Arch-Fur I I ' l ~ y ~ !(olY4.1F l 424: 152-8. Nakaznwa, I I ' l ~ i w o !( IYYO) 42R: 257-72 Zhcng, Cell Hlr11 (1991 ) 112 279-HK. Zoctcwcil, II~oclll,n~ J / 19L)2)2R8: 207-13. Spranzl, Iti 1 P!~(rrn~(~i-oI [ 1 9 ) I I 57. 507-1 5. Intluc, F u r I I'l~rrrnimt~ol ( 1902) 21 5 12 1-4 VonKugclxcn, 7'rrrirl\ I'hrrrmr~c'olScl [ 1 Y Y 1 ) 12: 3 1 4-24
Xiong, Physiol Lond ( 199 1) 440: 147-65. Merritt, Rimhem (1990)271: 515-22. Krautwutst, lliochem ] (19921288: 102535. Thomas, Icr ] I’hnrrnncd (1941) 103: 39&-9. Soltoff, Ann NY Acnd Scr (19901603: 446-?. Krishtal, Rr ] Phnrmocol [ lgXH) 35: 105742. Kennedy, Arch Int Phnrrnncodyn Ther (1990)303:,7040. Henham, Ipn I Phnrrnacol{ 1992) 58: 1’1 79-84. O‘Cmnnr, Trends Phorrnacd Scr (19YF) 12: 1.3741. Hurnphrcy, T r p n h I’hnrmacd S C [~lYY.3) 14: 233-6.
Edward C . Conley
Entry 07
AbstractJgenctral description 0741-01: Excitatory synaptic transmissinn in thc rnarnmahan ccntral nervous s v w m i s mcdiatcd prcrlominantlv hv ionotrapict glutamate receptorchannels (iGluR1 which arc sclcctivuly activatutl hy thc compoiintl z-amino~?-hydrt~xy-5-muthyl-4-i~oxazoIcpropi~inatc (AMFA\. T h e w rcccptor-channcls show fast aczivatirm and dcscnsitizationt kinctics, and In most (hut not all] ncuroncs m u characturizcd hy having high Na’/K’-perrneability and low Ca’+-permeahi~ity.
07-01-02 A t different aypcs nf ’kainate-preferring’ receptors, thc neurotoxin kainic acid [kainate) activates fast-desensitizzngt currents. Kainate also activatcq curwnts through ’ AMPA-preferring’ receptor-channels, hut thcsc pcrsISt in thc prcsvncc
07-01-04: Gcn cs cncoding ‘kainate-preferring’ iG1u R [ GluRS-GluR7, KA 1, K k 2 , 61, 62) dn not show m y sppsrcnt variation through altcmativc
splicin~,of mRNA. Hnwuver, some kainatc rcccptnr subunits (and AMPA suhimits) arc found in rnndificd forms produced hy RNA editingt, which is alsn dcvcl(ipmcnta1ly scnsitivc. In somc cases, primary scqiicncc variants introducctf h y RNA splicin~/cditinghave hccn demonstrated to produce important fun,ctional changcs (e.g mnic selectivityf and rcccptor dcscnsitizatinnT propcrticsI. 07-01-05:Whilc some high-affinity kainatc w h u n i t s dn not express functional
c h m n c l s In homomcric form, others display high-affinity for kainatt ant! suppnrt mpirlly dcccnsitizingl currents. Crwxprcssinn of thcsc ’inactivc’ suhunlt.; with other iGlvR Rcnc family mernhcrs fnrm heteru-multimcnc cornpScxcs with mndificrl propcrtics. Dcspitu this, thcrr is some uvidcncu t h a t ‘AMPA-prcfcrring‘ and ‘kainatc-pacfcaring’ suhunits do no! CMW assemble hut rathcr fnrm functionally independent cntitics which can cocxwt within smglu ccll.;.
entry 07
07-01-06: Snme iGluR suhunits appear tn ‘dominate’ the properties of heterornultimerict channel complexes. For example, when recomhinant channels are formed in vitrn from monomeric GhR-A, GluR-C or GluR-D suhunits, they show high Ca2+-perrneahilitywith doubly-rectifying1 [sigmoidj I-V rclationships. Introductinn of thc suhunit GluR-R in a n y crirnhinntion (GluR-A/ R, GluR-R/C, GluR-R/DI produces recombinant channels which clnscly match characteristics of native1 receptors [ 1.c. low Ca7’-pcrrncahility and linear I-V relationship). iGluR in nativct cell types which lack GluR-R [c.g. Rergmann glia cellq) also display doubly rectifying+ I-V relationshipst and high Ca”-permeability. Thcsc typcs of studies indicatc nativc rcccptors to he heteromdtirnmi of a t least twn different subunits.
07-01-07: A full appreciation of the functional roks of thc non-NMDA glutamate receptors can only he made by taking into consideration parallel interactions with other (co-expressed) signalling proteins. For example synaptic rclcase of glutamatc produccs an cxcitatory post-syna tic current f (EBSC) which can he rcsolved into a fast-onset1 and decay? component (mediatcd by non-NMDA ionotropic glutarnatc rcccptor-channcls) and a slow-rising, slowly-decaying component (mediated by NMDA receptorchannels - see ELG CAT GLU N M D R , entry 081. Glutnmatc can also initiate responses at rnetabotrnpict receptors, which may be indirccdy coupled to other ion channel activities (see Rrwurce A - C: protprn-linked
receptors. entry 56). 07-0148: Receptnrs for ionotropict glutamate receptor-channels [ iGluR] are expressed on ‘virtually every’ neuronal cell and soinc glial cells in thc CNS. Thc availability rrf subunit-spccilic prtihcs for distribution snalyscs and in vitro rnutagcncsist procct~urcs for protein Structure-function and
rransgcnict cxprcssion analyscs arc likely to turthcr clarify thcir precise roles in the ncwous system.
Category (sortcode) 07-02-01: ELG CAT GLW AMPAIKAIN; i.c. cxtraccllular IiRand-ptcd cation channcls sclcctivc for thc glutamate rcccptor agonists AMPA and kainatc. The sugqcsted electronic retrieval code (unique cmhcddcd identifier or UEIJ for ’tagging’ of new articles of relevance to the contents of this entry is UEI: AMPA-NAT or UEI: KAIN-NAT (for reports or reviews on nntivet channel pmperties) and UEI: AMPA-HET or UEI: KAIN-HET (for reports or reviews on channel pmperties applicable to heteroloRously1 expressed recomhinantt subunits cncndcd by cDNAst or genest). For n discussion n f the advantnges ol UEJF and girrdelines on their implcmmtatmn, s p e !he wction on
n
Resource 1 under Introdsiction and lnyorrt, cntry 02,and for fiirrher detmls, .we Ilcxoirrce - Senrch critctia d CSN development. entrv hS.
Channel designation Shorthnnd desipntinns in use 07-03-01: Ionotropict glutamate rcceptor-channels / iGlu R-channcls] tcrl by thu a p n i s t t ol-amina-3-hydraxy-5-methyIEsoxazoIesclcctivcly n c t i v ~
cntry 07
I-propfonic acid arc gcnvrnlly designaturl as AMPA receptors (AMPA-R]. Thcsv rvcuptorq show rulativcly hiRh affinity for AMPA (K,l in the nannmolar range) and h a w rclativcly Inw affinity for kainatc (K,,in the micromolar rangcl. Convcrscly, kainate receptor subtypes display rchtivcly high affinity for thc :~goniszkiirnate (kainsc x i d l ovcr AMPA. Siimc rcpnrts usc thc designation AMPA/KA whcn rcfcrring to rcsponsw froin thc broad class af AMPA/kainatc rcccptcirs.
Common nhhwvjrrtinns for /lip m d flop vnrionts 07-03-02: Narncs for the ‘flip’ and ‘flop’ alternative splice variants of CluR-A, C;!uR-H, GlrrR-C anti GluR-I 3 suhunits (lor du!nrls. SL‘C Gene orxnnjzntjnn, 0720) arc somctimcs rdcrrcd to sn ahbrcviatcd form: C.R. GluR-A flip varrants cnn hc wiittcn 3s GTuR-A, and GIuR-A flnp variants as GlrrR-A,,.
Designntion of sulscloss-specific properties w’thin this entry 07-03-03: In ordcr tn distinguish information spccific for glutamatc receptors selective for AMPA, an undcrlrncd prcflx ’AMPA:’ _ _ . _ is used within this entry. Prnpcrtics specific for high-affinity kainatc rcccptors arc dcsignatcd with thc prefix ’KKAIN:’ within this untqv C hi l p q y
of A M P A - mi3 kn ino te receptors ris h o n - N M D A ’ glu torn o t e
rc c ep 1 0rs 07-03-04: Srmilnritics hutwvcn thc p h y s i i h m and distrihution of AMPAsolccltiwu anti kainatc-sclcctivc glutsmatc rcccptnrs has tcrl t o frcqucnt coclassificatian i l s the ’nnn-NMDA receptors’ (filr iltpt(iilv, N C ~Rrr‘rpor rr”yrlrllFt.5, 07-SO).
Alternntivc nonrenclaturcs {urtrcnc%encoding A MPAIlininote stihunrts 07-03-05:Prcipcrtics nf recrmhinririt glutamatc rcccptors gcncrally make spccific wfcrcncc to thc subtype nf the Rcne(‘sl cncodlng thcrn. The nnincnclatiirc of prcscnily known GIuR suhztnits and aswciatcrl protcins is, hnwcvur, under t-c‘wcw’. Alternative nomenclatures fnr thc same Tubunit gcncs have lwcn suggcstcd h y rliffurcnt lahr~ratrmcs. For cxarnplc, E-icincniaim ;ant! coIIc:~gues’-l“ havc uwtI serial numbers for glutamatc rucoptoi gcncs running from GluRI through GluRn. Mishina and collahnratcm5 havu assigncd Greek Ictterq tn suhunlt famlllcs grouped hy aminn acid scqucncc hnrnnlngyt, and scrial numbers tci thc meinhers within cach fnmilv. Rcttkr cr ~ 1 1havc . ~ dcscnhcd a furthcr nomcncIatiirc for alternatively spliccdt variants of a given receptor suhiinlt In thc furm GluRX-Y where X is thc running numhcr of thc rccuptor suhunit and Y 1s thc running numhcr (if thc splicc variant. Finally, Scchurg and cr)llcag~icshavu iiscd thc alphabetical series GluR-A to GluR-TE for naming gcncs cncrding AMPA rcccptors. Exnrnplc.; ( i f cnch ot thcsc nomcnchturw arc prcscntctl qirlu-hy-.iicIc in thc listings vntlcr (;tun(> /(irnilv. 07-05,For nn lnturim r l i w z r s w i n (if thcsc alturnativc norncnclntzirvs, st‘c rvf.’ and the IUPHAR Nnmenclature Cnrnmittec recommendations via thc CSN (wi,F c d / w c k eJ (:AN ( i w i w , rJiirr 121.
entry OJ
Nomenclnt ure based on immunolqyical prnperrfes 07-03-06:A further nomenclature for AMPAJkainate receptors based on immunnhistncbemical assays of Co*+-permeability has huen defined to includc suhtype K1 (activated by kainate alone), suhtype K 2 (hy glutamatc and kainatc) and suhtype K,? (hy kainatc, glutamate and quisqualatc) (we sciectlvrty, 07-40).
Previous nomenclatures 07-03-07: AMPA receptors werc fnrmcrly designated as the quisqnalate or QUFS receptors. Sincc quisqualate has been shown to hc an a p n i s t t at mctahotmpict glutamate teccptors, this classification i s now loss frcqucntly used.
Distinctive nomenclature for G protein-linked glutnrnate receptors 07-03-08: In shorthand designation, metahotropict glutamate receptors‘ 1i.c. scven transmemhrane domain glutamate rcccptors which couple to C. proteins and do not contain integral ion channels) arc convcntionalty designated hy the ahhreviation ’mGlu’ (although they are often referrcd to by the older designation ’rnGluR’J. These desipations distinguish thcm from innotrnpict receptors designated hy the alhreviation ’iGluR’ (see o h Resourct. A - G protern-linkcd receptors. entry 5hJ.
Current designation 07-04-01: GcncrnIFy of thc form InK,,n,rt, c.g. lnMrn (lourc], IKhlN, ttc.
Gene family AMPAjkoinote receptnr genes form port o/ the ELC: channel Sene superfamily 07-05-01: Thc genes encodinR AMPAJkainate-selectiw~glutamate receptors forin part of thc cxtraccllular Iigand-gatctI gcnu supcrfnrnil y 1 (n’escrihed under ELG Key facts, entry 04). Thc cxprcsswn-cloningt of a suhunit of thc
h igh-affinity AM PAJlow-afhi ty kainatc rcccpmr-channel {reviewed rn ref.’) was an important stcp in determining that all currently known ionotropict glutamate receptors form part of the samc gcnc farnilyt (reviewed in ref?. ’). Subgrouping of AMPAJkainate receptor genes hnsed on sequence homology and ligand affinity 07-05-02The large numher of distinct genes encnding different suhunits of functional non-NMDAglutamatc receptors can he listcd in thrce subgroups according to thcir sequence relatedness (Tclhlcs 1 3 ) . Ordcring of szihunit Rcnc stmcturc In this rnanncr a l w enahles a functional subdivision nf thc encoded protein subunits OR thc basis nf agonint affinity. Thrce hroad functional groups havc emerged thus far (high-affinity AMPA/law-affinity kainate suhunits, high-aaffinity bainate/law-affinity AMPh suhunits and high-affinity kainatc suhunits (.WE Trihler Althnugh this classification is nnt ;~hsdutc, it providcs a tramcwork for undorstnnding of marc dctnilctl suhunit structurc-function relationships.
TI1e p TC se n t nrc c ssi i y Tor c I t ( I t ion of ( I 1t E t ri 11 t i vc no m r n c la t 11re s in pnmllel 07-05-03: A listing (if Kcnus encoding thc hroad class of AMPAlKAlN iC.luR suhunits 1 5 givt'n untlcr thcir rcspcctivu structural and functional subgroups in T l i h h Alturnativa nomcnclaturcs for thc sninc suhunits as usctl In thc litcraturz: (c.R.GluRI vs. GluR-A, si'c' Chnnnal rlcsrgnritwn, O7-OL1) h a w hccn pruscrvcd within this cntry (wcordInfy 10 thosr s t m t c d in ~ h rc q y n r i l r ' i t n ~ i o n This was ncccysary rn thc ahsuncu of ;1 universal nomenclature. Enr updatcs on ajyccd nnincnclaturcs, rcfcr to thc latcst IUPHAR Nomcnclaturc Cwnmittcc rccoinmendntions via thc CSN (scr FrrdIiock cd C S N I I C C ~ C Ccntry . 121
Subtype classifications Dependencc ufphmtmmdogical subtypes on sepornble gene products 07-06-01: Initially, iGluRs wcru classificd intci thrcc scparatc rcccptnr populations, each dcfined by its sclcctivc activation hy thc naturally occurring cxcftatory suhstances NMDA, AMPA and kainate which are stmctural analcigucs of glutamatc'5v I('. Propcrtics of cnch of thcsc hroad catcgnrics dcpcnti on thc products nt separate genes and their combination ((or li7tin.q of vcrtehatc AMPA/kriinntr a u l i u n i f s , .WP Tnh1e.c I-.?). Several w h u n i t cornhinations can hc distinRuishcrl fmm cine anathcr hy thcir distinct gating1 kinetics, cation permeabilities1 and conductancest, and their susccptrhility to a rmgc of organic and i n n r p n i c a n t a g ~ n i s t s '(src ~ other f w l d ~ )Thc . ~nolccularhiology of mornmalion glutsmAtc rcccptnr.~. channel suhtypcs h i s hccn r ~ v i c w c r i ~"-*".
llnitnry reccpt orx with 'in t e r c h r ~ n ~Isle e a ' sirhnnit.s 07-06-02: Twn vcrtchratc cxcitatory aminn acid ianotrnpic unitaryt wceptnrs [ I . c .IiavinR morc than 4)nc class ol uxcitatnry amino acid agmist specificity within rmc protctn oligomcr) Eiavc hccn purified from thc Xrnryu.? central ~ C T V I H I S systcniz'vzz Thcsu rcccptors consist of ( i ) unitaryf kainateJAMPA and [ i i ) kninatt./AMPA/NMI)A rcccptcirs with interchangeable subunits. Each iwlntcrl rilignmmcr ccintains 42 k D a suhrinits cif thc 'non-NMDA' Itgand-hintling typc, hut thc sccond typu has an additinnn! NMDA-rcceptorspccific 100 kna suhunit2'. Channcls rccrmstitutctl into hilaycrs can c h i t currcnts (similar to nativc non-NMDA CtluRsl in rcsponsc to low lcvcls of AMPA o r kainatc.
Trivial names O7-O7-Ol: Scrp Chrmricl dcrr~nnrion.07-03. Thc nirncs p v c n to nativc a n d rccomhinant GluRs ultimatc!y dcpcnds on thcir subunit mrnpasitian. As dcscrihtd in Tnhlcs I J , thusc ~ r o u p sconsist of [ i l 'AMPA-prekrring' iGIuR (high-affinity AMPA, Inw-affinity kainatc rcccptor-channels) and [ i i ) 'kainatc-prc~crrin~'iGluR (high-affinity kninatc, low-affinity AMPA rcccptor-ch~nticI~).
Table 1. Genes encoding high-affinity AMPA, low-affinity kainate receptor subunits ('AMPA-preferring glutamate receptors') (From 07-05-02) Subunit (equivalent nomenclature in brackets)'
Descriptionb
Other distinguishing features
IL
- -
Glutamate and AMPA are potent GluR 1 AMPA receptor High-affinity agonists producing desensitizingt (= GluR-A) iGluR for AMPA (Kd nM), low(a1 homologue) affinity iGluR for kainate (Kd p ~ ) responses from heterologouslyt expressed channels formed from the Alternative splice+ variants of GluR GluR-A to -D classes exist (see Gene organization, 07-20) Kainate is a low-affinityt agonist that activates non-desensitizing currents similar to those elicited in CNS neurones"
-
Encoding
889 aa Sig: aa 1-18
70% sequence identity to GluR2 N
Predicted mol. wtC (nonglycosylated)
--
99.8 kDa
(mRNAs 5.2 kb; 3.9 kb; 3.2 kb)
AMPA-preferring iGluR are strongly potentiated by concanavalin A but not cyclothiazide" (see Inactivation, 07-37) Glutamate and AMPA are potent desenGluR2 High-affinity AMPA, low-affinity sitizing agonists for GluR-A to -D classes (= GluR-B) kainate iGluR ( a 2 homologue) Alternative splice variants of GluR2 GluR-B confers native channel exist (see Gene organization, 07-20) properties 1e.g. non-permeability to Ca") in heteromerict recombinant receptors, (see Protein interactions, 07-31, and Current-voltage relation, 07-35)
862 aa Sig: aa 1-21
-
70% sequence identity to GluR1; 74% with GluR3
-
96.4 kDa
GluR3 High-affinity AMPA, low-affinity (= GluR-C) kainate iGluR ( a 3 homologue) Alternative splice variants of GluR3 exist (see Gene organization, 07-20)
98.0 kDa Glutamate and AMPA are potent 866 aa desensitizingt agonists for GluR-A to -D Sig: aa 1-22 classes 69% sequence identity to GluR2; 74% with GluR2 N
N
GluR4 High-affinity AMPA, low-affinity (= GluR-D) kainate iGluR (a4homologue) Three alternative splice variants of GluR4 exist: GluR4/flip, GluR4/flop and GluR4c/flop (see Gene organization, 07-20)
881 aa Glutamate and AMPA are potent desensitizingt agonists for GluR-A to -D Sig: aa 1-21 classes
98.4 kDa
Transcripts synthesized in vitro from GluR4c flop“ form kainate/AMPAactivated channels showing strong inward rectification when expressed in Xenopus oocytes
Supplementary information is given under the appropriate fieldname. For retrieval of full sequences for specific analysis, database accession numbers and references describing the cloning and molecular features of individual subunit genes, see Database listings, 07-53. “Alternative nomenclatures are used interchangeably in this entry, according to the original description or citation. For a short discussion on the parallel nomenclature in use for these GluR-channel subunits, see Channel designation, 07-03, and refs3713. bFor primary sequence discussions, see references in Database listings, 07-53. CEncodingdata show the number of amino acid residues in the specified channel subunit, with signal peptide residues denoted by the prefix ‘Sig:’.
Table 2. Genes encoding high-affinity kainate, low-affinityAMPA receptor subunits ('kainate-preferringglutamate receptors') (From 07-05-02) Subunit Description' Other distinguishing features Encodingb Predicted (equivalent mol. wt (nonnomenclature in glycosylatedt ) brackets) GluR5 (=01
homologue)
High-affinity kainate receptor subunit Alternative splice variants of GluR5 exist: the longer GluR5-1 has an ORF of 920 aa and derives from insertion of a 45 nucleotide sequence GluR5-2 variants (905 aa) lack the 45 base insert
41 % homology to GluR-A to -D; 80% sequence identity to GluR6. Homomultimers are weakly responsive to Lglutamate4 Kainate is a high-affinity agonist for recombinant GluRS-GluR7 channels, producing strongly desensitizingt responses. AMPA is inactive or acts with low potencyt Kainate-preferringiGluR are strongly potentiated by cyclothiazide but not concanavalin A" (seeInactivation, 07-37 ) N
GluR5-1: 920 aa Sig: aa 1-30 GluR5-2: 905 aa
100.9 kDa
GluR6 (=8 2 homologue)
High-affinity kainate receptor subunit
Shows high affinity for kainate, and does 864 aa not bind AMPA. The competitive Sig: aa 1-31 antagonist CNQX is substantially less potentt in blocking kainate responses in GluR6 compared to GluR-A. Kainate causes rapid desensitizationt
GluR7 (=8 3 homologue)
High-affinity kainate receptor subunit
Agonist-elicited current responses are 956 aa n o t observed in homomeric configurations of GluR7 subunits. Combined with GluR5 and GluR6, KA-1 and KA-2 (below),may form heteromultimerict high-affinity kainate receptors (see Predicted protein topography, 07-30)
96.2 kDa
-
Supplementary information is given under the appropriate fieldname. For retrieval of full sequences for specific analysis, database accession numbers and references describing the cloning and molecular features of individual subunit genes, see Database listings, 07-53. *For primary sequence discussions, see references in Database listings, 07-53. bEncoding data show the number of amino acid residues in the specified channel subunit, with signal peptide residues denoted by the prefix ‘Sig:’.
Table 3. Genes encoding high-affinity kainate subunits (Note: homornultirnerst do not appear to form functional channels) (From 07-05-02) Subunit Description' Other distinguishing features Encodingb Predicted (equivalent mol. wt (nonnomenclature glycosylatedt ) in brackets] KA-1 (=-y 1)
Recombinant high-affinity kainate receptor subunit
30% homnology to GluR-A to -Dj 936 aa high-affinity for kainate/low-affinity for Sig: aa 1-20 AMPA (Kd kainate 5 nM). Homomulutimerst do not form functional channels. Combination with GluR5 or GluR6 yields properties distinct from GluR5 or GluR6 homomeric channels N
- 105.2 kDa
N
KA-2 = 7 2 )
Recombinant high-affinity kainate receptor subunit
Highly sequence-related to KA-1, with similar pharmacological profile but a more widespread distribution in the CNS. Homomultimers do not form functional channel^'^. Combination with GluR.5 or GluR6 yields properties distinct from GluR5 or GluR6 hornomerict channels
979 aa
-
109 kDa
61
Recombinant high-affinity kainate receptor subunit
Subunit of unknown function
62
Recombinant high-affinity kainate receptor subunit
Subunit of unknown function
KBP-c Structurally related KBPs from brain KBP-f chick/ of chick (464 aa) and frog (487 aa) (no frog brain channel activity) kainate-binding proteins
KBPs may be truncated forms (see Protein molecular weight (purified),0722, Domain conservation, 07-28, and Database listings, 07-53)
1009 aa
chick, 464 aa Sig: aa 1-23 frog, 487 aa Sig: aa 1-17
-
51.8 kDa
52.5 kDa (mRNAs 3.9 kb; 6.0 kb)
N
Supplementary information is given under the appropriate fieldname. For retrieval of full sequences for specific analysis, database accession numbers and references describing the cloning and molecular features of individual subunit genes, see Database listings, 07-53. “For primary sequence discussions, see references in Database listings, 07-53. bEncoding data show the number of amino acid residues in the specified channel subunit, with signal peptide residues denoted by the prefix ‘Sig:’.
Cell-type expression index Ubiquity of iGluR cxpression in the CNS 07-08-01: Receptors for ionotropic glutamatc rcceptor-channels ( i C h R s ) arc cxprcsscd on virtually every neuronal cell and some glial cells in the CNS. Preparations which display unusual homogeneity or notahle absence ot rcsponses to identified classes of iGluR are hriefly described helow. Note: Electrophysiological studies in native cells may not always pcrmit clear dclincation of receptor subunit types, since co-expression of subtypes within single cclls is common (.see Sul~cellular locations. 07- 16, cind Proteiri internctiori.~,07-.31).
Evidence q a i n s t cross-ussemhly of AMPA- and kuinate-prefcrritig receptor subunits 07-08-02:Evidence from experiments cmploying cydothiazide blockade of rapid desensitization (having ahsolutc selectivity for CluRI-C.luR4 (AMPAJ siihunlt reccptors) sugRcsts independent assemhly of AMPA-preferring and kainate-preferring receptor-channels expressed in the same ccll, with littlc evidence for cross-assembly” rior dettri1s. set’ Profpin internctions, 07-31. und lnactivnrion. 07-37,.
Advuntasqes of subunit ,Sene-specificprobes for complex distribution
studies 07-08-03: The availability of subunit-specific probes based on single-cell RTPCRT, in . T i m hyhridizationt and immunocytochemistr/ permit dctailed mapping of subunit cxprcssion. Thc results from these appro;iches can confirm expected subunit distributions, but also provide dircct cvidcncc for cihsencc ot expression and can rcvcal patterns of ovcrlapping distribution hetwecn any number of subunits. Tablc 4 suminarizcs pattcrns of cxprcssion, co-cxprcssion and abscncc of cxpression for AMPA/kainate iGluR subunits in R range of ncuronat cell preparations. Furthrr details on these expression patterns and thc contrihutions of suhiinit-specitic properties to specific cell-type functions arc dcscribcd in othcr ficlds.
Differential expression of GluR-R in distinct hippocornpal ncuronul populations 07-08-04: ’Morphologically heterogeneous’ hippocampal type I neurones cxprc5s AMPA receptors wit11 linear o r outwardly rcctityingT I-v curvest 0.L).39.A population of and have law Ca”-permeability [Pca/Pcq hippocampal neurones in cultiirc which cxpress AMPA reccptors with inwardly recrifyingT I-V curves and high Ca”-perrneahility (P,-a/Pcs -. 2.3J”9have been referred to as type 11 ncurones. Morphologically, typc I1 ncuroncs arc of rcliltivcly snial! sizc and cllipsoid shapc. Typc 11 nciironcs do not express GluR2 [CluR-R] subunits, which inay explain their functional propertiesJo (see Selcctivitv. 07-40,. Since the GluR-R gene product IS a n important determinant of native properties of heteromcrict iCluR (e.g. impermcahility to Ca’+) it is to bc cxpcctcd that its Rcnc is subiect to ’tight‘ regulatory control‘”.
-
Table 4. Sumrnurr. o f AMPA Jkrrinnt c c11brrnlt cxprrcclon in ~ c l c c t c dncuronal preprrratron r (For ftlrth~r ~nformatinn.w e trlw mN,\TA distr~hutlc~n, 07-13, and Protcln d l
Berpann gEia
CAI pvramidal CA3 pvmmidal Dvntatv cells cellgranule cells
-
-
Inw ver
Yes low
Inw \'PC
Ccrcbcllnr ~ r a n u l ccells
-
Purkrnlc cells
Spin21 cord motirr neumnes
A
VPF
-
vec
ves low
-
+
yes vcs
vcs ves vcs
+
L
-
VC'S
vcs
yes
vcs
-
A
-
ves +
vcs vcs
vcs
ves
f in adult rat neiimnnl cell tvpcs fnr thc p n n c ~ p a lAMPA/kainate rGluR suhunit jicncs, hascd on available T h ~ table s Ilcts pattcrns r ~ r.xpreselon ~uhunit.cpecif~c prohes (ccc almvo - ~ndicatesu n a m h ~ m n u sor ahundant cxpresrlcm of thc named subunit gene, whereas = indicates POWabundance rxyresrjon The exprcw1on clt Flip/flop alternative spliceAvariants nt GluR-A to -D are 115ted ~ e p a r a t e l vwhere these have hecn identihed. ntherwtfe, immunacvtnchcm~cal,DNA clr RNA prohcs were used w h ~ c hci~dnnt d i c c n m ~ n s t ehetween splice variantr. s haced on the review hv P ~ e c h t t r whn ~ ' ~ wtth W Wisden collated the l o c n l ~ z a t ~ ndata n frnm the T h e arranEcmmt of adult rat ncuronal cell t h ~ e is tndicateti references
I
inhibitory and excitatory neurones express djfferent types of nonNMDA receptor 07-08-05: Direct comparison of iGluR channel activities in excitatory cortical newones (spiny, pyramidal neurones] versus inhibitory cnrtical neumneg [aspiny intcmeurones) indicate that different types of non-NMDA receptor-channels contribute to spontaneous excitatory post-synaptic currents (sEPSCs] in these preparations". Variable pararncters include ( i ] the sEPSC decay time constant - az 2.5 ms, this is faster in inhihitory aspiny intcrneurones than the 4.6 ms for excitatory pyramidal neurones; (ii]the rate OF desensitizationt in patches, which is faster in inhibitory interncurnnes (3.4 ms) compared with the 12.0 ms of excitatory pyramidal ncurones; and (iiiJ single-channel conductance, which is larger in the inhibitory aspiny interneurones (27 compared with the excitatory pyramidal ncurancs ( 9 p~)''.
Nun-NMDA iCIuR subtype cn-expression- QR example 07-084lr:Native cerebellar granule cells commonly express more than one type of non-NMDA receptorxhannel subunitq2 (see examples in Table 4). High-conductance responscs (activated by glutamate, AMPA and kainatej mediate most of the synaptic current in granule cclls and originate from a GluR channel complex displaying conductance levcls of 10, 20 and 30 pS. There i s also a distinguishable, low-conductance kainate response in these cells of approximately 1.5 pP2.
Possible equivalence of cloned and native GluR channel currents 07-08-07: ICAIN: .Notably, the pharmacology and channel Rehaviour of GluKS receptors has heen reported to be 'virtually adentical' to those of the
native higbmffinity kainate receptor on dorsal root gangliad3."'. Mammalian spinal cord C-fibre affercnts have been described as possessing 'pure' kainatc-sclective GluR receptor populations which are insensitive ta
AM PA"^. Functional homomeric kainate-selective iGluRs in cuJtured hjppocornpol newones 07-08-08: W I N : Combined patterns of distribution for the five kainateselective subunit mRNAs {GluR5-GluRJ plus KA-1 and KA-2) are similar to patterns of [ 'HI-kainate binding determined hy autoradiography4' (see Protein distribution, Q7-15). Although currents typical of 'high-affinity' kainate subunits havc been detcctcd in scnsory ganglia", rccrirdings in central neurones were not reported until: 1993''. In these studies, high proportions of rat hippocampal ncuroncs expressed functional kainateselective iGluR early after in vitro culturin . The kainate receptors displayed features of pronounced desensitization with fast onset and very slow recovery, and were activated hy qukqualate and dornoate, hut not by AMPA". The recordings obtained in this preparation were consistent with thc cxpression of native homomerict GluR6 receptors.
7
Cloning resource Vertehore hwin cDNA Iihrnries 07-10-01: Recavsc of the ubiqitous exptessian of thc iGluRs in the CNS, rDNA lihrarics constmcted from tntal hrain poly(A)’ mRNA havc hcen
used as the m y n resource for cxpresqion cloning1 strategies. Conventional cross-homology;yf,low-stringcncyT screening nf hrain cDNA libraries has rcsultcd in isolation of most of the known GluR suhtypes. For possible cloning resources n u f s d c the CNS, w e rnRN.4 distrihtrtion. 07-13.
Developmental regulation iGlu R Renes typically d ~ s p l n ydistinct temporol patterns of expression throughout development 07-11-01: AMPA: In addition to thc wcll-dcfmed spatial patterns of expression charactcrikic Gf iGluR gencs (see mRNA distriliutinn, 07- 75‘) developing
embryonic and post-natal brain tissues display dynamic elevations and reductions charactcri stic nf indcpcndcnt develnpmental gene regulation. For example, studies of GluRl -G!uR3 dcvclopmcntal genc expresqion show GlvRl to he prornincnt in cortcx and caudate-putamen at early ages, but thcsc fall to background lcvcls in the adult“. GluR2 mRNA levels in P4P21 rat hrain docs not cxcccd adult levels at any stage, cxccpt in ccrehcllum at P I 4 and in thalamus [where it diminishes to background levels after P41.The GluR3 gcnc shows notahly high lcvcEs of expression in striaturn (at P4F and in ccrchcSIum (at P7I4’.
Fimcrional swrtching of GluR gene exons by alternntive spliciqy during hmin development 07-11-02: ~AMPA: AMPA rcceptois expressed in the early hrain differ in molecular typc and functional properties tn thme in the adult brain: ’Develnprnentally early’ (pre-natal1 GluR-A-GluR-D receptor4 carry mainly ’flip’ rnodulcs, and thcsc forms persist throughnut dcvclopmcnt into thc adult ( f o r description oi ‘ f l i j P I h p modolcc’, see Gene organizoticm, 07-20).
Rcccpmrs containing flip modules appear to have slowcr dcscnsitmtwnt kinetics than adult rcccptnrs. The ‘flop’ modules increase only during early post-natal dcvcl(~pmcnt,whilc the flip module 1s expressed In an invariant pattcm, Icading to frcqricnt co-expression of flip and flnp in adult cclls. Glutamatc activaticm of flip versions produccs more current than thosc composcd of flop’A. For each rcccptnr variant, the altcrnativcly splicudt mcsscngcr RNAs show distinct cxprcssicln patterns in rat hrain, particularly in thc C A I and CAA fields of the hippocampus. These results idcntihy a ‘gcnetic qwitch’ in the molecular and functional propcrtics (if gIutamatc rcctptrirs opcmtmg through alturnativc splicingi .
‘Editing’ of GluK m R N A during development in determining functional propertius nf hctcromcric AMPA receptors, exists in ‘edited and ‘unedited’ forms which arc developmentally distinct (for details ol RNA editing processeq, TPP Gmo Organismiion. 07-20, and Domoin functions. 07-11-03: AMPA: - - Thc GluR-R subunit, which is dominant
07-2Y).
A conserved sequential pottern of ion channel gene expression in spinal cord development 07-11-04: In dcvcloping pcipulations of cclls from several regions of embryonic [ > day 1,3j rat spinal ctird, functional sodium channels appcar prior to GARAA receptors, which in turn cmcrgc prior to kainate-activated iGluR. This stereotypical pattern of sequential channel cxprcssmn during ricvclr~pmcnt occurs individually on most cells in all spinal cord regionsqy. Vcntmdorsalt and rostrncaudalT gradicnts of expression reflect known patterns of spinal cord ncur~gcncsis"'.
Multiple CQ-Ordinatcd iGIuR gene expression control in developing CNS and PNS 07-11-05: As with othcr iGIuR rcccptor gcncs, 61uR4 and GluRS show striking patterns of gene inductiont and gene silencing1 in the cowrsc of emhrynnic and post-natal CNS and PNS duvclopmcnt. Thew patterns h a w hcen tahulatcd for a number of hrain regions dorivcd from a systcmatic in srlu study of GluR4 and GluRfi gene cxprcssion (see ref.').
Intrinsic or highly Iocolized signals guide G h R 4 gene expression durinR devehpm en t 07-11-06: AMPA: Transcripts for thrcc vnriants of thc GlwR4 jicnt. (GluR4 flip,
GluR4 hlnp, GluR4c flop (we Gene orgnnization. 07-20] arc much morc abundant in thc cerebellum than in other hrain area?, and thcir lcvcls arc incrcascd during cerebellar dcvelopment. Maximal increases arc nbscrvcd hctwecn post-natal days 1 and 20, ages corresponding to the division and maturation of granule neurnncs. Granule cells alrcady express GluR4c flop in the prc-migratory ZORC of thc cxtcrnal granular layer, indicating that intrinsic or highly localized cues induce GluRrlc exprcssim before thesc cclls rcach their final psition".
GluR4 and GluR5 are expressed in meas of nenronnl differentiation nnd synapse formation 07-11-07: Thc GlwR4 and GluRS genes are expressed in subsets of neiirnncs throughout thc dewloping and adult central and peripheral nemaus systems (SCC thc systematic in situ hyhridization study in ref.'). Gcncrdly, thc distribution and cxprcssion level of GluRS mRNA is diffcrcnt from and quantitatively much lower than that of the GluRl-GluR4 (AMPA rcccptor]mRNAsd. During embrpogenesis, GluRS transcripts are dctected in arcas of neurnnal differentiation and synapse formation (synaptogcnesisj. 'Phylogenetically old' brain stnicturcs art. thc first to accumulate GluR4 and 61uR5 transcripts'.
Stage- and cell-type-selective agcinrst sensitivities within glia 07-11-08: Comparative studies of ionntrnpict glutamate receptors in cultured cerebellar gIial cell types (type 1 and type 2 astrncytes, oligndendrocytcs, 02A progenitors) show agonist responses that arc stagc- and ccll-typcspecific". Multiplc subunits can bc expressed in glial cells - for examplc, the 0 - 2 A lincagc display rapidly descnsitrzingt rcsponses to kainate and cxprcssion nf mRNAs for GluR6, GluRJ, KA-1 and KA-2. They also display rapidly dcsensitizingi rcsponscs to AMPA, and mRNAs for GluR-R, -C and
-D. Thus two rocuptor populations arc prcscnt in thcsc cclls, with hiRh- and low-affinity for kninatu and diffcrcnt scnsitivity for potcnttation hy concanavalin A and for hlock of dcstnsitizatmn by cyclothiazide5’.
Developmental regulation in (arypicol) cnlciurn permenhility
k oina t e rccep! ors
of
07-1 1-07 KAIN: Thc calcium pcrrncahlityt of karnic acid-gated rcccptorchanncls is dvpcndcnt on suhunit composition”, and thc gcncs cncoding
thew may he dcvclnpmcntally regulatcd5.3.Notr High Ca”-pcrmcahility is iisuallv associated wsth thc NMDA-scIcctivc rnnntropic glutamate rcccptor-channcls ( s w E L G CAT G L U N M D A , m t r y 08)
Drffcrentjn? cxprrssim of kninrrte receptor s u b m i t s during hrnin de velopmen r R7-11-10: KAINr From cmhryonic day 14 (El41 (inward, suhunit KA-1cxprcwing cclls can hc ol~scrvcrl in populations of putative ntruranal prtrcarsor cells linmg the ciorsalt aspect nf thc hrain“. Subunit Kh-2 inRNA is uhiquitnusly cxprcsscd at a hiEh luvcl throughout t h u cmhryonic CNS. From E l 4 through tn pnqt-natal thy 1, KA-2 transcripts arc abundant in spinal cord, brain and some areas n f the PNS. A t E17, KA-2 mRNA is prwcnt in all laycrs nf the spinal cord, mesencephalon and telencephalic structure^. Thc cmhryonic olf;ictnry ncuronos, nnwl cpithclium, dorsnl root ganglion and pituitary cxprcss KA-2 at a high Icvcl. ~
Ncurn? cell compel it ion, survivnl Lind d m t h during devclopmenr 07-1 1-11: Activity-dcpcndent, competitive synaptic interactinns [whrch stabilize w m u axon hrmchcs and dcndritus and rcmtivc others.) invdvc glutamntc ruccptor cxprcssion (.wc EL<: Key foct,r. enfry 04. rind rrvwwy5a5 (, ) In terms of brain rlcvcl{ipmcnt, thusc phcntimuna arc funtlsmuntal t u thc ordcrcd pmgreqsion of neiironal phenotypes and dumonstratc impcirtant intcmctions hctwccn rncmhranc signill transtluction and Tpccific pattcrns of ~ c n c cxprcwion. Thc physio1r)gical and pathnphvsiolr~gical roles of cxcitatnry aminn acids during dcvclopmcnt h a w hccn rcvicwcd5’ ( S P C ri/so I’heriotypic cxprercion under E L L ‘ CAT G L I I NMDA. #R- 14).
AMPAjkninrite iCluR rtcrivntrm in regirloricm of growth f m t o r gene t ra ns crip t io n 07-11-12: Non-NMDA rccuptnr xtivatirm zn hippocampal pyramidal ncuroncq has hccn shown to rcgvlatc ilrRNA lcvcls of twn gtawth Bactnts csscnt 131 to nuurona I survrval: brain-derived netirotmphic factor” 1 I D N F\
and nerve growth factorrR.5‘’[NGFJ.
Isolation probe 07-12-01: Thc first examples of cloncd glutamatc rcccptors wcrc ~solntcd using Pxptession-cloning f protocols (for rcfcrcnccs, scc ~n toirnsc liytinyi. 07-5,3) Ccnerally, othcr suhunit gcncq werc iso~atcdw4ng c D N A s a s pmhcs in Inw-stringoncyf hyhridizatinni protocols [e.R. GlztR1 cDNA prohc for ~;Iuti2nnri c ; ~ u R , ~ ” F .
entry 07
mRNA distribution Met hodelogicol notes for distribution studies 07-13-01: Immunocytochemical determination of subunit distribution is
generally more sensitive than methods hased on hybridization+ of radiolahellcd probes to low-ahundance RNA transcripts. Allthough problems of antibody cross-reactivity might exist, immunological methods do not depend on pratein turnover (unlikc RNA-based methods, which effectivcly measure the ‘synthesis minus degradation’ component of spccific molecules in the cytoplasm. The use of antibodies may report the position of proteins in transit,and it is common for large pools of receptor or channel protein to he present in the cytoplasmic vesicles where it has no (apparent) function. In sitn mRNA hyhridization is welt-suited for reporting regional and temporal variations in expressinn level hut is not ideal for detection of low-abundance mRNA transcripts {which may he the case for many ion channel types]. Note: Single-cell PCRt methods have the greatest sensitivity and highest resolution for RNA distribution studies, and can be expected to makc increasing impact for these types of study. Differences in sensitivity and resoIution in thew commonly uscd procedures tor localization nf inn channel gene expression may account for some lack of consensus between independent studies. For details on computer-based ‘expression atlases’ designed to consolidate gene expression patterns in brain and other tissues, see Resource I - Search criteria d CSN development, entry 65.
Generalized pntterns of subunit expression ot the mRNA level 07-13-02:The MPA/kainate-selectivc receptors GluR I -GluR6 cxhihit two patterns of mRNA expression: most ncurones express GluR1, R2 and RB, whereas only -20% cxpress significant levels of GluR3, R4 and RS (see also Table 45. Restrjcted distribution of ‘flip’ variants 07-13-03: AMPA: In general, AMPA-selective iGluR subunit genes display
differential spatial expressiw’.’. Tissuespecific expression for ‘flip’ and ‘flop’ variants in the AMPA family is of central importance in the function of these receptors“ [for a description of ‘flip/fhp’ variation, see Gene OrgQRization. 07-2Q).In the hippocampus, CA3 neumnes preferentially express the ’flip#version of GIuR-A, -R and -C, while CA1 neurones preferentially express the ‘flop’ versions of these receptors (see summary in Table 4 undcr Cell-type expression index, 07-08).
Differential octivotion and putative silencing of AMPA subunit gene expression 07-13-04: A M P A Independent studiesa have shown that cornhinations of
GluR-A to -D mRNA exprcssion patterns vary considerably according t~ location. For instance, GluR-I3 mRNA is expressed strongly within most brain areas except the Bergmann glia“. GluR-D mRNA is also widely distributed, although the expression lcvel is relativcly low. Several brain areas can he observed which lack {or express very little) GluR-A mRNA 1e.g. some nuclei in the motor and auditory systems). Similarly, snme
cntry 07
nuclei in the hypathalamus and gencral somatosensory system lack or cxpress very little GluR-C mRNA".
rn RNA encodinK functional GluR-chnnnels in
~S~TOCJJ~CS
07-13-05: Mcsstngcr RNAs encoding functinnal acctylchalinc and glutamatc receptors with similar propcrtics to those in ncuronts have been detected in human astrocytorna cclls". In contrast, human glioblastama cells lack thcse mRNAs"'. Now: "on-NMDA'-typc Klutnmatu ruccptors play an important part in dutcraining the rcsting potential of visual streak astrocytcs in situ and may hc of gcncral importancc for thc functions of astrocytcs in vivom.
Contrflstinx distribution pnttetns of KA- 7 nnd K A - 2 suhtlntt gene expression 07-13-06:KAIN: High-affinity kainatc rcccptor KA- 1 mRNA cxprcssion is high in thc CA.? rcginn and dcntazc m r u s of the hippncampus hut is 'virtually silcnt' in CAI cellsh5. Thc cxpression level of GluRS mRNA is gencmlly much lawcr than that o h the AMPA or GluR6 reccptor mRNAs, hut arcns of high cxprcssion corrclntc with amas of hiRh-affinity kainatc ~
binding in thc dorsal mot gangliaq4 ( w e helnw). Subunit KA-2 transcripts arc ahundant in the ccrchral cortex (psrticularly layers rF/rrr and Y/VI), pyrifnrm cortex, caudatc-putamcn, hippncampal ccimplcx (all suhficlds), medial Rahcnuta and granule ccll tayer nf the cerchcllurn, Thus, thc hiRhly sekctivc KA-1 suhunit mRNA distrihutirm contrasts with the wide distrihution of KA-2 suhunit mRNA'4nh5.
Combined kainate rcceptor m K N A distributions rnntch high-nffinity ~7~I-kninnte-hindinl: sites 07-13-07: Thc ohscrvcrl patterns of hiEh-afhity I'H]-kainatc sitcs in rat hrain overlap with thc crirnhrncd pattcrns of thu GluRS, GluR6, GluR7, KA-I and R A 2 ~ R N A s ' " Thcsc . sitcs rncludc thc laycr I snd thc inncr laminac of thc neclcortex and cingulate cortcx, caudatu-putamcn, thc CA,? region nf thc hippocampuq, thc reticular thatamus, the hypnthalam~cmcdian cmincnce
and the ccrchcllar Rranulc cell lay~r'~*''.
Co-distrihutirm o i GluK6 mRNA and koinnte ljgond-hindina sites 07-13-08: KAIN: Thc C,luRh suhunit mRNA is not expressed in the same c w r m l l pattern as that for KA-I in vivo, hut it is enriched in patterns simtlar to high-affinity binding sitcs of kainatc located by autorarlinFaphic tcchniques (the CA3 hippocampal rcgion, the caudatc-putamcn and ccrchcllar Rmnulc cells).
CJuR frrmily m R N A distribution patterns - the retina as on example
07-E3-09: In sitri hyhridizntion studies of retinal transverse sections show that mRNAs for seven rcccptnr subunits (G[uRI-GluR7]arc cxprcsscd in both cat and rat retinal tissueM1.Glutnmntc rcccptor subunits are employed at many of the retinal synapses, including the photoreceptor input tn the outer pkxifozm layer and the contacts of hipalir cells with the prnccsscs at the inner nuclear layer (INL). Prohcs fnr GIiiK1 and GluR2 m R N A produce labcllmg over thc vntirc INL 2nd ganglion cull layvr (GCLJ. GluKt3-GluR7 mRNA has a lrmited distribution, indtcativc of cxprcssion by only a suhsct of ncuroncs.
All of the subunits are cxprcsscd hy the cells at the inner cdEc of thc INL [wherc amacrine cells arc located) thnugh the laycrs containing thc horizontal, hipnlar and RanElion cells contain diflcrcnt subscts of suhunits. Ry hyhridizing adiacent semi-thin ( 1 jtm) scctions of thc cat retina with prohcs for G l u R l - G l ~ R 3 ,cn-cxprcssinn was shnwn of all thrcc subunits (nr of pairs of thew subunits) in cclls within thc INL and GCL.
Differential expression of iGluR rnXNAs in adrenal g l m d
07-13-10: AMPA: In in situ hyhndisation studics of thc rat adrenal gland", mRNA cncoding all GFuRl-GluR4 subunits arc h i n d tr) hc cxprcsscd in thc mcdullary ganglion cclls. Pattcms of hybridization surncst that difFcrcnt CCII populations of thc adrenal gland may cxprcss hrmomcrict forms of diffcrcnt rcccptor suhtypcs. The four rnRNAs nrc prcfcrcntially cxprcsscd 3s frdlnws: GhRP (zona glomcrulosa rrf the cortex\, CluR.? (remaining parts of the cortcx), GluR2 [adrenal medullary cells) and GluR4 (zona glomcrulosa at lnw abundance). Of RNA populations analyscd, the 'flip' splice variants of GluR2 and GtuR,? arc highly rcprescntcd while GluR2 mRNA is present in thc argininc-cncoding form" ({or signrhcancr, S E P Grnc nrgrrnizrrtion, 07-20), Nnte: Expression of innntrnpict glutamatc rcccptor gcncs outsidc of thc ncnrnus systcm has also hccn rcported in p19 crnhryonal carcinoma cells".
GluR4 and GluRS expression in the peripheral nervous system
07-13-11: Fn thc peripheral metVms system, Rcnc transcripts for GluR4 and GluRS arc strongly uxprcsstd in thu mural ganglia (if thc intestinal organs, and variahly in thc dorsal root ganglia (high for GluRS) and cranial ganglia (c.g.thc trigcminal ganglion and acoustic g a n ~ l i a ) ~ .
Restricted expression of AMI'A subunits in optic nerve white mot tcr 07-13-12: RT-PCRt studies have determined that only GlctRl and GluR3 subunits are expressed in the whitc matter of rat optic GloRl "flip' shows a rclativcly highcr [hut dilfusc) cxprcssion pnttcrn BS detected by Northcrnt and in s i t u hybridizationt annlyscs'".
Phenotypic expression Phenotypic roles of non-NMDA GluR-channelx 07-14-01: In the CNS, the principal mediators of fist excitatory nenrotransmission arc GluRs responsivc to AMPA. IntrinPiu channcl properties
arc primary dctcrminants nf the kinetics of thc fast cxcitatnry post-synaptic ~ dthc rntc of ncurotrnnsrnittcr clcarancc (see (wlrls currcntsj ", as o p p ~ to under tht. STR IlCTURE d FCINCTlONS nnd E1,ECTROPHYSIOLOGY secrions).
Cormlatinn of koinnle-selective GluR expression wirh regionnl nenrotaxicit y 07-14-02: KAIN: Kainic acid is a pntentt neurotoxin fnr certain ncurnncs. Thc high sclcctsvc-exyrcssi~,n nf MA-I mcssengcr R N A in the CA,7 region of thc
hippacampus (we m R N A d i ~ f r r h n t ? c m87, I,?) closely corresponds tn highaffinfty kainatc binding sites defincd hy autoradiogmphy. This correlation, as wcll 3s thc spccific patterns of neurodegeneratinn chscrvcd in vivo, sumcsts that KA-I suhunits participatc In receptors mediating nnc mcchanism for kainatc sensitivity"'. Nntp: A clinrcal syndmmc characterized hy seizurcs and hrain damage has hccn linkcrl to ingestinn of a kainatc reccptor agnnist (dnmnatcl found in cantammatcd mussel^".
‘Dc)wn-rquhtion’of GluR2 (GluR-RJ~ e n expression e prior io neumnnl detyenerntinn 07-14-03: Scvcrc, transient global ischemia of the hrain induces delayed
damagc to spccrfic ncuronal pcipulations. Suqtal’nrd Ca2’-influx through glutamatc rcccptor-channel5 is thought tn play a critical rolc in postischaemic cell death. Following scwrc, transicnt foruhrahn ischacmia GluK2 suhunrt gcnu uxprcswm I S prcfcruntially rcduccd rn CAI hippocampal ncwoncs ;It a time p i n t that prrrcdrs thcir Timing of thiq change in cxprcssion of kainateJAMPA rccvptor subunits coincides with rcported incrcascs (if Ca2’-influx ~ n t oCAI cclls, and may thcrclnrc rndicatc a ’causal’ role in post-iwhacmic ccll t E c ~ t h ~(For ~. significancc of GluR2 [GluR-HI suhunit loss on Ca?+ ctinductancc, sec SeEecIivity. 07-40.) N O ~ C Many : unzymcs arc Ca’*-activated and may thcrufort. contrihutc. t o cxcitAtory amino acid toxicity ( w e N~cnnrypp~c cxpres\icin under EL<; CAT U L I J N M R A , 08-14). An in v i m model nf rndziccd post-ischrremic dnrnage 07-14-04: AMPAJkainatc receptor activation c o n t n h u ~ e s to ischacmic damncc ind~icutlhy 5 rnm ot oxyRcn a n d glucnsc deprivation in the C A I rcgicin of thc rat li~ppocainpal slice, A rrcacrncnt that causes long-term synaptic transmissinn failure [ LTFI”. Howcvcr, 11 has hccn determined that whatever the effccts on thc AMPAJkainatc iGluR during this pmccss, thcy do rro[ involvc cnhanccrnunt of Ca”-cntry through thc chsnnc17.’. Nntahly, total ccll Ca”-prror 10 I.rchocmin was dctcrinincd tci hc adcquatc to cause' crimplctc LTF. Ca?’-influx during the ‘first 2; inin’ of Ischaunia dcpcntls untlrcly o n NMDA channcls (sw E I L CAT’ G L I I N M l l A , c n t r y O H ) , hiit NMDA channcl hhickurs h a w no cffcct during thc ‘scconrl 2 : niin‘ (an cffcct prtihahly linkcrl tn NMDAR dephosphorylatron]. Thc ~sch.?oinia-lnducctlCa” influx during thc sccond 2: rnin nf w h a c m i a crmuld hc attcnunzcd 25% h y nifcdipinc (SO /IM, see YLC: Cn. rnrry 42) anti An ndditional 35% hy thc Na+/Ca?’ cxchangc inhihitnr henzamil (100 p ~ ) ~ ’ .
Chantyes i n G h R - R flip m R N A level? flccompnnying kninmi e-induced cpilcpsy nnd ischnemsn 07-14-05: Kainatc-induccd epilepsyt induces 3 rapid hut transient increase [50%)of GluR-R flip mRNA luvcls in ’all suhrcgions’ of the hippocampus [CA1, C A , ~ ,dcntatc gyrrrs~’~.Seizuret-resistant CA 1 dcntatc gyms neurnncs s h r w a wlmqucnt, pcrsistcnt GluR-R flip increase while thc ‘scizuw
cntry 07
susceptible’ CA3 area displays a 35% decrease in GluR-R flip message levels. In thc suhiculurn and C A I (areas hypersensitive to ischaemic insult) the levels of GluR-R flip and flop variants are substantially reduced (90-100%] following global ischaemia, and this reduction takes ,place long before morphological s i p s of cetl: deathT4.
Potentfotion of zinc neurotoxicity by A M P A receptor activation
07-14-06: In contrast to its effect on nttenunting NMDA rcccptor-mediated excitation and neumtoxicity, extracellular Zn” increnses AMPA receptormediated toxicity7’. High extracellular K’ concentrations or kainatc also potentiates Zn” toxicity, however toxicity arising from AMPA plus Zn” is attenuated by raising extracellular Caz+, or by use of Ca2+ channel
blockers. Exposure to AMPA plus Zn2’ induces an increase in fluorescence from neurones loaded with the Zn2’-sensitive dye TS-Q and increases These ftatures may be of sipificance in suhsequent 45Ca2+accurnulati~n’~. understanding mechanisms of neuronal death associated with ‘intense activation’ of glutamatergict pathways.
Long-term effects of glutamate toxicity in pothologjcol conditions 07-14-07: Prolongcd receptor-mediated depolarization
[and associated elevated Ca2+-influxJcan result in arreversihle disturbances in ionic homeostasis’” which may promote or inhibit gene expression. Activation of non-NMDA receptor subtypes has been shown sufficient to induce thc rapid and dramatic increase of immediate-earlyt gene products (see ~ S ELG Key facts, entry 04.and ELG CAT GLU N M D A , entry OX).
Q
LTP and synaptic depression phenotypes associated with IIQTI-NMDA receptors 67-14-08: While Ca2’-influx through NMDA reccptor-channels is usually associated with the initation of long-term potentiation1 (LTPJ in hippocampal area C A I , no NMDA receptors are required to trigger LTP in hippocampal areas CA21CA3. Similarly there is nn apparent rcquirement for NMDA for long-term depressiont (STLIE phenotypes in cerebellar
h r k i n j e cclls. En the parallel fibre-Purkinje neurone (PF-PN) synapse model, €,TO induction requires activation nf hoth AMPA and mctahotropic reccptors, together with PN depolarization. Post-synaptic Na ‘-influx through the AMPA-associatcd channel is nccessary for LTD. While a portion of the Na+-influx is provided by voltage-gated channels, the AMPAassociated ion channel provides the most important (For illustrations of the mechnnisrns underlying LTP phenotypes, see Phenotypic expression under ELG CAT GCU N M D A . 88-14).
‘Differentialenhancement’ of the non-NMDA EPSC component durinx induced LTP 07-14-09: Pharmacological modulation nf transmitter concentrations in CA 1 pyrarnidd cells of guinea-pig hippocampal slices [using the GARAn agonist haclofen or by the adenosine antagonist theophylline) result in parallel changes of NMDA and non-NMDA rcccptor-mcdiatcd components of
EPSCs ovt‘r a 16-fold r a n ~ c Inductim ~~. of long-tcrm potentiation hy delivery of low-frequency synaptic stimulation In cnnlunctinn wtth depnlarrzatron to +37n rnV leads to differcntial enhancement nf thc nnn-NMDA receptnrmediated component of the EPSC. Stimuli inducing LTP dn not cause a sustained enhancement nf isolated NMDA rcceptor-mediated EPSCst cvokcd in thc presence of thc AMPA receptor antagmist CNQX7’.
GIuR octivniion linked t u secretory events 07-14-10: AMPA: L-Glutamatu has hccn shown to inducc immcdiatc, transicnt anti cnnccntr~tion-dependentglucagon release hy activating a rcccptnr of tlic AMPA suhtypc in isolatcd rat pancreatic C C ~ ~ S(See ” ~ . also
iG~uR-1Fs.FouIulrrlgrowth hnrmnrre release in chrorno/{jn cells tmder Aecrp f orlt rrFn.ducrr in trrrici Ions, 0 7-49]
Specialized jGIuii’s medinling complex anditnry informaiion 07-14-1f : Thc fast kinctics of rcsponsc to glutamate and kainate in cochlear newones enable perception of sound dircctinn hy discrimination of microsecond differences in the arrival nf sound at thc two cars. Thc ability to fire action potcntials prcciscly corrclatcd with synaptic input or ‘phaselocking’ can occur a t high frcqucncicq (-9000 Hzl in species such a s owls. In avian cochlca, AMPA/katnate receptors exprcsscd nn ncumnes in the nucleus magnocellularis [ nMAG) vxhihit unusiially rapid onset and tcrmination. For cxarnpk, in chickcn nMAG, 10 !TIM glutamatc-cvtikcd cwrcnt rn patchcs riscs [IQ-90%]in 0 3.3 f 0.18 ms (N - l,? patchcsl and desensitizes hiphasically (< 1% peak current) with a fast timc constant of 960 ps fit 22 C, dccreaqing to 570 [ i s at 3-3 CrZ7.
iGluR involvement in rnndulntrnn of rnhrhitory post-synoptic current 07-14-12: Monosynaptically uvokcd inhihitory post-synaptic currents in hippocampal pyramidal sSiccs diminish in thc prcscncc of CNQX (an AMPA rtctptor-scICctiw antagonist - wt‘ Receptor nntogonrsts. 07-51) and
APY (an NMDA-wluctivc nntngonist) following a train of action pntcnltials. Rcsprmws to GARA applicd l>y irmtophortsist howcvcr, do not change si~nificsntly{cf. Chrinnrl nrodttlmtron umkr ELC: CI GRNAA, IU-44).
Expression pottern of iGIriR suhuniis nnd sitqnullrn‘qfunctions in ~ I i n l cells 07-14-13: KAIN: Eergmann glial cells in mousc ccrchellar slices arc unusual in that thcy do not exprcss GIuR-R suhun~tsalthoughthcy docxprcss GluR-A and G 1u R-D s 11h 11n i t s 2 3 v . Rergmann g:lial cells express native komntr-type iG1uR with a characturistic (sigm(iid\ currunt-voltagu ralation”. Thcsc channels arc [atypically) Caz’-permeahle and can hc hlockcd by CNQX (see Receptor ontapnirtq, 07-51), Entry of calcium also leads to a marked rcduction in the resting [passrvcj potassium cnnductancc of the gliat cells. Purkinjc cells, which arc closcly associatcd with Rcrgmann glial cclls, may
‘
prnvidc an important stimulus through thcir glutamergic s y n a p s c ~ ~ ’ *(.rep ~” CurrPnr-vnltugr r~lmtron,07-35) Note. iGluR in hippacampal astrocytes can propagatc calcium waves in ccElular networks, a mechanism which may form part of a ‘long-range’Eli31 signalling systemR‘.
Protein distribution RadioIigend and immunncytochernical distribution studies 07-15-01: .KAIN: -_ High-affinity sites for kainatc binding (K,T 5 and SO n M ]
-
have been shnwn in the CA3 area of the hiypncamyal formation” and thc peripheral neuroncs of dorsal rnnt ganglia44. The kainate-binding protein from chick cerehellum is exclusively localizcd on Rergrnann glial membrane (in clnse proximity to established glutnmatcrgic: synapscs]. Monoclonal antihodics raised against pcptidc scqutnccs of thc chick KRP (KRP-c]scquunccH” (WE Darnhasc IE’stinKs, 07-52) cr(m-rmct with a 49 PcUa prntcin in ccrchcllar mcmhrancs”’. 07-15-02: Tahulatcd summaries of innotropic GluR cxprcssion in sclcctcd ccll typcs nf the rat CNS have appcmd”*” (,we also m h l e 4 and the methndrhgi c nl m i e under mRNA distrrhlion, 07-73).
Subcellular locations Segrqatran nnd clustering of ionotropic GluRs in tar hippocampol nezmnes 07-16401: Clustcrs of AMPA-sclcctivc GtiiR 1 and GluR21.3 channcls colocalize in culturcd rat hippocampal newroncs and nre rtstrictcd to a suhsct of port-synaptic sites". GluR 1 a n d G l ~ R 2 / , scgrcgatc 1 to thc somatndenddtic domain within the first week in culture, even in the ahsence nf synaptngencsis. Glutamate rcceptor-enriched spines develop later and arc prcscnt only on presumptivc pyramidal cells, nnt on CARAcrgic mtcmcuroncs. Thew is preliminary, hut there is direct evidence fnr dendritic location of GluRS-G~uR7subunits in primate cortcx and hippocampus (cited in ref.”).
Genernl pnttern of iGluR prorein /[lrni?y distrihution 07-16-02: AMPA and ”IDA GluRs are gcncrslly post-synaptic’”’”‘ and arc closely appnsetl ( 5 2pm] to transmitter-release sites or cxprcsscd nn glial cclls. Kainatc rcccptor-channcls have hcen localizcd to prc-synaptic sites.
Orientation of jGluR N-nnd C-iermina?domains in the post-
synaptic face 07-16-03: GTuR-A anti-N-terminal antibodies show im munoreactivity to the synaptic fncc of thc plasma mcmhranc, while anti-C-terminal antibodies immunolocalize to the intraccllular part of thc pst-synaptic incrnbrant?. Cornpnrfirivc note: Snmc pmtcin domain topnlagy models place thc Ctcrminus of iGluR subunits nn thc cxtracclluh facc (we / P E T M I , Fig. 3 ) .
ImmunocytocherntcaJ dist rihutinn studies 07-16-04: GluR-R and GluR-C subunits Arc nhunrlantly cxprcsscd on Putkinje cell bodies and spines. Hnwcver, no AMPA rcccptor bmmunorcactivity IS dctectcd at thc parallel f i l m synapse that mediates excitatory post-synaptic curtents from granule cells on to Purkinie c ~ l l s ~ ~ .
1 / I , . $ 1 111/)131' j l ' l j 1 r~,!th!n this rrnd tho nexl scctron dejcnoles nn rlltrsfnrtr,~iIvrrtrrrr, o r ] tJ~cc l i r ~ n proterr~ ~ ~ ~ l (Iornnin topogmphy nzodcl (Flat31. Nor,,
Chromosomal location Predrcted phenotypes n!' rnrlrnnt (cJysfunciionn!)iGluli genes 07-18-01: A l t h r ~ u ~no h dcfinitivc linkagcl of iGlrtR ~ c n udcfccts has hccn rnnilc tr, any known ~nhcritctldiscasc, inutations in iGluR gvnus w o ~ i l dh t prrrltr*tcrl to IcarE to phcnotypcs such as epilepsy, cell death, tlcfcctivc neural development or psychzatric syinptoms (st,(: I'lienotypiu exprc.\';ic~n. 0 7 - 14). T h c tiunnn AMl%hJlow-affintty kainatc rcccptcir srihunit gcncs havc Ilcun mnpptrl tr I spccif sc chromosr,mcsnp, a5 sun~marlzcdin Tahlu 5.
Table 5. Chmmnwrnul locrrtions of the htrrrrr~nAMl'd/low-c~ii~nrry kuino!r rccepftrr rul>ran~l~ c n r q "(Fmm 07- 78-01 )
Glult-A gunc homclloguc C;luHI (- 97'Xbtduntlty with rat GIURI 1 G1ul.t-13 gunc h n m o l o g ~ ~ u GluR-C genc homologuc GluR-I3 gcnc homologuc GluKS gcnc h r ~ m o l o ~ u t . HRC;III '
H 11~;~ 2 " H Rc: ~ 2 '
Human chrr,mnsolnc 5 Human chn,mosomc fiq,Mh
Human chromosome 4q.32-.33' Huinan chromosome X Human chrr~mt~soinc 1 1'' Human chromnsornc 2 142 1.1-22.2" Human chromosome fiq,? 1.,3,2,1.,3 ( f r o n ~ (:cnHrrnkH entry) Human chrr,mosrlme 5~1.11..7-.1,7.3w Hum:~nchrrimoso~nc4q25-.34..7"
"Erir dctcrminatlon of chromosotnal 1oc;uions of AMI'A siihunit gcncs, suc rcf '". "AS dctcrmtncd by fluorcsccncc In s f l l r hybridization [FISH\. ' T h ~ location s cxcli~rlt..;Cli~li-!< as a wnditlatu gcnu involvcd in Huntingtcln's rliscasu. ,I T h e rt'xlon containing thc G l u R - n gcnc has hccn assnciatcd with linkage tn schiznphrcn~aand rnnior dcprcssinn. ' T h c CluRS Rcnc is In thc vlcinitv of thc Rvno linked t c ~familial amyotrophic latcral scl~ro.;i.;""~~. 'Isoletc nainc, . ; r ~Drrt(rhrr+c I~sr~n$.c, 177-53
Encoding 07-19-01: For prcdictcd clpcn reading framct lcngths dctcrrnincd from =DNA scrlucnccs, scc. the rcspcctive suhunit gcnc namc in Td-rlcs 1 , 3 rlnder Ckne {(i~njly,[17-05
Gene organization GluR-channel diversity vja the alternative splice variants ‘flip’ ond ‘flop 07-20-01: AMPA: In the GluR-A t~ GluR-D family, a 115 hp segment preceding thc gcne region encoding the predicted M4 transmembrane domain (see IPDTM], Fig. 3) has been shown t~ exist in two versions with
different amino acid scqucnces. These 38 amino acid modules, designated ’flip’ and ’flop’, arc encoded by adiaccnt cxonst of thc rcccptor gems and impart ditltcrcnt pharmacological and kinetic propcrtics on currents evoked hy t-glutamate or AMPA, hut not thosc cvokcd by kainatc (for dctnrk see Receptor ngonfsfs,07-50), ‘Flip’ and ‘flop’ ditkr in only a fcw (9-11) of the 38 amino acids. A pentapeptide occurring at a comparable position in each suhtypc is consistcntly diffcrent in the ’flip’ and ’flop‘ versions (see Fig. I, and /I’DTM], Frg. 3).
Comparison of cDNA and genomic sequences encoding AMPA receptors 07-20-02: The switched versions of ’flip’ and ’flop’ are generated hy altemativc aplicinft ot the two regions which are on adjacent e x c ~ n s ~separated ~‘ by an intron of -900 hp (see Fig 1). For each receptor, the alternatively spliced rnRNAst show distinct expression patterns in rat hrain, particularly En thc C A I and CA3 ficlds of thc hippocampus (see m R N A dnrrihution, 07-13). Thus, thc flipJflap modules cnahle functional properties of glutamatcactivated currents to hc controlled hy alternative splicingt events. (For the phcnotypic conscquenccs of flip and flop modules, see Inactivatron, 07-37.)
A note on splice variont nomenclature 07-20-03:The names ‘flip‘ and ’flop’ may imply that thc alternative cassettes arc in ’reverse orientation’ to each other, hut this is not thc case (see FiR. I for clnrjficntionl.
Existence of further nlternative splice vnrinnts 07-20-04: A third typc of transcript (GluRQcflop) derived from the GluR4 gene hy differential RNA pmcessingt has been isolated fnllowing screening a rat ccrchullar cDNA !ibray‘2. GluR4c transcript encodes a protein with a
‘ f f ~ p ’module hctwccn transmcmhranc regions 3 and 4, but with a C-
tcrminal segment of 36 amino acids different from the previously descrihed GluR4 tlip/floy, cDNAs. Transcripts synthesized in vrtro from GluR4c flop” form kainate/AMPA-activated channels showing strong inward rcctiticationt whcn expressed in Xenopus oocytes.
Alternntive splicing in the GluRS gene 07-20-05:Alternative splicct variants of thc high-affinity kainatc receptor subunit G h R 5 h a w a l w hccn dcscrihd‘. Thc longtr GluRS-I variant has an open reading frarnct (ORE] of 920 38 and dcrivcs Imrn inscrtion of a 45 nucluotidc sequence within thc first third of the N-terminal {cxtracellwlar) glutamatc rcccptor domain. A full open reading frame1 tor the shorter splice variant (GluR5-2, lacking thc 45 base inscrt for a prcdictcd 905 aa ORF] was not d a t e d in the original study4.
-Region
encoding putatrve transmembrane d m i n s I to II'
'Flop'
exon
b Linear n?pmw?tat/onof part or the gene encoding
-38- aa
1
i
the GIuR subtypes A-D
! \,
in trnn (*YO0 h p ) I
-
-
'Flip' exon ,38 . - .aa t
---. .
,
1
\
Figure 1. 'Flip' and 'flop' alternatjve splice voriantr in AMP.4 receptor genes. (a) Sequencing o f cDNAs for the four subtypes of she AMPA receptor revealed o 1 1 5 hose sequence encoding n 38 oa segment which existed in two sequence versionF designated 'flip' and 'flop' Ib) Sequencing I R the reginn of genomic DNA precedinx that cncodmg domain IV revealed the 'fhp'and 'flop' segments to be encoded hv two separate exom. flanking OR mtron, as shown. Alternative use of 'flip' and 'flop' exon sequences in AMPA receptor genes confers different kinetic properires of currents evoked b y glutemote or A M P A on G h R - A to GluR-D subunits. For details, we Gene organization, 07-20. and Receptor ogonists, 07-50,Within the alternative 38 aa flip/flop module (occupying equivalent residue positrons En the cDNAs), flop versions of the Tubunits contoin a conserved SGGGD motif, while flip versions are variabte ot single residue (nrrowed) in the subunits. (From 07-20-02)
GluR subunit sfmcturnl changes via I2 NA editing mechanisms 07-20-0(,: A further possible mechanism for generating hctcrngcncity in thc channel coding regions nf G h R transcriptst is RNA editingt. In thc case of
thu GluR-R gcnc, i t was found that there had been a nuclcntide change [an adcnosinc-to-guanosinc transitiont 1 ohscrvcd hctwcen the scqucnccs ohtaincd from gcnornict DNA and O N A t sources" (note. normally thcrc is perfcct agrccmcnt of scqucncc). It has bccn propnscd that ‘editing’ of An adenosine to inosine givcs risc to an Arg [R in singlc-lcttcr mdu) mstcad of a Gln (9)rcsirluc a t the critical QIR site (for the consequences of rhis chnnge, see Domain functions, 07‘-29).
Subunit R N A transcript selectivity for editing ~ T O G C S S E S 07-20-07: A ~ t h o u hthe GPuR-R suhunit transcript appears to undergo editing In > 99% of isolates found, the GluR-A, -C and -D subunit transcripts [sharing identical domain M2 scqucnccs) dn not appear to be edited. In transcripts for GluR5 and GluR6, cdited and nnn-cditcd transcripts are found at different The principle nf RNA cditingt is illlistrated in Fig. 2. Note: R N A editing has been shown not to he a RenerclI cellular mechanism for GluR diversity (see mRNA distribution under ELG CAT GLU NMDA. 08-13].
Rnxe-poired intron-exon sequences are required for GlnR-R RNA editinx 07-20-08: Low RNA editing efficiency is observed in GluR-R gcnc constnicts rnodificd in sequences at the proximal part of the intmnt downstream of the
.-
-
dr RNA-spsEifre adcnoslne dsamlness (see Gens o r g e n h f l o n )
(3) In edited RNA,
adenosine ( A ) deeminntes to an
inosine (1) mwmmmmal CfG cA[Is m m w m m m i
(4) In unedifed RNA, adenosine (A) is unchanged
mmmmmmi (5) Resultant
R
Q
in pore-llning domain M2 {see the PDTMJ (R = Arg; 0
CM -3
Q
.nmml
Q
= Gh)
Figure 2. R N A e d i t i q RS n mechnnism for Renerating hesero
entry 07
GluR-13 iinctlitcd cxnnict sitcYu. Perfect intran-cxonf sequencc base-pairing (if this rcgirm is rcquircd for cfficiont sitc editing. [n thc native ~;Iuil-lt gunc, this portion of thc mtron contains an irnpcrkct invcrtcd rcpcat prvcutlinr: a 10 nuclvottdc scqucncu with cxact complcmcntarity to t h u C X ccntrcd ~ on thu unutiitud cotlon. SinKlc h a w substitutions in this short intronic scqucncc (nr its cxnnic complcrncntl inhibit Q/R situ cditing, hut this can hc rccnvcrcd hy rwtnring cnrnplcrncntarity 1 in thu rcy-icctivu partnur rtrantl. Rasu-paircd scquunccq in thc C,IuR-R suhunit prntcin coding rcRicin appcar tci dircct h a w convcrsiiin hy n nucluar adenosine deaminase spcci fi c ior douhlu-stra ndctl RNA''".
u/n
Editirix of G h R h R N A trnnscripts - comhrnntorifll vririnrions f o r udired fr1rrn.s 07-20-09: In atfrliticin to transmumbranu domain M2 QIR site editing, GluR6 has two additional positions in dmiain that arc niorlificd by !IN,%cditinRt ( w e hclow and Domain fzmcticins, 07-29) GluRh thcrcforc can exist In tlic gcnnrnict [uncditcd~form ant! in w v e n diffurcnt cditcdf forms, dcpcndinR on thc comhinatinn nf cditcd or uncditcd rcsiducs. Thc origin of thcsu cnmbinntions ant! notcs on thcir differing Frcqucncics of occurrcnw rn dic
CNS Are shown in Tahlc 6
Anttlyiicr~Inuie: Reprcscnt(i1F
nizn t I
I
rind cinci?yysisof complcx j yn e orgrr-
O ~
~
07-20-10: Usc n f apprtipriatc datahaw ncccssiiint numhcrs and iinportation of raw data into scquuncu analysis programs porrnits a gruatur apprcciatian of hoth markcti ant1 suhtlc variatinns in inn channel gene nrganization by mcchanisms such a< altcmativc splicingt ant1 RNA cditingl (For nn rxrimplP, w r thr C O O ~ I I O ~ C t o fhr: tohlc in Drrtnhnw liYtrn,qs, 07-53 &wr]hiqy tliu ciySiznizir!iori oI IJIP G1trR-R Scnr.) Standard databasc cntry hmmats ciftcn accr)rnnicirlatc data for loci of kcy splice sitest and altprnativc exon t i s a g ~ l and can hu iiscrl intcractwcly with ori~inalIcwmnl artrclcs.
I
Homologous isoforrns 07-21-01: For names of equivalent subunits isal;ltcd in dilfcrcnt spccics and those in the samc spccic.; by different laboratories, see T(ililes I L T , 7 nnd nrii (I 1x1w lis Im g s . 0 7-5,1.
1
Protein molecular weight (purified) Moleculcir size of imrnunoprccipituied recnrnhinnnt subunits
07-22-01: AMPR: Suhunit-specific antibodies against pcntamcric channcls containing GlL;!<-A to -D subunits rcabgnizc il typical suhunit M, of - 1 OX kDaYS.For thc AMPA rcccptors, immunoprucipitatirIn data do nut sum:cst any suhunit othcr than GluR-A tn -D within precipitated complcxcsYr.Jam spider toxin [JSTX)has hccn L I S L ' ~tri purify an AMPAhinding protcin of M, 1.30 kWa frnm nativc r n c m h n v s ( s t y Illnckcr?, 074.3, rind Li,yrinds, 07-47)
-
Table 6. Combinororid variations in Glur 6 intmduced b y R N A editing. (From 07-20091 Unedited genome Unedited Ile in M1 Edited Ile in M1
-
Val
Unedited Tyr in M1 Edited Tyr in M1
- Cys
unedited I
Edited form 1
Edited [om 2
Edited form 3
unedited
Edited form 4
Edited form 5
unedited I
unedited I
Edited form 6
edated edited edited I
v
V
V
unedited
unedited Y
uneditcd Y
Y edited C
edited Y
uned~ted
Y
edited C
edited C
Edited form 7
edited C
Unedited Gln in M2 Edited Gln -- Arg in M2 Frequency in CNS
edited
-
10%
remaining
25% ot occurrcnccs
edited
edited
edited 1
hi",, --
Footnotes: The M2 Q/R position can only influence ~ a " - ~ e m r e a b i l iwhen t ~ the M1 residues are in their edited form (see Domain functions, 07-29). GluR6 (R-edited]channels show a higher ~ a ' ' - ~ e r m e a b i l i than t ~ G h R 6 fQ-uneditedl channels. Co-expression of GluR6 (R-edited)and GluR6 (Q-unedited) forms with forms 'fully-edited' in MF. produces channels with low Ca"-permeability, and the influence of the QJR switch is low when the M1 domain positions remain unedited [see also Domain functions, 07-29).GluRS also shows a diversity with respect to kdited' and 'unedited' versions of its Q/R siteq3.
cntry 07
Cornprisons of nntivc chunncl srzes h y immtinoprr~cipitcrrrnnon$ rodl'nli,qond n f f i n Sty -luhciltntq 07-22-02: ~AMPA: When synaptic plasma rncmhrancs arc soluhilizcd, highaffinrty AMPA hinding and GluRl imrnunorcactivity cn-rniRratc nt n natavc gl ycnprotcm of M, of -6 10 kDaHn.
Crosx-renuiivrfyof nntrfiodius t o kcitnrrte-hindlnR protejns 07-22-03: KAIN: Antihndics against the frog kainate-hinding protein ( K l { P / , see 1hrnhrisc jrsrrngs. OJ-5,7) whnch has a predictcd M, of 4X kDa from its cDNA scqucncc], cross-react with a nativc protcin of M , -99 kDa in rat brain preparations. Thew antihodics r c c o p i z c this cruss-rcactivc rnatcrial in thc sainc hrain rugirms whcrc GhR-Kl'" (3 GluR-A hornthgue) has hcvn localizedY'.
Sequence motifs Sequence motifs for N-glycosyhtion 07-24-01: Thc numher nf potential N-glycosylatiant sites per suhunit in each cDNA cloncd thus far arc shnwn in hrackets: Rat GluRl (61;GluR2 (4); GlvR3 [fij; CIuR4 (51;GlliRS ((1);GluRh [ h ) ;GIuRJ (6);KRP-f (21; KPP-c (2);KA-1, KA-2 (R-101.The positions of thcsc N-glycnsylationt motifs? in thu individual subunit see [he rr{prrncrf mnd occrwmn numbers jiivrn undrr Rntuhow listin,q.q 07-ri.7. Differing cxtcnts nf N-glycosylatinnf may account fnr the scIt'ctivity of certain lcctins such as concanavilin A fnr dcsensitizatinnf at karnatc-prufcrring recuptors (lor h r t h e r dutnilx, see Innativrrrrnn. 07-37).
N-Ierm I R OI clusr eriqy oC N-tqpSvco.sylationmotifs
07-24-02: In i s d a t c HRGR-I (a human hnmrhogue of rat T.luR1Ip" the Ntcrminal segment prccctling M 1 contains a11 potcntial h'-~lycosylation sitcs (aminn acrtis 45, 2,j 1 , 23.9, ,?45, ,183 anrl ,?RX].
U ni ver,wJ p rew n c c o/ si,q n( I I sc q tien cc ,s 07-24-03: Signal peptidest of 17-31 aa rcsidues arc present at the N-terminus of all glutamatc rcccptor-channel subunits cloncd thus far (we JPnTMJ.Fig. 3 untf Tirhlr. I).
1
Southerns
07-25-Of:It has hecn vcrificd by Southernt analysis (performed on DNA of the s ~ m mouse)"' c that multiplc gcncs do nnt exist for GluR-R, GluRS an! GluR6 subunits. This supports thc conclusion that nwleotide exchanges' found hetwcen genomic and c n N A sequences arc duc to R N A editing' processes (sec C h w c q o n i z(I f IOR , 0 7-20).
02): 1. Hnllmann, Neirron 11 994) 13: 133 1-43: Experimcntal approachus hnsud on N-glycosylationt site taggingt as rcportcr sitcs tor cxtracvl~ul~r Iocations of protcfn domains in GluRl show: ( i ) the N-tcrminus is cxtrnccllrihr; (ii] thc C-terminus is intrclcellulnr; ( i i i ] only t h e tmnsmcmhranc domains arc prcscnt, dcsignatcd TMD A, TMD R and TMD C (corresyontbng t o M 1 , ML3 and M4 as dcscrihcd in thc ‘finalizcd‘ miry); (iv) contrary tci carlicr nicidcls, tlrc putativc chmncl-lininE dnmain M2 docs not span thc mcmhranc, Ixit lics in close proximity to the intracellnlar face of thc plasma nicinhranc ( i r loops into thc mum’hranc without transvcrsing it; (v) thc rcgiciii hctwccn M3 and M4,in prcvims models hclicvcd to hc intraccllular, IS an cntircly extraccllwlar domain. 2. A rcfincd structural model of thc glutamatehindinp; site nf innotrnpict glutamate rcccptnrs, liascd nn cxchmging pnrtinns of the AMPA reccptnr sulmnit GtuR.3 and thc kainatc srthunit GluRh has also hccn puhlishcd {Stern-hch, Neuron ( 1 994) 13: 1,145-S7]. Thcse new intcrprctations underline the difficulties nf accuratc domain topography modelling from indircct data.
Amino acid composition 07-26-01: Ry hydrophohicityt Ihydrnphilicityt criteria, all GluR suhunits nf -900 aa rusirlucs in lcngth arc predicted to contain four rncrnhrancspanning rcgians. Thcsc arc variously rufmcd to as domains TMI-TMIV, M 1 -M4, TM 1-TM4. Thc ‘four tranwncmhrmu domain’ modvl should hc seen 3s tentative (see ELG Key {ncfs, entry 04). Note: Thc version of thc iGluK mnnnmcric protcin domain tapngmphy modcl (Fig. 3) shnws an additional transmemhnne domain interposcd hctwccn M,? and M 4 [partly rcplacrng thc large M3-M4 (pntativc) intracellular loop typical of other modcls fur ELG-gatcd channel^, d.Fig, I undcr F I L : CAT .5-HT1, entry fJ*5).
Domain arrangement General a r r n n p n e n t of suhunits 07-27-01: Thc four prcdictcd membrane-spanning rcgians (predrc~edi n some models, see ohow) dctcrmmc the monomeric subunit architecture of iGluR.
Structurally, thc channcl suhunits arc arranged ‘barrel-like’ around a ccntral conductive pore. in cnmmnn with othcr mcmhcrs nf thc cxtr~ccllularl i p n d gatcd cation channel supcrfarnilyt, nativc channels arc likcly to he formed from five mmnmetsYR(we I P D T M ~ , Fig. .q).
Subunit nrrnngements i n native recepror-channels 07-27-02:In cnmmnn with similar studies on other ELG-type reccptnrs (e.R. see E L G CI GARAA, entry 10, nnd ELC: CAT nAChR. entry 09) comparison of cmrcnt-vnltagc rclatinnsf for nativct channels vcrsus thosc cxprcsscd frnm cci-cxprcsscd rccomhinantt suhunit comhinatinns prcdict nativct rcccptors to he heterornultirnerict, composcd nf at least two diffcrcnt rclmtl’nns, 07-;J5).Immunosuhunits (srimmnrizerl rrndcr Currenr-~ol~rn~yc chumical stutlics with G l u R suhunit-spccific nntihodics suggcst a pcntamcric, hctcrtl-oligcimerict asscrnhly of suhunits in nativc channels"' (hiit SUP
I h t r i n interrwtions, 07-,71).
i'rt>pcriirs of KA- I IKA-2 suhuniis wilhin heturornuric ctrmplcxes 07-27-03:K A I N : Atthnugh thc Kh-I or KA-2 subunits f c ~ i lto f o r r functlclnal chnnncls w % cxprcssutl a< h r t m t ~ m u l t i r n c r s ' ~ ~thcy ' ~ , do cxhihit a highabfinitv for kalnatc. ~ u t u r o r n c r l c i cxprussmn c ~ fKA-2 with thc distantly rclatcrl G l ~ ~ l ior f r G l i ~ l i hs u l ~ ~ ~ nIcatEs t t s t c ~forlnatlc~nof functional channcls with novel p r o p ~ r t i ~-sc . ~AMPA . actlvatcs C;Iulll,/KA-2 channclq hut docs no! activ;~tchoint~nlcricCiluRC, rcccptorsr4.
Domain conservation Conservnrion of xicynml sequences nncJ s r ~ k r l n ~armn
Conservarlon
131
the ELC: ul~nnnel'core sequence'
07-28-02: Thc. corc sequence 1s dcfinctl a s thu rcgion af the cxtraccIlular IignntP-gntcd channcls which showr; thc h i ~ h c s t sequence similarity. C;unt.mllv, thu 'corc .;cclucncc' inclutlus thc doinains M1-M4 (/1'13TMj, Fyy. 3) T h c suhz~nrt.;diffcr most ~n t h c ~ rN-tcrm~na!-470 rusirlllcs, o.g. w6icrc C;ItiR-A to GlrlR-I3 sharc -C,O'XI ant1 GluRS ant! GhiR6 sharc -75'L sctll~cncc idcnt~ty. Pairwire comparlrrms hctwccn thcsc twt, groilps of rcccptors rctlucc iclcnt~tvto -25% In thc N-tcrrninnl cxtraccllular rcccptox domain (II'IITMI. Y1.y 31.
Posrtions oC rrmirro rrcid chnngcs cIrlc to rrftcrnniivc spllcinx 07-28-03: S p l ~ c cvari;tnt~Trtf inntitrop~cfglutamatc rcccptclr can Kcncr;ltc inscrbionu or s ~ ~ l > s t i t l ~ t i 111 r l nscvcraf s rcclons (.wr II'DTMI, FIX.,3 rind Grnr clrgrrnlz(lrlon. 07-21)). T h c 'flip' and 'ilopr forms (if the A M P h rcccptnr s ~ ~ h u ~ iarc i t s gcilurattd hv alternative sFlicingi hctwccn rcgrons cncoding trnn~i~lclnhranc t l r t r n a ~ nM.3 ~ anrl M4. Variant s u h u n ~ ttcirmr ;llri) d c r ~ v c ~ l frt~nialtcrnat~vusplicrng1 u ~ i In ~ tthc N-tcrrninal dornaln (c.g. the G I U R S ~ and thc NR- I suhun~t.;l.(For rhc phcnotypiu ronrrqrirnccs of C I I ~rin~lF IJop nrodlrlr~r.scc 11111~tivot1on. 117-371. Seqrience hornolqyy het ween !he iGluR nnd ~ l u t n m i n e - h ~ n d t n , ~ prof eins 07-28-04: Thc isolates C h R - K 2 ant1 G h R - K . l (ccirrcspclncEing tn the 'flip' vcrsion of GluR-H ancl GluR-C) havc hccn rcpc.~rtcdov as showing sign~ficant sctlilcncu con.;crvation with the ~lc~tnlninc-hintl~ng component nf thc glutamine petmcasc of F i.oli
Kcrlncitu-A M P A suhunir srrnilr~rirics 03-28-85: KAIN: RA- I , thc first high-affinrty kalnatc suhunit to hc cSoncdn5 I I ; I ~ ;I ;30'X, scclucncc sirn~laritywith thc AMPA ruccptor s u h u n ~ t sGluR-A to -1). Not(' Thc frca ;~ntl c h ~ c k brain k a ~ n a t c - h ~ n d i nprtitclns g [scr
P u M m glutannH-hmdlnp donulnm brrd an p.riW to pmkmyolk prrrramlnr mnrpurrers
Fllp / Flop module Cm'
. nomalogy
, '." 3 U t
QrpaOiut'onl
w4,ri
-
Lw?;
--. r-z'ly3u4
y3 H i Mb
Extracellular Psntamarlc arrangemsnt (pu ta tl va)
(a) Monomsrfc dornalns FntraceEIuler
-
o l l g o ~ r l ca t w m
(prramr-seemeiwds
-8?enpememd
,,
-coon \
deiemlnmtc
0-R .It.
( m n G m orp8nhilm and Dmnmlrr htncrlunr # M s ) -a . P uiun-5 Q or a
-u
'
R
-c
'
Q
'
-D
'
Q
'
' 9 '
.
.
QluR-b a.11111-1
bl hi
+
.
Q or R
.
. *
0 - 9 ' ' 9 '
-
Addmmut -them#} mnwn h r r o d u c d In moms to pol^ mod.lm lu n l a h cenmlnnncy wlth Itrueturs/lwncllon data ln Suburg (lee31 Tfmnds Pk.mr8cd Scl 14: 297.303. Comp8n W t h a)l.m.tlva model. wflh larps ( U 3 - M ) Imiwesllulsr h o p and rmdcwIIuNr CODH
-
I8//
ELO CAT
s-H~)
Chmnol aymmbol Wa.K, ICaF
n /OlU.t.rlng
mm
ol *-'
CQI..nwU.
RM
nhlnLvAdtm Pronln p l m @ ~ l I n n )
m
In A Y P W l n l m IWuR r t h c h d b* dlnruthn Iklng wnntm (mOm. arpmnlzaHonJ
NOTE:
All d # v W pOSlt/On6
Of
mOflh,
doomeln Bh8peS #kM ere dfagrarnmatlc and am sub/ecr to re-lntarpretetton
-
'" \
I
I
ELO
__
v
Figure 3. Monomeric protein domain topography model (PDTM] for AMP~lkainate-selectiveronotroprc glutamate receptors (iGluR). In press updates [see criteria under Introduction d layout of entries, entry QZ): A modified 3-irmsmembrane domain model based on N-glycosyletion site togging data has appeared in press. For brief details, see the rnsert before Amino Q C I composition (07-26). For further details, refer to the entry update pages via the CSM. (From 07-28-02)
~
Table 7. Extent of amino acid sequence identity (%) amongst members of the cloned ionotropic GluR subunit family and kainate-binding proteins (From 07-28-01)
GluR-A GluR-B GluR-C GluR-D GluR5 GluR6 KBP-f KBP-c KA-1 KA-2 -:
GluR-A
GluR-B
GluR-C
GluR-D
GluR5
GluR6
100
70 100
69 73 100
68 72 73 100
40 40 41 41 100
41 41 42 40 81 100
information not found.
KBPf 38 -
42 43 100
KBP-c 37
KA-1 35
-
-
38 40 56 100
42 44 35 34 100
-
-
KA-2
07-53)appear to lack thc first -350 aa nf the GluR-A protein ant! possess only -25% amino acid identity when compared to thc N-turminal half of GluR-A. Dn!nhnsc Irstings.
Domain functions (predicted) The QIR site in the M2 domain is crittcnl for regulntion of ion permeahiIisy and I-V re?oiionships
, I
07-29-01: Thc M2 domain scqucncc shows variability at n position known as thc glutarnlnc/argininc sitc or QIR site (see [fie sequence alrprncnt helow and lPnTM/ F F ~ 11. m.wt). GliiR-R possesses an aegininc (R) a t this sitc comparcd tn a glutamine (Q) hcing prcscnt in thc liomologous pcisitmn in GluR-A, GluR-C and GluR-D. Significantly, thc argininc cndcin is not found in thc GluR-R gcne, hut is introduced hy an RNA editing process (see Gene orgnnizoticm, 07-20), The Q / R site also detcrrnincs thc Ca2'-permeabilityt of thc channel (for detarls, sec ScI~ctivity. 07-40'". I"'). Site-dirccted rnutagenesist of the QJR sitc has shown that thc single amino acid difkrunce in the GluR-R subunit also dctermincs thc I-V rclationshipt of hctcromcrict channclsFo2(fordetails, see Fig. 4).GluRh also occurs in two forms with rcspcct tn thc amino acid residue occupying thc Q/R For comparison, the scqucncc alignment helnw also lists thc cquivaknt (aligncd) aminn rcsiducs of thc NMDA rvccptor subunits NRI and NR2A (see ECG CAT GL1J NMDA, entry O X ) .
Scqzicncc ali,ynment-i in rG1riR suhunits surmundrnR the Q I R site in the MZ domI1in GluR-AFGI
F N S L W F S L G h F M B Q G C 1 I I
S P
GlitR-B F C I
F N S L W F S L G A F M m Q G C D 1
S I'
T L L N S F WI: C Y G A L M
0 C . S E L M P
GluRh
F
KA-2
Y T L C N
11
A T L H S A I
NRI
L T L S S A M W F S W G V L C
N S G I
NRZA
F T I G K A I W L L W G L V F
m N S V P V Q N
S
L W F 1' V C. C. F M
Q G S E I
MI'
W I V Y G A F V ~ Q G G E S S V C E G A
1 Thc Q/R
Fite
Control of Co''-pcrrneohi/jty of koinnte receptors determined b y R N A editing 03-29-02: KAIN: __ Ca2'-pcrmcahility of kainatc rcccptcir-channcls can vary dcpcntling nn 'editing' cif RNks cncoding both M 1 and M2 transmcinbmnc domain sequences".'. In addition tci cditbng a t thc critical QIR sitc in M2 (src rrliove mnd ref."*), scqucnccs forming the GItiR6 putativc transmcmhranc dnmnin MI, arc also divcrsificd by RNA editing (sec /IJ13TME.FIR. 3 rind thr,
wvrn J ~ ~ I rdj!(,(I ~ . ~ ~ ~o r ~~ ~~ ! ~ ~ j nP t ~~111der r j o n v<;rrl(>orpin:z~z!~on, U7-20). T h ~ s process can gcncratc c ~ t h c irs o l c ~ i c ~ noru v a l ~ n cin nnc and tymsinu o r cysrcinc in thc other MI ~ l o m ~ iposition. n In GluRh channels thu pruscncc 4)f Q (glutarninc) at thc domain M2 Q / R site forms channc!.: with low ~ a " permcahility [in contmst with AMPA ruccptor-channclsl. An srginino s t this pnqttlon determines a higher ~ a " - p c r ~ n c a h ~of l i tGluRh ~ channuls ~f domain MI IS 'fully-edited'. In thc 'uncditctl' form of C.l~tR6donlam M I , Ca"pcrmcal~illtyis lcss dcpcnrlent cln thc prcscncc ot cithcr ~ I u t a m i n cclr ar inmu In domain MZ'". Thcsc rcs~tltsraise thu possih~litythat RNA utEiting m.ly morlulatu ~Iutnrnatc-activatcti~ a ? ' - i n f l l t~hxr o u ~ hC;luRh rn vlrJo.
P ,
A$oni.~tt-hindinGysire 07-29-03; Thc region prrt.r~din,y pu tat ivc rransmcmhranc scgtncn t M 1 clf GluRs I S wcll-conscrvcd arnnng sithunits and ha4 hccn propc.,scd tn cunstitutc a part c ~ fthc aganisz-hinding.site (see r~lsoothrr FCC: en!riesl. Fnzroihrction nf pmnt mutations Into chargcrl rcs~tluusof thc rnousc AMPAsclcctivc T I suhunit (Glu.7YX Lys,TYX; Lys445 Glu445) arc sssociatud w ~ t hc h a n ~ c sIn thc KG,, valrlcst with dihfcrcnt agclnists, indicating t h c ~ r involvcmcnt for a~r~nist-sulcctivc interactions of the C;EuR ~ h a n n c l ' " ~ .
-
-
Supplerncrr t nry not c for c~hovc t -Glutal~~;ltc, kalnatc a n d AMPA I~lnd tc~ rl~//cri>.rcnr receptor substructures on rccomhinant AMPA rcccptorsfNY' ( ~ 1 . 0 Ecluilihr~um rl~ssoarir!lr~~? c r ) n s r ( i l l f .07-45, r~ndI,EprrrrJs, 07-47)
07-29-04:
Predicted protein topography Common r~sstlmptionsof hydrophobic seqrlencex as slrr~ctz~rnl domains 07-30-01: L ~ k cothcr incinl>ursof tliu cxtr~cullularlignnrl-gntcd [ELG)family, ;111 ~lutalrn~arc rcccptor-channcls display frmr prrrl~c.rrcl rncmhranc-spann~ng hydrt~phohrcrcglolls ( M1-M4l, ; l l t h o u ~ hsomu modcls Ie s.rr~l."l pnjposc an ;iildlti(~n;il'conjcct~iaal'translncrnhrane domain between M,? and M 4 ( w e I I T I T M j , F1.y. 3 ) . Howcvrr, the l;lck clf direct structural data docs nnt vct ;illow ;Inv firm ctmcl~tsrt~n.;to hc inarlc regarding 'trctc' transmcmhranc ~vntuin.tt~pogmphy(.rcr FLC: Kr.v frrr.tr, rntrv 04) Irr-l~russrrprjrr!c~.Sec ~ l o r cr ~ l ~ o v r~c.l(i r 07-2h
Protein interactions Common srrhlrnlt n.sancinrions with GluR-R n7-31-01: AMPA: GhR-R subunits dominatc prtlpcrtics of
I U ~ I C flow In hctcromcrici k l u ~c.c,mPlcxcs5'~'". For cxarnplc, co-cxprcssinn of thc GlnR-R flil>~tnit wtth c k t h ~ rCIuIt-A, GluR-C cir CrlttR-D forms rccomhinant chnnncls which closely match charactcsistics of natwct rcccptnryY, 1lnpPy1111: thnt inclst nntivct xcceptors arc hctcromuttlmcrici and lncovoratu thc C;luK-I< suhttnit. GluK-I< anti GluR-C ~rnrnunclruact~vity has I~ccnd ~ o w ntcl co-localize with thc mctahrttrt~picirnGlulr rccvptor at thc clirnhlng fihrc synnpqc in ccrchel!umHn.
cntry 07
Evidence against crass-nssernhly of 'AM P A -pteferting' nnd 'kainatepreferrinx' suhunits 07-31-02 The 'absolute selectivity' of cyclothiazide and concanavalin A for respective block of fast desensitization1 of AMPA-preferring and kainate-
preferring receptors (see lnflctivation, OJ-37) has hccn used to monitor subunit assembly patterns in functional recombinant iGluRs" . In all CBSCS, asscmhlv of to-cxprcsscd suhunits from the two difkrcnt families suwcst independent assembly of functional AMPA and kainate receptors without any cvizlcnce for cross-family assembly of subunits.
Common functional interfictions at synopses 07-31-03: Diverse classes of extracellular 1igand-gated channcls commonly interact to shape dcpolatizing pnst-synaptic potentials (DPSPsl 1n the CNS. Fnr example, DPSPs of granulc cclls in lthc dcntatc gyms is part-mcdiatcd by AMPA, GARAA and NMDk receptor proteins (see Protein intercrctions under ELG CI GAR&, 10-31, and Fig. 4 under ELG CAT GCW N M D A ) . Postulation of inhihitory proteins associated with native AMPA recepi ors 07-31-04: The existence of an inhibitory protein (with a pnssihlc 'negative rcgulatory' function] has hccn invokcd to explain the observation that AMPA K,I valucs hccomc much lowcr (i.c. AMPA affinity Increases) upon various mcrnhrane trcatmcnts and upon purification".
Protein phosphorylation
.r
jG1uR.Y display multiple putative phos horegdatory motifs
07-32-01: A nurnher of potential consensus phosphorylation sites for protein kinases [e.g. protein kinase A, protein kinasc C, tyrosinc kinase and casein kinase Ill have heen found in AMPA- and kainatc-sclcctivc GluR suhunit cDNA sequcnccs. Only a minnrity of these have hcen shown to hnvc {unctionnl roles to date. Note: Locatinns of cach putative regulatory sitc can he traced by refcrencc tn citations under D n ! a h o ~ eEr'qtinRq, 07-5,7. PO t en t in t inn of Co2+-fluxes 07-32-02 Ca'"-fluxes can pass through non-NMDA glutamate receptorin thc abscncc of channels cornposcd nt the subunits GluR-A and GluR-C [it. GluR-B] (see Selectivity, 07-40), Calcium flux through open KA/AMPA reccptor-channels can he potentiatedt by phosphorylation mediated through protcin kinasc A as part of a CAMP-dcpcndcnt sccond rnessengcr system"'.
Enhancement of native E'GluR rcsponscs thrmqh ptotcin kinuse A phosphoryln tion 07-32-02 Non-NMDA channels cxprcsscd in culturcd hippocampal pyramidal ncumncs arc suhicct to neurnmodulatory rcRulation through thc adenylate cyclase cascadc. Thc wholc-ccll current rcsponsc to glutamate and kainatc 1s cnhanccd hy fnrsknlin [an activator of adcnylatc cyclascl. Singlc-chmncl analysis has shown that rotein kinasc A incrcascs thc opcning frcqucncyt and the mean open time of non-NMDA-typc glutamate rescptor-channcls.
f
Forskolin, actmg through PKA, incrcascs thu amplitiide and dccay time of spcintanacius cxcitatciry pnqt-synaptic c ~ r r c n t ~ ' ' ~ .
htentifltinn of recornhfnrrnt iGhiR r r s p m s c s h y prntejn kinase A 07-32-04: KAIN: Channels cxprcsscd from GluR6 suhunits (when trans~cntly cxpresscd In mammalian cclts) hevc h e m shown to hc dircctly phrw phorylated hy PKA. Appjrcation of intaacellular PKA incrcasc.: thc amplbtudc (if thc glutamatc rcsponsc25. Site-directed rnutagcncsist nf thu scrinc rcsiduc (Scr6H4J rcprcscnting a PKA conscnsuq site complutcly eliminate4 PKA-mcdiatcd phosphnrylation nf this s i t ~a4 wcll as lthc potcniimont of thc glutamatc rcspnnsc25 (FPP [PDTMJ, Frg 3). ~
PnstuJoted frincfionnl roles of rGhR phosphorylntim 07-32-05: Pmtcin phasphorylatinn of glutamatc reccptorq by protein kinase C and CAMP-dcpendcnt protein kinasc has hcen suggested to regulate their functinn in synaptic transmission (see possibly playing a prominent and lonpterrn depressiont "". Far role in long-term potentiationt additional note4 on thc hroad roles of protcin phosphorylation in thc ELC channcl family, sec ELC: Key /ours, critry 04.
'"."'
Activation AMPA-srlectivrr iC3uKs mediate 'fost' excitcItory sign:nolliqq in the CNS 07-33-01: AMPA rucuptors mcdiatc thc most rapid synaptic excitatory neumtransmissinn and conduct mainly Na' currentq. For example, hrief (-1 ms) appIicntions of glutamatc on mcmhranc patches exciscd from ncurrmcs in thc rat visual cnrtcx producc a rapid rcsponsc that mimicks thc time coursc of mrniaturc cxcitatory pnst-synaptic cuncntst (c.g -2.4 ms for AMPA-cvnkcd EPSPS"~). Thc rate nf cmsct of dtscnsitimlti~m1 is much slowcr than thc dccay rate of thc rcsponw (we Inrrctivntinn. 07-37], implying that thc dccay of miniature EPSCst reflects channel closurc Into a statu road1 Iy avaiIahIc for rc-activation"'.
Rrsc times for iGIuR current nctivotron
07-33-02 Rricf pulscs (< 1 ms) of glutamate ( I mM) on AMPAlkainate rcccptors in granulc cclls of dentatc myms and pyramidal cells of CA.7 and CA 1 hippocam pal reEions activatc patch currents which rise and dccay rapidIy"'. Thu 2040% rise timuf of thcsc GluR-mediated currents is typically -0.2-0.6 ms. At -50 mY, peak currents vary from 10 to 500 pA in diffcrcnt pstchcs.
Ka in R t e R Is r) act ivn 1 es 'AMPA - prcferrinK ' recept o r-c hn nn eIs 07-33-03:Kainatc also activates nnn-desensitizing 1 currents q i r n i h r to those clicitcd in CNS ncuroncs through 'AMPA rcccptor-chnnnclt frirrnutl from suhunitq GIuR-A tn -no. lo.
cntry 07
Current-voltage relation Arnctjonnl evidence for heterornultimers based on shapes of F-V re lo t ion s 07-35-01:AMPA: Thc maiority of nativct ncuroncs cxhihit AMPA rcccptormcdiatcrl inward currents with Fincar or riutwarrlly rcctifyytnKt currentvoltage rclatronshipsjY. When Xenripu.5 nocytcs arc inlcctcrl with RNA encoding GluR-A alonc, AMPA agonists cvnkc ;1 smonth inwardly rectifying+ current unhke thc lincar I-V rclatinnship sccn rn V I V D . Howcvcr, whcn thc cnmhinations GluR-AIR or CEuR-RJCarc co-cxprcssurlyy,a nonrectifying inward current ( I c. lincar and ohrnict) is uvcikutl hy kninatc o r AMPA (closcly rcscmhlinR currcnt typcs sccn in nativc ncumnal cclls). In cnrnmtln with similar studics on othcr ELK-typc rcccptcirs re R. see ELC: C? GARAA, cntry 10, and ELG C A T nAChR, cntry 09) thcsc rcsults indicatc the native receptor tn he a hctcrnrnuftirnert of a t kcast twa diffcrcnt suhunits.
Dominant characteristics of C:luR-R subunits 07-35-02:KAIN: Bergmann glial cells display a kainatc-type glutamatc s sigrntiid (doubty-rectifyingt1 current-voltagc r ~ l a t i o n ' ~ and receptor w arc pcrrneahlc to thc dwalont cations Mg' and Ca"' . Note: Rcrgmann Ella1 cells arc unusual in that they do not cxprcss GluR-B subunits"' (see Cell-type expre~sinn Index, 07-08) Hornomeric GluR-R clranncls (or hctcromcric channcls containing CluR-R as dcscsihcd in the prcviaus paraRraph) cxhihit near lincar 1-V relations and havc low clivalcnt catinn pcrmcahili tics.
''-'"'
Lncolizntion of k e y amino ncids determining I-V re?fitionshjp% 07-35-03A single amino acid difference in lthc GluR-R suhunit dctcrmincs thc I-V rclatirinshipt of hctcromerict AMPA-selcctivc NMDA rcccptorchanncls'". Thc putativc transmcmhranc domain M 2 scqucncc is idunticnl in cach of the GluR-A tn -D suhtypcs, with the cxccptiun that Gh1H-B has a pnsitively chargccl argininc (Arg, R) rcsiduc in sa prisition 5x6 (cf. thc ncutral glutaminc [Gln, Q) rcsiduc n t aa 586 in GluR-A, GluR-C and GluRD. Exchangu of Arg58h GlnfiM in GluR-R and a correspnndmg Gln 4 Arg txchangc in GluR-D hy sitc-dircctcd rnutagcncsist rcvcrws thc shapcs of the I-V curvcst cvokcd by glutamatc in thcsc channcls formed by the hnrnomcrict suhunits"' (see Fig. 4 )
-.
Dose-response Routex nf Cn2'-influx dependent Purkin je cells
OR
ogonist mncentrntims in
07-36-01: Inn channcls integral to non-NMDA rcccptors on immaturc Purkinic cells [%tO-day-ald rats) nrc pcrmcahlc to Ca?', Nn" and Cr?' Incruascs in I&* 1, induccd hy rplotivelv Iriwcr oxonis[ conuentrrilrms arc larguly dcpcndcnt on Ca2+-influxthrough voltage-scnsitivc Ca2+channcls, which arc thcrnsclvus nctivatcd hy a largc Na*-influx. Hi&r concenrrritinns of agonists dnsc-depcndcnt!y incrcasc [C?'], (untlcr ctmditions in which activation of vciltagc-dcpcndcnt Ca?' channcls and NMDA channcls arc hlockcd), intlicat mg a Ca' "-influx thrnugh t hu Ron-NMDA rcccptt~r-chnnncl'".
'".
D t f f c r e n t ~ asensitivity l to eihr~ntlldependent on uppllrd cz~onisr concenfrotion 07-36-02: KAIN: Rcspc~nscs prr)rluccrl by low or high conccntrations of kainatc arc diffcrcnt~aflyinhihitctI by acutc cxposuac of kainatc rcccptnrs tn ethanol whcn cxprcsscrl from rat hippoca~npal nlRNA In oocytcs '". For cxamplt, 50 mM ethanol inhih~ts 1L.5 I I M kainatc rcsponscs hy 45% comparcrl to only 15% inhihitinn nf 400 I I M kainatc rcspnnscs. Ry contrast, acutc cthanol cxposurc inhihits response4 stimulatctl by low and h ~ g h cvnccntmtlons o f N-methyl-11-aspartatcto B sirn~lart!cgrccl". Note. For an ~Ilustrationof thc rclativc potentiating effects on GARA,-~ncdiatcd C1 -flux
high [Ca7'l
high I M ~ ' ' ]
-
-
I-
-
Figure 4. Compr~rrsnnn/ elcctrtlphy~E'olo~icnl properties lrom horntlrneriu ~C:I~~K-r.h(lnnelq c-onto~nin.~ Q, R clr N res~ducsat thc Q/I< site. Top row. P h n V ~ P W nl Q / R sl!r positinn m pcntnmcriu nrmn,Tcrncnt o f cllnnnel ~~rhunrts. M ~ d d l c row. Typicr~E I-V selotionship~. Ifottnm rt~w: TyprcoE whole-cell c u r r r n t a c'll'rjtrd h y glntarnatc under t11e strrted ~xtrrrccllu~lor icln~uuonillr~onsIIrrr ,700 m ~ Note: . The experrrnenfr~lf u l ? ~ t r t ~ ~ to?fnon n osporagine (N) In! n t hc gl:~atomine/arginine(Q/R)site (I! AM PA receptor s u k ~ ~ n gcnPrrilec ~r+ chflnnrlc drsplrrying ri crlectrve prrmeohility for ca7' ovrr M
-
and the depressive effects on NMDA-, kainate- and voltage-gated Ca"-flux, see F i g 5 under ELG CI GARRA.
Selective responses from iGluR populotions dependent on n p n i s t dose 07-36-03: In dorsal horn neurones, where mixed suhtypcs of glutamate receptors are expressed, Rlutamatc responses a t concentrations Iess then 3 J ~ M are due exclusively to NMDA receptor activation"'. At higher ,yhtamnte concentrations, intraccllular responses are mediated by both NMDA and non-NMDA receptors"'.
Inactivation Ph ysiologicnl roles of desensitization 07-37-01: Ionotropic rcccptnr desensitizatinnt properties help govern the strength of fast excitatnry synaptic transmission in the brain. Under equilihriurn conditions, >9Q% of available receptors are desensitizedt, although thcir affinity to glutamate is much higher than that measured prior to dcsensitizationt lm.Desensitization at AMPAlkainate receptors has been proposcd to contribute tn the Iast decay of excttatoryt synaptic currents.
Desensitization kinesics 07-37-02: Kainate receptors g e n e r d y desensitize$ only 'extremely slowly', whereas AMPA receptors (with rare exceptions, see rcf.'2'\ undergo this transition relatively rapidly and in a concentration-dcpendent manner (,we examples bclow). Note: Kainate has also heen shnwn to cause rapid desensitizationt of hornorncrict channcls cxprcssed from subunit GluR6.
Utility of 'absolute selective block' of mpjd desensitization for AMI'A channels by cyclothiazide 07-37-03:Potentiation by cyclothiazide (CYZl of recomhinant glutamate reccptur rcsponscs via an allosteric Hock of rapid desensitization shows absolute selectivity for GhRlLGluR4 (AMPA] receptors when expressed in X U I Q ~ U Soocytest1.Rapid dcsensitization in HEK-29.7 cells transfected with AMPA receptors is also Mocked hy CYZ, but is nnly weakly attenuated hy concanavalin A (Con A). Conversely, desensitization+ a t kainate receptors IGhRS-GluR7 and KA-l/FCA-21 is hlocked hy Con A hut unaffected by CYZ". Note: Cyclnthiazide is a henzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide.
CYZJCon A-sensitivity phenotypes can report iGluR subunit assembly patterns in viva 07-37-04: Nativet cell types shown to predominantly cxpress kainatepreferring subunits (e.g dorsal root ganglion ncuwnes, mainly expressing GluRS) show an expected Con A-SensitiveJCYZ-iRsensi~ive phenotype". Conversely, nativet cell types which preferentially exprcss AMPApreferring subunits leg. hippocampal ncurones) display a CYZ-sensitive/ Con A-insensitive phenotype. Table R summarizes findings of comparative studies on nativet ccl! preparations,
entry 07
Table 8. Cyclothinzide ( F Y Z ] vrrwq i+oni*nnuvolinA (Con A ) sensifivity phentitypcF /nnr AMPA-ceki*tivr iGlwR-ohonnelc exprcwcd in native neiirtines (From 07-,37-04)
Preparation
Cyclothiazirlt. phcnotypc
Rcfs
Hippocam pal spinv 'mossy cclls' versus aspiny hilar in t u r n curones
A greater sensittvity tn cyclothiazide in hippocampal spiny 'mnwy cells' v e r w s aspiny hilar intcrncuroneq has heen reported [with half-maximal rcmoval of desensitization hcing 90 WIM and 200 mM, rcspectivel yl
122
Hippocam pal SliGUS, glutamcrgic neurmes responding tn glutamatu
Cyclothiazide ( C Y Z )reduces rapid desensitization, enhancsn): thc steady-mtc and peak current pOduGL't! by 1 r n quisqualate ~ with EC5nvalues of 14 and 12 I'M rcspcctivcly. CYZ causcs glutarnatc to induce long h u r w of channel openings, and greatly increases the numher of repcatcd npenrngs. At 110 I'M CYZ docs not have mcasurahle cffects on the fast componcnt of dcactivation nor docs i t h a w statistically significant clfccts a n thc distributirm (if the faster campancnts of glutamate-~nduccdhurst duration
Hippocam pal neuron es rtspnnding tn kainatc
Responses of hnppocampal neurones to kainatc are strongIy pntcntiatcdt (3100%)hy cyclathiazidc, which is cnnsiderahly rnorc effective ['cnmpletc hlock nf descnsitrzatinn') than annracetam in rurlucing dcscnsitization evnked hy glutamate
Dorsal root ganglion neurnnes compared with h ippncampa 1 neurones
Cyclothiazidc cnmplctcly blocks dcscnsitization produccd hv 5-chlnrowillardiine in hippocampal neurnncs and strongly potcntiatcs rcsponscs to kainatc (thc action of aniracctam is similar hut much wcakcr). In DRG ncuroncs, cyclothiazidc and aniracctam has no cffcct on dcsunsitization hut prtduccs wuak Inhibition of rcspcmsc~to kainate
Cultured ccrehcllar glial cclls (aligodcndnicy t L' lincagc, 0 - 2 A progenitors]
T w n receptor populations arc prcsent in thcsc culls, with high and low affinity for kainatu shriwing ditfcrcnt sensitivity for potvntiatien hy concanavalin A and for hlock desensitization of cyclothiazidc
'"
A mechanistic basis for kainate subunit-selective phenotypes of concnnavdin A 07-37-05: Cnncanavalin A (ConA) is a lectin which can hind to glycosylatedt mernhrane pmteins with high affinity. Expression of rccomhinant iGluR subunits which display II higher prnpnrtinn of glycosylatedt N-termini [extra-
cntry 07
cellular\ might bc thcrcfore expected tn hind Con A with grcatcr affinity. Although both AMPA and karnatc cDNAs show N-glycosylation motifs (see Seqiience motifs, Q7-24)treatmcnt of glycosylatcd G l u M [ kainatcprcferring) with N-glycosidase" induces a 13 kDa shift in M, comparcd with a shift of only 5-6 kDa for AMPA-preferring suhunits'"~FZ".Thcsc analyses sugqcst kainate-prcfcrring iGluRs are glycnsylatcd to a grcatur extent than AMPA suhunits, and may thcrcforc explain thc highcr sensitivity of the kainatc suhunits to lectins like Con A". Note: Thc flip/ flnp variants (SEC helmv) show no apparcnt diffcrcnces in sensitivity tn concanavalin A.
Desensitizing effects of cyclothiazide vary in fliplflop splice varinnts of AMI'A receptors 07-37-06: Although the molccular basis for 'absolute selective hlnck' of rapid
dcscnsitization in AMPA suhunit channels IS unknown, the 'flop' splice variants show much less potentiatinn hy cyclothiazide (22 f 4-fold fnr gkutamatc responses, 4.2 f Q.7-fold for kainatc rcspcmscs) than their 'flip' variants (130 80-fdd for glutamatc responses, 12.4 f 2.7-fold for kainntc rcsponsus). Similar properties are ahserved with tliplflop variants in hctcrorneric comhinatinns (e.g. GluR-A, + GluR-R, V V ~ S U SGluR-A,, + GluRRJ. Thcsc results are consistent with a rolc for thc Fliplflop locus in regulating desensitizationt (see helow nnd rc(-i''* "3.
+
Desensitizatinn plateflux in alternative splice vorionts flip and flop 07-37-07: AMPA: . - -- Upnn fast applicatian, glutamatc clicits currents at AMPA rcccptorqhanncls which exhihit a fast rise timet and then dccay to a plateau value in the continucd prcscnce of agonist. Thc platcnu i s morc prtinounccd with flip- than flop-containing GluRs (see Gene orgnnizatinn, 07-20), Thus thc differing dcsensitization kinctics shown by receptors containing flip and flop mtidulcs a k c t thc 'peak : steady state' cnmponcnt nf iGluR forrncd from AMPA-prcfurring GluK-A-GluR-D suhunm. Note: Kainatc cvokes dcntical nondesensitizingt currents in hnth flip- and Ilnpcontaining GluR rhanncls formcd from thc GluR-A to -D classes.
Moda 10 t ion n/ desensit izn t inn 07-37-08: The nnotropict drng miracetarn, wheat germ agglutinin, and concanavalin k act via separate mechanisms tn rcducc ricscnsitizatinn evnked hy L-glutamntc in rat hippocam pal neurnncs'". Thc dccny of cxcitntciry synnptic currents, and miniaturc excitatory post-synaptic currentst (EPSCsJ cvnkcd hy sucrose arc slowcd 2- ta Xfold hy aniracctarn. Animcctaiii also incrcascs the rnagnitudc of glutamatc-cvokcd EPSCs 1 .Pfold, prnhahly via a posl-svnnpric mcchnnism (if act inn. Aniracctnm increases the hurst lengthf and peak amplitudest of Lglutamatc-activatcd singlc-channel rcsponscs'". Simulations suggcst that sniracctam eithcr sl(iws cntry into a rlcscnsitizcd statct or dccrcascs thc closing rate cnnstantt for ion channcl gating1 . Cornpnrrrtive note: Whcat germ amlutinrn and cnncanavalin A ruduce EPSC smplituJc via a prcFynrrptrc rncchanism (we helow). Diazoxide cnn also rcducc dcsensitizationi of ~iippocnmpa~ AMPA rcccptors to AMPA, glutainatc and q uisq ualatc.
"'
llr(fcrslnt nr~trorrrrl~.r~?ls show tlif/crcn! nrtcs d:.rensSt1zr1tion
o(
rr'crwrry f r r ~ r ? ~
07-37-09: In nativc rlcnt:~tc EyrLls, hippocampal CA.3 and CAI ccll pntchcs a p p l ~ c a t ~ o nofs I m h ~cliitarnato of 10I1 nls r1ur;ltkon sht)w tltnc constants f for dcscnsitization of L1.4 2. 7, *I 1.3 t 2 H, and 9.3 i 2 H t n y ruspcct~vuly'14.
Dcscnsit~zntinntime c o n ~ t a n t s inru only wc:tkly dcpcndcnt on glutamatc conccntratlon (200 /rM ;ind 1 r n ~ lfor thc thrcc ccll tvpcs. Urluhlc pulse :~pplication.;of glutnm:~tcinrllcatc that 1 ms pulse of 1 inM glut;unatc cause part la1 GluR c11;lnnt.l rlcccn5itiz;ltlon~(-h(l'X,). Thu tfmc colrrqc ot rrTcovrrv from rlcscnsitizaticln I S xl(~wcrin rlcnzatc E Y T I ~ Sgran~rlcccll pi~tchcsthan in CA.7 or CAI pyr;~mirlalccll p3tclrcs1t'. N o t r . Sprci311~0tfIGILIRS~nctliatrng cnmplcx auditory information in cochlcnr nciironcq
Ilecrrp time ct?rzstmn~ < rlrr ~ndepcndenrof mcr~~hronc potcnticrl tin(? ngoni
Selectivity Principr~lionic select i v ~ t vrl~ffrrcncecbc t wern iGlu1Z-channels 07-40-01: AMPA/kain:~tc rcccpror-c11;lnncls in nativc tiswcs havc ~ ~ s u n l l v hccn cln.;sifictl a s nicrlr:~tinl:;in influx c ~ frnnnovalcnt catinns (in contr;lst to NMI>A rcccptors whicll nrc thought t o nicrl~:ltc thcir physicdo~ical rcsprmw rnainly throligh thu ~nflux of cxtmc.cllular c a l c ~ u m ) .M(~wt.vcr, I J ; "-sclucttvitv ~ 15 now known to hc infliicncc~Zh v ~ ~ ~ h composltlon u n ~ t l>r~lowl. Thc Ilcrnlcatlon pathways of a r:ingc of nuurr,transrn~ttcr-gatutl ion chnnncls h:ls hccn rcv~cwctl"". (rrpr,
TIT(.C J u K - H ~ u h t r n ~c1r)tnirzr~~c.s t propcrtir~ot JonIc tlow 67-40-02: AMPA: -~ t t c r o i n t . r i c t A M PA rcccptor~ cantazniny: thu GluR-ll subunit display law divalent Eon pcrmeahilities. Howcvur, rccrlmhinanti AMI'A roccptorr lacking the GltiR-R subunit are ca2+-permeable n t physiulogical C;J" coilccntratlorlr [ c . ~CiluR-A, . C;luIl-C or GluR-A t CiluRC In ~ ~ > n ~ h ~ i ~ .H ~C 't~ ~C ~c Ol I nO ~~~C~LXI~~F~~~C S~ S )I I.ofI ~ CIZIR-RmRNA prcrnlxcrl In diffcrcnt tnnlar mtios with rnRNAs cncodlng GluIl-A, GluR-C or GluR-12 sliow ;i I,~rxcr;lnglh 01 ~Ii~t;~matc-;~ctivatcrl (:n"-pvrtncnhil~t~cs1llfl .
Srdc-chhirrrlciza ir~ltichr~r,yerrffcct dfvr~Irtr1j?c>rrncrihilityrn rccomhir7ont C:?FII<~ 67-40-03: AM PA: Followln~: cxprrs.;ir)n 111 W EK-29-3 cclls, hclrnornrrrct aswmhlic.; of C - ~ ~ I R - R ( S Xchanncls ~RF (rcr I?omrrrn C~rnc*tionc, 07-20) display
perrneabilityt, whereas homomcric GluR-B(fiR6Qj and GluR-D channcls exhihit a high divalent permeabilityt'8". Mutational analysis has shown hoth the positive charge and the side-chain size of the aminn acid located at the Q / R site (position 586) control the divalent permeahility of homometic channels. Changes in GEuR-B(586R) expressinn are therefore capahle of regdating the AMPA receptor-dependent divalent permeability of a ceI~"""". a Inw divalent
Ca2+-infhxthrough I I Q ~ V M Dreceptors A in native tissues 07-4044: Ca2+currents through native mammalian non-NMDA channels
h a w hccn reported in retinal bipolar cellsr3o, hippocampal neuroncs'"', ) ~ 'type-2 ~ ~ ~ astrncyacs (when Rcrgmann glial cells ( G I ~ R - A / G ~ U R - Rand activated hy kainate, see refs m"").(See (ilso Cell-type cxprcssron index. 07-08, nnd Dose-response, 07-36,)
Differences in Ca2+-perrneabilitydeserrninnnts /or kninate receptorchnnneh 07-40-05: KAIN; In general, the Ca"-pcrrneahility of kainic acid-gated receptnr-channels is governed hy their subunit composition'' and this may hc developmentally regulated. In GluRA homomeric channels, the presence (if IgEutnminc] in thc domain M2 Q/R site produces channels with low Ca"-pcrmcahility (in contrast with AMPA rcccptor-channels - for delni1-i. SCP rilcn Domain functron?,07-29).
u
Separate determinants ni rectificnrion ond divalent ion mobility in selectivity filters 07-40-06: Different amino acid rcsidues control (i)the ability to pass outward current (rectificationt properties] and ( i i J divalent ion mobility (pcrrncahilityj properties] in the selectivity filter1 of GluR-C and GluR6 nm-NMDA . Mutagenesis at (or near) the QIR site in GluR-C indicates that li) the position of the argininc is critical to function and ( i i ] thc ability to pass outward current is not neccssarify linkcd to low harium permeability.
lielnntive Ca" fraction through nntive NMDA ond non-NMDA rCCEpfoT-ChflnTl@lS
07-40-07: The Ca2+fractinn of the ion current flowing through ghitamatergic
NMDA and AMPAlkainatc receptor-channels has hcen compared directly in forchrain neuroncs of thc medial stptum'". A fractional CaL+current nt I .4% was dctcrmined for the linearly conducting AMPAJkainate reccptor-channels fnund in these neurones137.In comparison, at ntgativc mcrnhranc pntcntrals ) Ca" fraction of thc (cxtraccllular free Ca2+ concentration of 1.6 m ~ the current through thc NMDA rcccptor-channcls is -6.8%, or -2-foM lawcr than prcvirwsly cstirnntcd from rcvcrsal pntcntialt muasurcmunts.
hlovel jrnrnunochernicol ossays detect ins tiivnlenr inn-perrncnhlc koinnte or AMPA receptors 07-40-08: Glutamate analogues stimulate uptakc of cobalt ion into ncuronal
cells in cell culture or tissue sliccs. Sincc CO" -permcable channels arc also Ca"-permcahle, the precipitahle Cnz* ha? hccn osed to quantitatc agnnist
Table 9. General features relevant to the conductance states of A M P A - and kainate-selective iGluRs (From 07-41-01) iGluR type
Features
Refs
AMPA channels
Distinct single-channel conductances were determined for non-NMDA receptors for (inhibitory) aspiny interactions (27 pS) compared with (excitatory) pyramidal neurones (9 pS) (see Cell-type expression index, 07-08).In cerebellar neurones, multiple conductance states are observed with quisqualate and kainate agonists (mainly below 20 pS with the predominant conductance state for AMPA channels being 8 pS). A rapidly inactivating, high-conductance state (- 3 ms, 35 pS) associated with the fast (quisqualate)receptor-mediated EPSCt has been described.
:138 &-
-
-
Kainate gates primarily low-conductance channels in neurones of the hippocampus, spinal cord, cortex and cerebellum, e.g. outside-out patches from these membranes display a principal conductance of 4 pS with an open time of 0.5-3 ms for kainate agonists. Noise analysis indicates that kainate may activate a 140 fS channel in some preparations High-conductance High-conductance non-NMDA channels, such as the 10-30 pS glutamate receptor-channel previously channels in granule characterized in granule cells, carry the majority of the fast component of the EPSCt at the cerebellar cells mossy fibre-granule cell synapse. Low numbers (- 10) of non-NMDAR-channels appear to be activated by a single packet of transmitter. Conductance estimates for the non-NMDA receptor component of synaptic currents activated during EPSCs at this synapse show a mean single-channel conductance of approximately 20 pS Analysis of whole-cell noise in the presence of Mg" induced by glutamate, quisqualate and kainate in Low-conductance channels in goldfish the retinal horizontal cells of the goldfish indicate all three agonists activate channels with a retinal horizontal conductance of 2.5-3 pS. Note: Vertebrate retinal horizontal cells appear to lack NMDA receptors cells Kainate channels
-
Affinity-purified, reconstituted iGluRs
-
Excitatory amino acid receptor proteins purified from Xenopus central nervous system using domoate affinity columns followed by reconstitution into lipid bilayers exhibit variable single open channel conductance levels depending on agonists used to elicit current. These have been measured as 6 pS with AMPA, 9 pS with kainate, and 50 pS with NMDA. Occasionally, unitaryt channel openings of up to 400 pS are observed, suggesting that reconstituted receptors may form functional aggregates
-
-
-
i:; 139
140
141
'4j
0
affinities. Three types nf Coz+-permcablekainate rcceptors have been defined using these assays: K1 (activated by kainate alcme), K2 (by glutamate and kainate) and K3 (by kainate, glutamate and quisqualate)'=.
1
Single-channel data
AMPA- and kainate-activated channels have distinct conductances 07-41-01: A number of general features relevant to the conductance states of AMPA- and kainatc-sclcctivc iGluRs arc sumrnarizcd in Table 9. A minority of studies havc conccntratcd on dctcrmination of sinxle-channel propcrtics under dcfincd conditions, so only a fcw examples arc quotcd in the table.
Blockers Open-channel blockers isolated from spider venom
07-43-01: A component of the spider venom from drgrope lohatn, argiatoxin, has hccn characterized as a n antagonist of hornomerict and hctcromerict glutamate-activated receptor-channels. Argiotoxm acts as an open-channel blocker in a voltage-dependent manner and discriminatcs ~ . dctcrminant in the M2 domain for between AMPA r ~ c c p t o r s ' ~ A divalcnt cation pcrmcahility also dctcrmincs argiotoxin sensitivity (see Selectrvity. 07-40), Subunit-spccific diffcrcnccs in tirnc courscs of argiotoxin block and rccovcry demonstrate that hetcromeric AMPA receptors can assemble in variable ratios".'. Notably, the spider venom toxins argiotoxin and lorn spider toxin (see below) have higher potency at NMDA r c c e p t o r ~ ' ~ ~ . /om spider toxin hinds to the pore domain of iGluR subunits nt a glu tnrnine residue 07-43-02Jom spider toxin [JsTx)is a potent non-NMDA receptor antagonist which can cxcrt A subunit-specific Mock at suhrnfcremolar conccntrations'"5'. Rcccptor suhunits with rectibying L Y relationships (GluRI, GluR3, GluR4 and GluR1/3] are reversibly hlocked by JsTx. Receptor subunits forming a receptor-channel with a linear I-V relationship (GluR1/ 2 , GluR2J3 and Gl~iRh]arc not affcctcd. JsTx hinds closc to the central pore region of thc channel - a single amino acid position (a G h - 5 8 6 a t thc U/R sircl appcars critical for the JsTx h l o ~ k " ~(SCE Current-voltage refnlion, 07-35 and Selectivity, 07-40), Note: Philanthotoxin is also known to block a Drosopoyhiln kainate-selective glutamate receptor-ch anncl "'.
Channel modulation Modulntion of GluR qyonist responses hy nootropic drugs 07-44-01: Micromolar concentrations of piracetam, aniracetam and oxiracetarn cnhancc AMPA-stimulated Ca"' influx in primary cultures af ccrchetlargranulc ~clls'~'.Such nootropict drugs incrcasc the cfhicacyT hut not thc potcncyt of AMPA, and thcir action pcrsists in thc prcscncc of thc voltage-scnsitivc
c a l c l u n ~channel h1~1ckt.rnifedipine. I'iracutam, anrracctam and clxlracctarn lncrcasc thc i n ~ x i ~ r m dcnsity ~l r d thc spccif~ch~ndingsttcs for ['HI-AMPA in .synaptic rncrnhr;~ncsfrom mt ccrcl,ral cortcxf4'. Anjrncctaln ( I - p - ~ n i s t ~ y l - 2 pyrrolldinnncf allostcricallyi potcnt1:ltcs ionotropic quisqualatr (iQA) rcsponscs t n d ~ ~ c c~n t l Xrntlpr~vorlcvtc5 cxprcssctf from r;lt I~rainm R N A In a rcvursihlc ~nanncr"* /vr'n mlco Inrlc-r~virr;r,n. 117-.37).
07-44-02: N o l c . Fi~rtlicr~ . x i ~ n i p lof c s1GIuK channvl modulation arc ilcscrihcd ~intlcrI'hunc~fvl~ic. rpxprcvsrc>tl,07-F4 onrj /'rot crn p h n ~ p h o r v l r ~on, r 07-,?2
Equilibrium dissociation constant !dish-nffinrtykrrr~lrrtchlnding sites in nolive t i ~ s u c s 07-45-01: KAIN: Kninntc-binding sitcs that d8ffi.r from high-affinity AMPARintlinc, srtcq h ~ v uhccn tduntlticrl hy T~gnnri-hntlings t u d ~ ~'Class~cal' ~ ' ~ ~ . high-affinity kainatc sitrs (K,i 5 anrl 50 n u kalnatc) exist in t l ~ cCA,? arca of the hippocampal forrnatnon. -+
Sirnilflr ngonrsi nffi~lrtics for nrrtrvr! rind recomhinont krilnntc? reueplors 07-45-02: Thc '~~hartnacolngica1proftlc' of cxprcsqcd rccomhinant KA-1 ~dctcrmincttin h i n t l ~ n cxpcrimcnts ~, with I 'HI-kainatc) differs from that of t h c clonctl AMPA rcccptors, hut is sitii~larto thc mammalian high-affinsty rcLcptor (kainatc > quisilualstc > glutamatc b' AMPA, whcrc kainntc K,, ,k.l,n.l,rl 1% -5 nnh5) In t-onip,lristln, thc inhil~itoryconstant ( K , ] v~lzlusfor cliusq~~alatc,I -glut;lmatc ;tnt! AMI'A arc. I X 200 snd 5000 I I M r ~ s ~ ~ . ~ t ~~'$>tr. v ~ . Rccomhinnnt l~". KA-2 s u h u n ~ t sd o not form channcls In h ( ~ ~ n n m u l t i i n c rI. ;~~t uxh~hrt t h ~ g hnfftniby fclr k a i n ~ t u[KLI 15 ~ I M ) " .
-
-
Incroriserl' mgon i f ! cdfinl! ips oh~rrvrdfor purified iGI11li 07-45-03: AMPA KLIY;IIP~CS havc Iwcn ohscrvctl to hccomc ' ~ n u c hIowcr' upon various iiltii~l~r;inu trc;ltmcnts ant1 followlnr: protcin pllrif~cat~on (r;r.r, Prorcin rrit~,ri/r[ion\,07-&?
K,, volrlr's for rlnltrfrvt rocrpIorc 07-45-04: In Xr-t~oprrvI~rain,kainatc- and A M P A - h ~ n d i ns ~ t c co-cxist s En a I : l ratin snrl crrnnot I>c vcprinrt r ~ r lhy physical a n d chcmtcnl f r,rctionatir~ns*',~~ ( S C ~ ,S ~ i h f j , ~pE~~ I S C I ~ C ( I ~ I O I ~(17-0(>). S, In thcsc protcins, AMPA and kainatc arc mutually and fully competitive+, wlth K , valucs idcntlcn! t o the K,, valucs for thc radioligand (AMI'A, 34 nM: kainatc, 1.5 n ~ ]Channcls . rcconst~tutctlrn hilaycrs can clicit currcnts (sirnl1;lr to natlvc non-NMDA GI~IRsFin rcsponsc to low lcvcls o f h M P h o r kainntc.
Cillrtmmr~tc-. AMPA- r~ndkminr~tc-bindingsltcs 07-47-01: From pharrnactdogical considcratinns, L - ~ l u t a m a t c kainatc , and AMPh hind to tirffcrcnt reccptnr suhntructures on rccc~nhinant AMPA rcccPtors'"" Rcccptnr I,~nd~ng/nuzr~rarl~ographic appn)nchcs to chnrnqtcrizat~rlnof cxcltatnry n n ~ i n oscitl rcccptnrs hnvc hccn rcvicwcd"".
cntry 07
Avniln hle radiolignnds 07-47-02 AMPA: [3AI-AMPA and i3H1-CNQX. Use of AMPA or CNQX can define the nnn-specific binding of ["HI-glutamate. Note: CNQX also hinds to thc glycine site and possibly thc NMDA iGhR. Radiotigands for kainatc sitcs include f3H]-kainate and [3H]domoate (but note the hezcrogcncIty sf domoatc-affinity purified products - see SingIe-channeldata. 07-4 11.
Affinity purificatinn of A M P A receptor-channels using /'H]-AMPA and lor0 spider toxin 07-47-03:A glutamate reccptor has hecn pusificd from Triton X-10fl-solubilized hovtne ccrehehm rnemhranes hy affinity chromatography using a spider toxin 1Jorospider toxin; JsTx,irnmohilizcd on a lysinc-agarosc column followed by a Mrmo Q anion exchange columnlr5".The active fraction purifies an AMRAbinding prnteirt of M, 130 kDa. Lineweaver-Rurkt plots indicatc thc protcin to h a w a K,f of 12.7 n M [%]-AMPA in the purificd fraction. In rccnnstituted liposomes, the purified protcin yields a glutarnatc-activated channel which can bc inhibited with JsTx"" (sce Blockers. 07-43),
-
ReceptorJtransducer interastinns Involvement of second messenxers in excitatory nrnjno acid signal transduct ion processes 07-49-01: Excitatory amino acids {EAAJactivate second rncssengcrt systems via rnotabntropict receptors i n nddirion to the dircct gatinx of 'intcgral' Thraugh these (ionotroyict ) rcccptor-channcls (rcvrcwed in ref. 15'). 'indirect' rnetabotmpic pathways, EAAs are capable of activatmg lmth adenylate cyclase and guanylate cyclase and also to induce phosphoinositidet 11'1) turnover (see, for exomple, Fix. 4 rmder ELL' CATULU NMIlA crnd tnhScs in Resource A - G protern-linked receptors. entry (56,
Calmoddin-dependent inhibition of post -synoprfc vnltagc-gnted co2+currents 07-49-02: Glutamate-evokcd Ca2'-influx through both NMDA and nnnNMaA rcceptor-channcls in rat hypothalamic ncurnncs inhibits highvoltage-activated {WAJ Ca2* channels (see VLG ~ n entry , 42) via a ca3modulin-dependcnt mechanism 52. A pee-synaptic glutamate receptor agonist (L-2-aminQ4-phosphonobutyricacid1 and a sclcctive rnctabotropiq agonist (trans-ACPD) arc ineffective in mimicking the W A Ca" current inhibition prumoted hy glutamate. Inhibition IS also dependent on the presence of extracellular Ca2+,and can hc hlocked hy internal perfusinn of thc cclls with BAPTA. The calmodulin antagonists tdfluoperazine and qalrni-
dazolium completely prevent the inhibition'".
Links bet ween Glu R 1 a,qonism and hormone secretion by heterologous gene expression 07-49-03: Co-expressinnf of a plasmid1 construct encoding growth hormone and a plasmid cncoding a non-NMDA glutamate reccptor, GluRI, yields
chmmaffin cells in which Ca"-depcndcnt growth homonc sccrction can ho stimulatcd hy kainate.
entry 07
Receptor agonists (selective) 07-50-01: Nntr. Availability of sclcctivc antagonists ' and agonislts havc heen central to the rccngnition nf receptor suhtypcs underlying nativef iGluR rcspnnws. Thc hasic fcaturcs of thcsc and nthcr agonists are listed in Tahle 10.
Agonist nffiniiies of recombinant GIuRs 07-50-02: Rcccptors generated from thc GluRl to GluR4 c D N A s ~have higher apparent affinitv for AMPA than for kainatc. Whcn homomerici ruccptora o f thc GluRh class arc cxprcssctl: in Xcnopris mcytes, thc rcccptors are activated by kainatc, quisqualatc and L-glutamate, hut not by AMPA. Furthcrmorc, the apparent affinity for kainate IS higher than for
rcccptors from thc GluR I-61uR4 class2.
ECsn vnlues for I G ~ F ngonists R 03-50-03: Typical EC5[, values fnr recornhinantf GluR-AJR receptors cxprcsscd in oocytcs arc 3.,3l S I M (AMPA); 6.16 I I M Ifilatamatc]; 57.5 / I M (kamatc)"'". ECqIFvalucs for rccomhinant GluR-R/D receptors expressed in cincytcs arc 5.01 prvr (AMPA];32.3 (IM (glutamate); 64.6I'M /kainatelfM.
Agonist riffinities and potencies ot GluX-channel splice variants 07-50-04: Gcnc cxpressnon cnntrnl of thc altcmativc splice variants 'flip' and 'Flop' (ser Gene ciyyunirriiion, 07-20] can confer different kinetic properties on thc currcnts cvtikcd hv the aRnnists glutamatc or AMPA, but not on thosc c w k c d hy kainatc. For both '(lip' and 'hp' vcrsions (if GluR-A to GluR-D, kmnatc cvrA-5 a nrm-tlcscnsitrzingt current whereas both glutamate and AMPA C ~ W C :in initi;il bst-duscnsitizingt current followcrl by a steadystate plateau Whuicss glutamate, AMPA .~ndkainatc cvtikc currents cif similar amplitiirlu in 'flip'-cxprcssinK cclls, kainatc-cvokcd currents arc much largcr than thnrc cvokcd hy gI iitamatc or AMI'A in rflop'-cxpress~ng cclls Clutsmatc sctivntcs channels 4-s-timcq mow cffcctivch whcn acting at the 'flrp' vrrsion of thc GluRs2".
Receptor antagonists 07-51-01; Scvcral stutiics havc indicated that antagonism at NMDA receptors (.we E i L CAT GLU N M D A . entry 08) is only partially protective in stimc r n ( i h ! s of local ischaemia and may hc ineffective in global ischaemia (scc Phencuyyiic vxpressrrm. 07-74 nnd disciissjnn m ref 1571. Dcvclopmcnt (if AMI'AJkainatc-sclcct ivc antagonists (particularly NRQX a n d t h n w nf the 2,3-hcnzodiazcprnc class) havc indicated thcir valiuc as 'neumpmtective' and anti-cnnvulsant agents. Thc hnsw propcrtics of these nntnpnists art' listcd in Tnblc 1 1 in coinparison with rjthcr (less-sclcctivc) nnt:igrmists in u s c
Table 10. Common agonists of AMPAlkainate receptors and their features (From 07-50-01) Agonist Features
Principal agonists AMPA Kainate
Willardiines and bromo-/chloroderivatives
For constitution of 'AMPA-preferring' versus 'kainate-preferring' iGluRs, see Gene family, 07-05. For reported activities of AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), see paragraphs below this table and other fields prefixed with 'AMPA:' Kainic acid (kainate)is a full, non-desensitizingt agonist at subsets of iGluR assemblies (see 07-05). A functional kainate receptor has been cloned which is insensitive t o quisqualate/AMPA. Conductance responses evoked by kainate at the GluR channels are competitive with those evoked by AMPA and are not additive. Note: Domoate has also been used as a kainate receptor agonist and affinity ligand (eg. see Table 9). For further activities of these agonists, see paragraphs below this table and other fields prefixed with KAIN: The (S)-but not (R)-isomersof the naturally occurring heterocyclic excitatory amino acid willardiine and 5bromowillardiine are potent agonists for AMPA/kainate receptors. Willarhine [( S)-1-( 2-amino-2-carboxyethyl)pyrimidine-2,4-dione] produces rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine(ECS0,1.5 p ~is ) 7 times more potentt than ( R , S)-AMPA(ECS0,11 p ~and ) 30 times more potentt than willardiine (ECS0,45 P M ) ~ ~ 'Note: . Willardiines are the first compounds characterized in which simple substituent changes in molecular structure are associated with marked ddferences in the ability of agonists to produce desensitization of AMPA/kainate receptors L-Glutamate and L-aspartate are mixed agonistst of the AMPA-, kainate- and NMDA-selective receptors and their effects are partially inhibited by all selective antagonists. In dorsal horn neurones of the rat spinal cord, millimolar concentrations of L-proline elicit an inward current that is partially antagonized by strychnine, APV and CNQX. Thus, L-proline is a weak agonist at strychnine-sensitive glycine receptors and at both NMDA and non-NMDA glutamate receptors. The ability of L-proline to stimulate CNQX-sensitive Ca2+-entryfollowing activation of excitatory amino acid receptors implicates L-proline as a potential endogenous excitotoxin Quisqualate (QUIS)was formerly used as a principal agonist for AMPA receptors, but it has also been shown to be an agonist at metabotropict glutamate receptors. N
Mixed agonists L-Glutamate L- Aspartate L-Proline
Quisqualate
Amino toxins of plant origin BOAA
Refs 161
16'
N
(BOAA)is associated with incidence of neuroThe amino acid toxin p-N-oxalylamino-L-alanine lathyrism (a spastic disorder with acute and chronic onset) associated with consumption of the chick pea, Lathyrus sativus. BOAA appears to act as an excitant on spinal neurones via agonist activity at AMPA receptors
163
82,174
rM Compctittvc antagonists with diffurcntml wlcctiviay for rcctmhinant CluIts inclurlc. 6nit r r ~ - ~ - s i i l p h a m c h ~ c n z o - ~ - q r ~ ~ n o x s l i n c - ~ , ~ ~ , - d i o n c INRQX, PA? 7.1 ). Thc potency of NRQX for hlockinr: currcnts mutlintud hy GluR-A/!3 rcccptors c1i;lnRcs dcpcnding [in thc aRoniqt u w d to activatc thc rcccptors (FA7 valucx 7 2-1 1 0 (11 For hlock OF kainntc rcsponscs; 6.78 1 0 02, for hlock of L-gliit;lnintc rcspnnscs; 6.95 ? 11.112 for hlock 0 6 AMI'A rcsponws]. Ihffurcnccs hctwccn sgonists arc. luss inarkud in cells cxprcssing GIuR-R/U rcccptcm (pAJ valucs: 7 2X t 0 01 fnr hlock of karnatc rcsprmscs; 7 -70 t 0 02 for hlock of I--glutamate rtsponsus; 7 3.5 0.01 for hlock of AMPA rcsprmws). NRQX acts a s a potent and sclcctivu antagonist a t rccmnhinant rcccptors, h u t i t 5 action c:m hc ovcrcomc hy increasing a p n i s t conccntratrtm 1i.c. it is. crlmpctrtivct). NHOX has anticonvulsivu propcrtics in scvcral scizurct mrdcls 2nd i s ncuroprtituctivc in brain isch:icmini models. In native mumhrancs NRQX i s 5lHbh)Id inrirc ScIcctivc for A M P A w c r NMl3A ruccptcirx (crmpnrc thc rclntivc nrv-sclcctivitv of CNQX and IFNUX at
+
-5
rcctiiaihin;lnt rcccptors helriw]
T h c homnphthalazinc t / - ]-GYKI-524(16 1 5 a Rcvlcw' highly-sclcctivc AMPAJkainatc antagonist (1C5rl for kainatc 7.51mJ and docs n n t significantly Affect NMDA, n ~ (~ncta~,otropict ~ u glutarnatcl, o r GARA, ruspcinscs. GYKI-52466 hinds t c i hnth opcn and clmcd recuptor-channels, and is vr)ltnRc-indcycndcnt in its antngonistac cfkcts. Thc action of GYK1-5246h cannot hc m e r c n m c h y raising agrmist conccntmtirms [ I . c .i t is noncotnpctitivc I]. GYKI-52466 1s s h a d - s p c c t r u m nntictinvulsnnt. Thc mcthyl-carhamnyl dcrivatlvc GYKI-53655 i s scvcral-fdd more potunt thnn LYICI-52466. Thc 2,3-henzndiazcpinr class of AMPA/kainatc rcccptcir nnncompctltiva antagonists may h a v c therapcutic application.; ~n cpi Icpsy, i rchacniin, ncurorluKcncratmn nnrl Pairkinson's discnw
-
cntry 07
Tahle 11. Continued Antagonist
Lipophilic competitive antogonisis
DDHR and dcnvativcs
InhaInl ionnl anoesthcrios
Enflurane
Rrphenyl clcrivcltive of NDSA Evan4 blue
Features
Rch
In addition to their spccific antagonistic ctfccts 'Oli on neurnnal GluRs, quinoxalinedioncs (c.g. CNQX) have also hccn shown to hhckglutamatcinduccd rcsponscs mediated by recnmhinant A M P A I M rcccptor-channels when expressed in hetcrologoust systcms (c.R.GIuR-A/R and GluRR/D receptors). Antagonism occurs iwcspcctive of the particular subunit composition and displays little sclcctivity hetwccn AMPA and kainate receptors). 6,~-Dini.aroquinoxatine-2,,7-diflnc (DNQXl,a h shows relatively !ow sclcctivity A class of glutamate receptor antagonists that show cornpctitivei action, significant potency at multiple sites, and a high degree of lipophilicizy are the substituted henzazepines. 2,s-DthyrEro2,s-dinxn-3-hydmxy- bI-bcnzazcpinc (DDHR] and three suhstitutcd derivatives, 4-hromo-, 7methyl- and R-rncthyl-DDHR, block thc activation nf non-NMDA rcceptors by kainatc and L-glutamate (see oho Reccptcjr rmto~onrsrs under F I G C A T GL[J N M D A , 08-51) y-Glutamylaminomethyl sulphonate (GAMS) has .". *", been uscd as .a partially selective non-NMUA receptor antagonist which offers pmtcction against audiogcnic seizurcst. Other cornpetitivc antagonists at thc AMPA-binding sitc arc GDEE and DGG Enflunne a t anaesthetic conccntrations ( 1 .X mM) ' 5 7 inh ihits AMPA-, kainate- and NMDA-induccd currcnts exprcssed in oocytcs hy 29-10%, ,1&%3% and 20-27%, rcspcctivcl y. Inhibition by en flurmc is independent of thc cmctntrations of thc agonists [NMDA, AMPA and kainate] or thc NMDA-coagonfst (glycinc]suggesting that enflurane inhihition dmx not result from a compctitivu interaction at glutamate- or glycinc-binding sites 154 Selectivc blockade of A subset Qf AMPAIKA receptors has been rcported for the noncompctitivct antagonist Evans blue"'. This hiphcnyl dcrivativc (if naphtha1 cnc di sulphnn ic acid hlocks ( a t low crmccntrationns) kainatemediated rcsponscs of the snhunits GluR-A, GluRA,R, GluR-A,C, and ChR-R,C cxprcssud in Xenoptls nocytcs hut nni respnscs of GluR-C or GhR6
'"
'"
entry 07
Table I € . Contrnued Antagonist
Fca t w rt‘s
Refs
-
355 nM for thc subunit combination GluR-AJR). The blocking action of Evans hluc is partially revcrsiblc and docs not cnmpete with the kainate for thu agonist-binding site (ICir,
Nnncompetitive nnragonr cz Riluzole
Responscs faom katnic acid-cvokcd currcrtts in Xcnopus oncytes injected with mRNA from rat wholc brain or cortex can hc nan-carnpctitivclyf ’hlockcd hy the anticonvulsant and ‘ncuroprotcctivc’ compound riluzole (ICrrj 167 I‘M cf. CNQX: Kicl 0.21 I‘M an& NRQX: ICio 0.043 /[MI.Riluzolc is marc potent at hlncking rcspnnscs to NMDA [ICW 18.2 JIM cf. lthc crimpctitivc NMDA rcccptor antagonist 2APV: IC5n 6.1 )/MI
-
-
-
Spider nnd wosp venom toxin?
Is’
-
For thc non-sclcctivc actions of certain spider venom toxins [argiotoxin and roto spider toxin) and the damcr wasp toxin philanthotoxm, we Riockers, (I7-43
Database listingslprimary sequence discussion 07-53-01; The relevon! dotnhase IS indicated by the Inwer cnse prefix ( e , ~ . gh:). whrch chntlld not he typed (see introduction & laynut nf entries, entry 02) Datuhnse riccrscjon numllirrq imrnrdintdy follow the cnlnn. Note thrit o comprehensive listin,F of nll avarlnhle accession numbers is superCluous /or Jocntim o f rctevant seqiicnces in GenRank ‘‘ resources, which are nriw r~vnilnhlem t h powerful in-hurlt neighboaringt analysis routincr (!‘or drsrnptron, we the Dcitrihnsr listingc field In the Introductron (inn lavotit of entries. entry 02). For exumple, sequences of cross-specieT VflriflRtA or relnfed g n e /flrnilvT rnernhers con be rendily occesrcd h y one o r two rounds of neitrhhourm.rt nnnlysis (which lire hnsed on precomputed nlignment 7 performed usmg the RLASTt algorithm h y the NCHlt). Thrc featnre I S mosf useful for rctrrcvnl of sequence entries deposited in dntnhnses later than thow listed bclnw. Thus, rcpcscntntIvc rnernhers OT kiinwn scqiwnce hornolrqp proupi~gsnrc listed to permit rniilml direct rcrriewls b y QCCCSS~OTI number, authorlrcfcrencc or nornench t ure. Fnllow~ng direct accession, however, ncighhounngt annlysiq i q strrintyFj7 r e u o r n m m d d to ~ d ~ n t i npwly ly reporred and rClru/pd .wqllenLc~
Nomenclature Species, Enon-systematic] DNA source
Original isolate
Accession
Sequence/ discussion
Rb: S9437I
Gallo, Neiirosci (1992)12: 1 01 i M t 3
GluR-4~flop
Rat Sprague885 aa Dawlcy; alternatively spliced, cerebellum, cDNA
GIuR-A
Rat forebrain 889 aa g h Xt7184 CDNAJ (clone GluRcxprcssionK1 clnned in oocytes. Equivalent to GluR-K1 4n0w renamed GluRl o r GhR-A)
Hollmann, Noture (1 989) 342: 643-8. Hallmann, Cold Spring Harbor SYW Qnnnt R i d
(1990)55: GhRI (human isofnrm)
Human hippocampal cDNA library
HBGR- 1
Full-length human
[human isoform)
GlnH€ I=GluRl homologue)
'Glutamate receptor I'
888 aa [mature]
gh: X5R633 gh: S40299
hornofogue of GFuRl [or the flop version of the GIuR-A clonc) Human brain 907 aa gb: M64752 cDNA lihrary; M, 100 kDa chromosome 5 97% homologous to rat GluR-A Mouse, brain 908 aa gb: X5J49J cDNA sp: PL381R
-
41-55 hticr, DNA Seq (1992)2: 21 1-18 Sun, Proc Natl Acad Sci USA { 1992) 89: 1443-7
Pwckett, Proc Nail Acad Sci USA (1991 88: 7 5 5 7 4
Sakirnura,
Neuron (1992)8: 267-74
GIuR-A GlnR-R G1uR-C
GluR-D
Rat brain cDNA library
889 aa
gh: M36418 sp: P19490
Keinanen, Science
Accussion
Scrlucncc/ discussion
GlrtK-A flclp GlttR-R flop GILIR-C:flop GluR-I3 flop ClttR-A flip GlvR-It flip GlulZ-c fltp GILIR-I3f l ~ p
Flip nnrl flc~p; gh: M.764 18 ccll-spcclflc gh: M,16439 gh: M.76420 tunct~rlnnl switch {vrxr, ~ h M,16421 : ( ; V r l i * or,Lygh: M,?HOhO gh: M38Oh I nizr~tron, 07-20) gh: M,3X062 ~ hM.3 X O A 3
Adl~ltrat forchr;tin cDNA library
~ h MX50,35 :
I3ollltcr,
sp: 1'1949 1
Sc~bncb (1990) 249: 1 &33-7
'Glutamatc
gh: X57498
receptor 2'
sp: P2.3810
Sakirnura, Netlrnn 1 1992) R:
G h R - A to
GluR-D: Flip
and Flop variants
26 7-74
Glull2 [ - C;luK-HI
Rcntmllct"
GluR2
MUF ~ C I F ( : L I / I I S cxt,nt 2 (strazn RhLRJc, s~thspccaus
Kochlcr, M., Komau, H.17. and
~ O I ~ Ci cS l I~ c )
Scchur~,
tnalc liver DNA
P.H. ctnpcthlrshcd 1 19941.
~ u rntlvctllt~s v cxonl 3
C;luR-~tl (stram RALIIlc, K ~ n o m ~ ~ T ' lsithspcctcs dornesl~cuq] male livcr DNA
:[
GIuR2 [ = GluR-R) Rcnnmlct''
gh: L321YO gh: L32 15 1
Mus rnuscr~lus cxonf 1 sntl {stmrn RALRlc, prornotcr sithspcctcs rcgion ~lornc.st~uusl rnalc livcr DNA
~ h L321Y : 1 gh: L72 IS 1
Ki>ch!cr,M., Knrnau, H.C. : ~ n d Scchurg, P.H. unpuhlashcd 119941.
gh: L32 1 HY gh: L32 15 1 gh: W2152
Kochler, M.,
Knrnau, M.C. and Scchurg, P.H. itnpuhlishurl ( I994J.
entry 07
Nomcnclaturc Species, [non-systematic) DNA sourcc GIuR-R GluR-6
to
genomic sequences
QriRinal
Accessinn
isolatc
Sequence/ discussion
Sorn m c r, Rh: MJ64?7 Cell (19911 gb: M7h43R 67: 11-19 Rh: M764d9 gh: M76440 gb: M76441
Mousc gcnnrn1c
GluR-R, genomic GluR-C, gcnomic GluR-D, gcnomic G~uR-5,gennmic GluR-6, gcnornic
gh: L2OX14
HRGRZ (human Human brain glutamate cDNA lihrary receptor 21
Sun, Neororeport (1993)5: 441-4.
orininal gb: M8SQ36 GluR3 (= GluR- Adult rat sp: P19442 forehrain cDNA pulilished CJ library sequence narnc: RATGLWRq3;
Roult cr, Science ( 1990)249: IO.3.3-7.
$189 aa
hGhR3 flip Ihumanl
hGluR3 flip [human)
Stratagem 895 an cDNA lihrarics 9.36205 and 93 (1206
gh: UlO.701
Stratagenc 895 aa cUNA libraries 986205 and
gh: UlOX02
itn puht ish cd
p19941.
The long (putatively1 intracellular loop Of
R a t t 11s
hmpcrsarl,
Y. ~inpublishcd ( 1 994).
936206
GluR4a I=GluR-Dj
Rampersad, V.
90,3
33
gh: MtlfiQ37
norvcgr’cur [strain SpragucDawley) N.R. Subcloned nuclentidc for phosphory- coding latian studics scqziencc segments
Rcttkcr, Neuron 119901 5: 5 an -9 5.
gh: SS6679 g h S56890
Wright, J R ~ c e p lRes (199,?j 13:
653-65
AMPA-sclcctivc
KluR Kim: 4053 12 Morita, M o l IIrnrn Rcs
(1992114: 143-6.
cntry 07
Nomenclature
Species, [non-systcmatic] DNA sourcc
GluR.5 has two splice variants, GhRS-1 (920 aaj and GluR.5-2 (905 aal (see GCRCo r p nization. 07-20)
Original
Accession
Iw1at.c
GluRS-I: gb: M83552 920 aa (a full open reading frame for the shnrtex GluRS-
Sequence/ discussion Rctth,
Neuron (2990)5: 5 88-95.
2 splice variant WHS
nnt found]
Rat ccrchcllum 884 aa cDNA library
not found
Egcblcrg, Ncrture (1991) 351: 745-8.
GluRJ 61
not fnund
956 aa
not found
Mouse delta-!
1Q09aa
PIR: JH02hh Yamazaki, Rrachcrn Hrophys Res Commun (1992) 183:
GEuR chain precursor
not found
886-92, 52
Mausc delta-2 G h R cham prccursor
72 ( = K h - 2 hornologue)
Mouse GluR 979 Ramma 2 subunit sclcctivc fnr kainate
GIuR-K~
Rat hippo-
(= fhp
campus and curchral cnrtcx cDNA
v m i m cif
GluR21 GIuR-K~ (=flip vcrsinn of GIuR3I
humEAA2
Human hippocampal cDNA library; strucrurally related, though not identical to KA- 1
not
1
found
gh: DO 127.7
gh: XS46SS
R8d aa
gh: X54656
962 aa plus 18 aa signal scqucncc
not fr~und
-
kDa
Sakimrrra, FEERS Lett (T090)272
J3-80.
888 aa
M, 107 176
Lomeli, FERS Lett (l5)93\ 315: 318-22.
Nakanishi, Neuron (t990) 5: S69-8 1.
Karnhoi, Md
Phnrmacol (1992)4 2
10-15.
Nomenclature Species, (non-systematic] DNA source KA-1 I=31
homoleguc 1
KA-2 {=721
KBF-C
KBP-f
Original: isolate
Accession
Sequence1 discussion
Rat brain 956 aa EDNA, highaffinity kainate; homomeric assemblies do not form channels
gh: X5999h
Werner, Nature (1991)351:
979 aa Rat hrain cDNA, highaffinity kamate; homomeric asscmhlies do not form channels
em: X59996 Herb, em: ZI 1581 Neuron gb: X59996 (1992)8: gh: Z11stEl
775-85.
Chick cerebellum cDNA kainatc binding protein
KA binding (channel inactive) 464 aa
not found
Gregor, NCltslrc ( 1 989) 342:
Frog brain
KA binding
not found
cDNA/kainate- lchannel binding protein inactive) 487 aa initially purified hy dornoic acid affinity chromatography
742-4.
684-92.
Wada, Not lire 11989)342:
684-9.
"The complex alternative splicing+ patterns ohserved in the GloR-B gene [Koehler, M., Komau, H-C. and Seehurg, P.H. unpublished, 1994) can be traced from splice site joining data' accompanying certain databasc entries. Importing sequences in a standard file format can he intcsprctcd by some sequence analysis programs and incorporated in an interactive fcaturc tahlc. 'For example, the GluR-R short-splice form entry /gkW2204/gh: J-32151) contains thc dollowing 'joining' protocols: mRNA join (I-32189:1075..8594,LKl190:1 ..141,LA2191: I ..240,~32192:1.. 197, L3219,3: 1 ..54, L32194:1..1h2,LV 195: 1.. 1(18,L32I96:1..lOs,W2197:1__ 1 11, L72 1% 1 ..2O7, L32 199: 1 ..371,L32200:1__ 199,W22O1:1..248,L?2202: 1 ..I 1 5, W2203:2.. 1 15,1..249] and CDS join (L32189: 1507.. 1594,L32190:1 .. 14l,W219 1 :1..24O,LE 192:1__ 197, L321Y.3: I ..S4,L32144:1..162,W219!i:l..lh8,W219~:€..105,L~2197:1 ..111, L32 158: 1 .,207,W2 199:1..37 1,W2200: 1..199,L.3220 1 :I . .24R,L-32202:1 ..1 1 5, L32202:I..1 15,1..2461 which rclatc the contcnts of database entries ('L-nurnhen' in the example above) to desqwted splice points.
Sr~urcesof ~n/orrnnt Inn on orher glrlrrrrnnte rucuprnrs 07-53-02: N o t r . T h c gcncs anrl cl3NAs tahvlatcd ahovu uncodu s ~ ~ h u n l t s f o r i i i ~ n ~ionotrnpict : glutamate rccrptors (iGluRJ. In datahasc scarchcq, tPicsc shoultl not hc ctlnfuscd with thc Fcnc noinunclaturu llsctl to dcscr11,c thc r n r t a h ~ t r n ~ i c tglutarnntc rcccptors such 21s thc n~C:Ftr,-n~C:lu, sCTIC'Sh. 1hd- ~ ( l.h rnGIu rrccptor functions through C, protclns and cnositc~l
turnover'" arc c l i ~ r a c t c r ~ z cby d sclcct~vu actlvatlcln w ~ t h 1 ainzntl-cyclopcntyl-1,,7-d~carhrlxylatc [ACPDF. Ry gatlng of K t currcnts via rnctal,~)trop~c glut:im:~tc rcccptors, excitatory atnino acids can also act as tory tmnsinl t t crrl" (ccrb R r * r * i ~ ~ ~ ~ r ) r / ~ r rrnt ~ nrrrlc*t s d t ~Ionq t-~~r slow ncrtro~nr~d~lln unrJrr I L ( ; K Clr, 27-49) phosphate.
1 Related sources & reviews I
I
07-5h-OE: Maror ql~r~tutlsol~rccs"" 't"~mY; C;lr!Rs
In h~ppc~campnl r~curr~ncs"~; rnnlccular ncurahlolnm of G ~ U R S ' ~'H-ZN; "~ m(11~~11lar h11110gy 06 lonotropic glutarnatc rcccptrlrs In ~ l r r ~ s r ~ ~ h i l rpcrmcation r'~'; pathways of ncurotransmittcr-gateti ion channclsUY; phystc~lngicaland pathophysiological rthcs of cxcltatory amino acids during dcvclopmcnt"; singlcchanncl recording from i ~ l u ~ " ' ; thcrapcut~c pntcntial of sclcct~vc AMPA/kainatc rcccptor antaRon~sts'57;non-NMnA glutamatu rcccptnrs in g l ~ a lcell siLmallinRH";rolcs of GluRs in CNS f u n ~ t i r ~ n ~ ~ ~ .cxcitatory ~"~'"" arnlno aclrl.; a s cnt!r,gcnt*l~s functional ncurotransrnlttcrs"7T; cxcitatory arnlntl A C I ~actlvatlon of sccontl Incsscngcr systcms In a d d ~ t ~ ot on a tTiruct gattng of Ion ~ h n n n c l s ' ~ ' .Srxrj c ~ l r r ) ~ h r 'Ilr5ct~urr*rF. - lor1 rhhrrnnel I ~ o n k r c { ( l r v ~ w i , v tbrlr , rv h0
Feedback Error-cnrrcc! Ions, enlrunucnrent ond r ' x i ~ n s i c ~ n r 07-57-01: Plcasc nntrfy spccsflc urrnrs, ornlssinns, uptiatus and crlmments o n t h ~ scntry hy c o n t r ~ h l ~ t l n to g ~ t se-mail feedback file (for d c t n ~ l s ,src Rr~sozrrr.t,I, Srmrch Criteria c4) CSN llrvc/ilpmrnr). Far t h ~ scntry, scnd umail mcssagcs Tn: [email protected], i n d i c a t i n ~thc appropriate paragraph hv cntcrlng ~ t ssix-figurc index number (xx-yv-zz or othcr idcnt~frer)Intn the Suhieca: ficld of the IncssaRc [c.g. Suhlcct: OR-50-07).Please fccdhack on 41nly one specified paragraph or figure per message, natmaEly hy sending a cnrrpctid -- replacement . .-- -- acct~rding to the p ~ i d c l i n c sin Feedhnck CSN A(,crcv . Fnhi~nccmcntsand extensinns can alqn hc supgeqted by this rnutc [ ~ k l 1.d Not~ficd changcs will hc indcxcd via 'hotlinks' frnrn the CSN 'Homu' page ( h t t p : / / w w w . t ~ . n ~ . i t k / c sfrom n / ) mid-1996.
Entry slrpport ,yronps otld e-mnrl newslet tets 07-57-02: Authors who havc cxpcrtisc in nnc or rnrjrc flclds nf this untry [and ;Ire willinl: to pnwldc utIitr)tinl or othor quppnrt for dcvclnping its contcntq1 can Iotn its .iupport group: In this casc, scnd a messaRc To: CSN07@?1e.ac.uk, (ontcring thc wortls "support groi~p"in thc Suhlcct: field). In the intsraFc, pluasu intlicatc principal intcrcsts (scu f~cklnnrnrrhrrlurrrz In I /I(' Iri!rorluc!~on(or r.ovrrr~,qc) t o ~ c t h c rw ~ t hany rclcvant http://www site
cntry 07
links [cstahlishcd or proposed] and dctails of any othcr possihlu cnntrihuticins. En duc coursc, support group mcmhcts will (optionnllyl receive e-mail newsletters intcndcd to co-ordinate and develop the present (tcxt-based) entryJlicldnarnc frameworks into a ’library’ of interlinked rcsources covering inn channel signalling. Other (more Rencral) information of intcrcst to cntry contributors may also hc scnt to thc ahovu addrcss for group distribution and fccdhack.
Snrnmer, Trends Pharmacn! Sci 41992) 13: 291-6. Egehierg, Noturc 19911 351: 745-8. ~ n uter, I Science (1990) 249: P 0.3~3-7. Rettler, Neuron (1990)5: SRd-95. Sakimura, Neuron (1992)8: 267-74. Schoepp, Trends PharmacoI Sci (19931 14: 13-20. Gasic, dnnu Rev Physiol { 1992) 54: 507-36. Nakanishi, Science (1492) 258: 597403. Innas, 1 PhysinI (1992) 455: 143-71. I’ Patneau, Neuron 19Y 1 1 6: 78.5-98. ” Partin, Neuron (1993) 11: 1069-X2. Gallo, J Neirmscr’ (1992) 12: 1010-23. 1 3 Kcinancn, Science [ 1990) 249: 5 5 6 6 0 . Hcrh, Nezirnn (1992)R: 77S-R5. Maycr, Prog Neurohiol (19871 28: 197-276. MacDcrmott, Trend.7 N r u r s c i (1987117: 280-4. I’ Wisdcn, Curr Elpin NeurohioI 119543) 3: 29 1-8. Scchurg, Trends PhnrrnncnI SCF(199.37)14: 297-371k7. l9 N ~ C ~~hy-irnI I I , ~ e ( IvW O ) 70:513-65. Rarnard, Trends PhnrrnncoI Scr 11990) 11: 500-7. 21 Henlcy, Proc Nnll Aced Scr USA (1992) R9: 4806-10. 22 Hcnley, New R m l (3989) 1: 15t%H. Mnnycr, Netiron (1991 6: 799-810. Shcn, Riol Chem [ 1994 268: 190JoL5. Raymond, Nature (19931361: 6,3741. Sommer, Science 19903 249: 158CLS. 27 Mdler, Science (1992)256: 1 5 G M . 28 Rettler, Neuron (19921 8: 257-65. z9 Iomcli, FEBS Lett (1992)307: 1,1943. Larnholez, Neumn (1992) 9 : 247-58. Monycr, Scicncc I19923 256: 1217-2 1. Mcgurn, Nature (1992)357:JM. I S ~ ~ I RroI , hem 19931 2h8: Z H ~ W ~ ~ X R4 Mariynshi, Niture (1991)354: 31-7. Comeli, FEBS Lerr (1993) 315: 3 1 R-22. 36 Wisden, Nciirnsci (1994 (citcd ns in prcss in sourccI. 37 Tiillc, Nerrroscr 11991 1 (cittd ns in prcss in source).
’ ’
’’ ‘‘ ‘’ ’‘
’.’
‘’ *‘
”’ ’’
entry 07
".
---
Kuhsc, FEH.7 1.r'tt [I991 ) 283: 7.3-7.
" "no, I I1hysiol (1990)424: 151-65.
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41
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''
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'I4
entry 07
Molnar, Neuroscience ( 1 993) 53: 307-26. Martin, Ncnron ( 19921 9: 259-70. RY McNamara, 1 Nenrosci 1992) 12: 255542. 9o Sun, Prnc Nut1 Acad Sci USA (1992189: 1443-7. 9' Euhanks, Pmc Null Acnd Sci USA (19931 9.0: 1782-87. " Pnticr, Genomics (1993) 15: 696-7. 93 Sornmcr, Cell (1991) 67: 11-19. Higuchi, CeIf (19%) 75: 1,361-70. R i d Chcm [ 1992) 267: 501-7. 95 Wcnthold, p/, Hollmann, Cold SprinR H o r h S y m p Qunnt H F (1990) ~ 55: 41-55. '' Wada, Nnture (19x9)342: (1x4-9. '' Unwin, Cell (1993)72: 3 1 4 1 . Nakanishi, Neuron 11990) 5: 569-81. Rurnashev, Neuron [ 19921 8: 189-98. Hume, Sciencp 1 1991 1 253: 1028-3 1 , I"' Verdonrn, Science [ 19913 252: 17 15-1 8. 'm Kohler, Neuron (199.1) 10: 491-500. 1114 Egehjerg, Proc Nntl Acnd Sci USA (1994 90: 755-9. Ins Uehino, FERS Lett 19921 308: 253-7. 1m Stein, Mol Phnrmacol (1992) 42: 864-7 1. lo' Kcllcr, EMRO J (1992j 11: R91-6. ' O R Grccngatd, Science ( 1 99 I ) 253: 1 I~V-X. ' 0 9 Raymond, Trends Pharrnacol Sci (19%) 147: 147-53. Madison, Annu Rev NeurnscE (1991) 14: 379-97. I f ' Linden, SCI'L'ITCC (1991)254: 1656-9. Sturn, l'hysiol (19921441): 24J-7H. 'I' Hcstrin, Ncnmn (1992)9: 991-9. Cnlquhoun, J Phyal'ol (1992) 458: 261-H7. Rumashcv, Ncuron (1992)8: H-20. Rumashcv, Science ( 1 992) 257: 1415-1 9. '17 Cnle, Nfllure ( 19841 340: 4744. Dildy, 1 Ncumchern ( 1 992) 5R: 1 ~ - 7 2 . '19 Rcichling, I Physid (194,1] 469: 67-88, Vyklicky, Neuron (199117: Y71-84. ;*I ~ n r i Brain , R C S (1988) 457: 3 % ~ . Livscy, Neurnsci 1199.11 13: 5,72633. Yarnada, hleurosci (1993) 13: 3904-15. Patneau, N m r s c i 11993)13: 3496-504. wan^, Mol PharrnacoS {lS93l44: 504-10. "ti Hullehroeck, Brain Res I19921590: 1x7-92. *27 Raman, Neuron ( 1992) 9: 173-Rh. Isaacson, Proc Not] Acad Sci USA (19911SR: 1093640. Lester, Annu Rev RinphyT Rinrnol StruC (1992) 21: 267-92. Gilhertsnn, Sclcncc 11991) 251: 161,1-15. I.'' Ozawa, NerirophysinI { 1 9 ~ 166: ) 2-1 1. 'XDingledine, { Ncurosci (1992) 12: 4080-7. '33 Schncggcnhurgcr, Neuron 11 Y9.1) 11: 1*3343. '" Pruss, Neuron 1991) 7: 509-1 R. CollinRridgc, Physiol Rev (1989140: 145-210. R7
INI
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"" Cull-Cnndy, Ntrture (19K7I 325: 525-X. Aschcr, I'h!ls~al( 1 488) 399: 22745. Tang, Scrcrluc (19x9) 243: 1474-7. Sansom, Int Ilrv Neurnl>rt~l(1990132: 5 1-106. ' 4 0 Traynclis, Neuron ( 19'1,71 11: 279-89. khida, Proc Nrltl Acrrri SCI IlSA (1YHS)82: 18,1741. Kcrry, Mnl Phrirrnnctd (I 9Y<3)44- 142-52. Hcrlitzc, Pdrrirr)n [199,3) 10: 113 1 4 0 . Pricstlcy, Rr I Phrrrrn(r.cn/ 11988) 97: 1,115-23. 145 Blaschkt, IJror*Ntltl Acrlrl Scr U S A 11 99.71 9fl: 6528-32. "& Wltsch, h,>rocNrltl Acrzd .Cc3 I JSA (1992) 89: 104848. '" Copani, Netrrot.h~m) 1992\ 58: 1190-204. ' 4 R it(], J I'hys~ol( l 9 Y O ) 424: 53.741. Young, Trrnrfs Phormacol Sci (1990) 11- 126-3.1. Shhirnazaki, A t o l I l r a ~ nRPC (1092113: 33 1-7. Is' Smart,Ccl! Mol Ncnrohic~l(1989) 8: 193-206. 152 Zcilh{~fcr,Nrwron ( 19931 10: 879-87. Rogawski, Trrndc Pht~rmt~cr~l Sr-i [lr)93) 16: .US-11. '" S w ~ r t z Mo! , IJhijrmirr*ol[ 1992)4 1 : 1 130-4 1. $55 Lr>dRc,TrcnrE\ I)hnrmncr)lSr*r ( I 9901 1 1 : 8 1-6. 156 Chapinan, Nrr~rr>rcrLet( (IYHS)55:325-30. L m , FASEII I (IBY,?\7: 479-85. R~cller,Proc ~Zrr~tl A c d Scl I JSA [ 1993) 90: 605-9. "" Dclchano, Errr Phrlrrnr~col(1993) 235: 2K7-9. *"' Jackson, Trenrl~Npurmc.~(1988) 11: 278-83. HolPmsnn, N{~trlrr(1989) 342: 643-8. Patncau, N t ~ t r r o r c( ~1 902) 12: 59.5-606. 86%Hunzt, Mol I)hr~rmnc*oF( 1992) 41 : 70.7-801. P M Tanahu, Neuron (1902) 8: 169-79. 165 Sugiyame, Nrasrrr)n( 1 989) 3: 129-32. "" M ~ l l c r ,Trcnrlc Phnrrnncol Sci ( 1 991) 12: Su~lyama,Nrlturc (19871325: 5.11-3. "" Charpak, Nr~trrrc(I9YO) 347: 765-7. 16'1 Hunnchcrry, IIroossovc 1 1 9921 14: 465-7 I. $70 Ozawn, Ipn I'hyz104 ( 1 9031 43: 14 1 -50. I71 Rutz, Trcrndr !'hrrrmrrt.ol Scr (199.71 14: 428-31 I72 Cull-Candy, Trends I'hrrrmr~r-olSci (1987) R: 218-24. Hvadlvy, Trrndc Phrlrrnrlco! Sr! 119901 1 1 : 205-1 1. '74 R T ~ ~ ~ c Ns~,T I T O I (\1(YS4) 9: 2073-9. "' Part~n,Mol iJliomilir.rrl ( 1 9041 10: 129-AX.
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Edward C , Conley
Entry 08
Abstral:t/general description 08-01-01: Prc-synaptic release of glutamate produces an excitatory post-
synaptic currentt (EPSCI] which can he: resolvcd into (i) B fast EPSC component whcrc the onset and decay i s mediated by nnn-NMUA ionotropicj glutamate receptor-channels (see E L 6 CAT GLU AMPAIKAIN, entry 07) and (ii] a slow [or ’Tong-lasting’) EPSC component mediated hy NMDA-gated receptor-channels 1 NM DARsJ. OR-01-02:Responses of the NMDA receptor-channcl are characterized hy a slnw rise and decay, a large Ca2+-permeahilityi,voltage-dependent Mg2’ black and a rc uircment for glycine (nr a glycnnc-like cndngennus rnoleculej as a co-agonist .
9
08-01-03: Genes encoding cation channels selective for the glutamate receptor agonist N-methyl-D-aspartate(referred to as NMDARs in this entry for convenience) foran part of the extracellular ligand-Rated channel gene superfamily ldescrfhed under ELG Key fncts. e n v y 04). Functional NMDA receptors have been shown to be composcd of a Fundamental subunit, encoded hy the NR1 subunit gene (or cquivalcnt name) and its potentiating subunits (cncodcd by thc NR2A-NR2D gene harniFyt ). Whcn hctcrohgoous!y cxprcsscdt, the NR 1 subunits form hnrnomeric receptnr
08-01-04: NMDA rcccptor activation is also cssential for neuronal differentiation proccsscs and cstahlishmcnt or elimination of synapses in dcvcloping brain (see Developmental re,Tulntion, 08- I I). Transfcnt incrcascs in NNDA rcccptar gcnc expression occur during dcvcloprncnt, and application of NMDA-rcceytor-sclectrvc antagonists can hl ock experiencedependent plasticity+ in the adult. NMDAR densitics appcar tn bc modulntcd by developmental stimuli, and influx of Ca” throuxh thc NMDAR and L-type Ca” channels of hippocampal ncuroncs has hcen shown to rcgulate gene transctiptionilt evcnts in thc nucleus. 08-01-05: The involvement of ” I D A reccptors in several neurophysialogical and pathophysiological processes has hccn rlcmonstratcd, including normal information processing of auchtory and visual signals, activation and
maintcnancc of mOtQr rhythms (c.g. in respiratory control), central sensitization during nociception (pain rcccption], cardiovascular pressure control (baroreception and control of vasomotor tone), development of motoneurnne disorders, cpil cptic states and post-ischaemic brain damage (glutamate toxicity). There is a large lircrnturc rclating the function of the NMDAR to roles in synaptic plasticityt and by impllcation, learning and memory+ processes (see Phcnotyprc expressinn, OX- 14, nnd the next pnrograph).
largc number of associated signalling components and mechanisms hare been shown to modulatc mechanisms of long-term potentiationt (LTPJ a long-lasting increase in the strength of synaptic transmission due to brief, repetitive activation of excitatory afferent1 fibres. As a striking example of synaptic plasticityt, the experimental induction of LTP and other distinct forms of synaptic potcntiatinn have been shown to he reliant on molecular propcrtics (if the NMDAR-channel. Principally, this involves activation of NMDAR by synaptically released glatamatc with concomitant post-synaptic membrane depnlarization which rclieves a voltage-dependent, extraceIlular Mg2’-hlock of the NMDA rcccptor ion channel. Removal of Mg”-hlock, present under resting conditions, allows calcium to flow rnto the dcndritic spine (for details, see I’henotyprc expression, 08-14). Notably, several NMDAR-independent pathways for synaptic potentiation havc also heen characterized. 08-01-0h: A
08-01-07: Comparison of genomict and cI3NAt sequences for genes encoding NMDARs has revealed that alternative splicingt prmesses affecting the fundamental INRI ) subunit can Kencrate a numher of functionally distinct NMDA receptors. In common with other ELG receptor-channels, there is strmp, evidence that protein phosphorylation events can modulate NMDAR functional rcspclnsw in VIVO. 08-01-08: In addition to their susceptibility to vohage-dcpcndcnt ME’’ block, B key molecular fcaturc of the NMDAR series is their Ca2’-perrneability, which IS central to their proposed phenotypic roles such as synaptic
potentiation, glutamate toxicity and information processing. The molecular determinants of hoth Mg2‘-sensitivity and Ca2+-permeahility have heen ‘mapped’ ming dcfined mutants, ant1 amino acid residues in a position horno1op;nus tn the site in the M2 rcgion lQ/R site) of AMPA receptors have heen shown critical for dctermination of Ca”-permeability (compare the Domoin {unctions, 08-29, In this entry and Dornmn functions under ELG CAT GLU AMPAIKAIN, 07-29).
OR-01-09A large numbcr of endogenous modulators and pharmacological antagonists have heen characterized for the NMDA receptor and appear to act at several distinguishahre ’sites‘ including the NMDA (or glutamatel site, the glycine [co-agnnist) site, the Zn2’ [ modulatoryy) site, the redox modulatory site, the polyarnine site, the Mgz’-hlnck site, and sites for H’ ion modulation and those involved in binding athcr open-channel blockerst (see Receptor ontognniTts, Q M t , incIudmR FIR. h and Tdhte 10).
Category (sortcode) OR-02-01: ELG CAT GLU NMDA, 1.e. extrace!!ular Iigand-gatcd cation channcls sclcctivc for thc glutamate acccptnr agonist N-mcthyl-a)-aspartate. Thc sumcstcd electronic retrieval code (unique crnhcdded identifier or UEl] for 'tagging' of ncw articlcs nf rclcvance to thc contents of this entry IS UEI: NMDA-NAT (fnr reports or rcvicws nn nativct channel propcrticsl and UEI: NMDA-HET (for reports nr rcvicws on channcl propcrtics applicablc t o hctcrplogousl y t expressed recnmhinant' suhunits cncnded by cDNAs' or gcncsi). For o d ~ s c r ~ w od n the odvontngcc of W E I s nnd gi~idelineson their ~mpJernen~otron. see the sectinn on I
Channel designation Ds'stincrion of ronntroprc from rnetohotropic Rlutnmnte receptors 08-03-01: NMDA receptor-channels are frcquently rcfcrrcd to ss NMDARs. !onotmpic~ glutamate receptor-channels in general (sep a h ELG CAT G'LU AMPAAJKAIN. entry 071 arc sometimes designated as 'iGluR' as cnmparcd to thc metahotropicT GltiRs such as the rn61trI-rnGItr7 seriesf4 whlch arc linked to G proteins and do nor contain intcgral ion channels. Dcpcntlcnt on thcir molecular suhtypc, mGlu rcccptors can cithcr x t i v a t c
phnsphoinnqitidct mctaholism via phospholipasc U activation (subtypes rncIul, r n ~ I u ~ultimately I, rclcasin~Ca'+ from intmccIlrrIar storcs ( w e ILC: Cli In.#?, cntry Is), or inhibit thc activity of adenylate cvclasc via a pertussis toxin-wnsitivc .G protein Ircrnnining suhtypcsl ( s w Rrcourre A U prorcm-linked receptors, entry MI. The rnle of ~lutamatc-reccptnr-Ilnkcd second messenger generation In rcguhting mtraccllular Ca" has hcen rcvicwcd5.
NMDAR suhunft/,pene nomenclature 08-03-02 Rccnmhinantt NMDA receptnr suhunits arc dcsignatcd according to the name of the gene that encodes them, e.g for rat - NMDARI, NMDARZA, NMDAR?R, NMDAR2C and NMDAK2D (see Gezic fnrnily. 08-0.5). Hvphenatcd forms of these RcncJsuhunit narncs arc also in cnminon usc (i.c. NMDAR-I, NMDAR-2A, etc.]. Qthcr nomenclatures in use arc listcd in Tahlc 1. Mouse equivalents of the NMDARI (NRI1 and NMDAR2 (NR2F serifs h a w hccn dcsignatcd by Greek letters. (For hirfher discussion oC nomenclnturc5, sce Gene Inrnilv, 08-05, and the I I J P H A R Norncnclatrire Cnrnmittce recommpndutinnq viri t h p CSN 'hnrnr pngr' lsrr Frrdhick d C S N i7L'cC~r.cntty 121.1
Current designation c.g. INMllA,or hy spccificaticm of the current follnwing cn-cxprcwion nf namcd whunits, C.R. I I ~ ~ l , 'n7 A.~ ~. ~
08-04-01: Convcntionallv of thc form
cntry O H -
Gene family Distinction hct ween Rrncs m c o d i t i ~'firndmmeninl' n ~ d
'potentintinc~' NMDAI< suhunits 08-05-01:Thv guncs cnctiding NMDA receptor? fnrm part of the extraccllutar Iig,snd-ptcd channcl Rcnc ?upcrfamily (Inirorlticrd under ELG Kcy f m t s . cntry 041. Functinnal NMDA rcccptors hsvc hccn shown t o he composed d a fundamental subunit, cncoilcti hy thc NRI subunit gcnu ( o r trlulvalcnt namc! s n d its pntentiating whunits (cncodcd by t h c Kcnus NR2A-NR2D).
AIietnm tive nornmcImires Tor independently iwlntcd NMDAR s 1I 11 ~1nit tqe nes 08-05-02 Thc NMDA rcccptnr-channcl suhunit gcncs havc hccn classifrctl into the c p d a n ( E , fundamental) and zeta I[, potentiating] families nccrmlinl: t o rho rlugrec of shsrcd amino acid scqucncc ({or clrrrr/rcation, see TlIhIe 1). Indcpcndcnt dewription nf thc samc rcccptor suhtypcs hns led t o thc proposal of scvcrsE alternative nomenclatures which arc compared in Tahlc 1 ant! untlvr Drrtnhnsc / ~ ~ ~ z q (18-53. ys, In thc ahscncc of a universal nomenclature, sprcics-specific isoform data arc quntcd using thc name rlcrivct! from thc originnl citnticin - c.g. NMDAR I , NMDAR2A-2D/NRI, NK2A-213 (gvncrally froin Tar) and zcta-l (;I 1, rl-r4 (guncrally dcrivcd from rnouscl. For hrcvity, rlcwriptions (if nit whunits arc citc'd in thc ah'nruviatud fmni (c.g. N R l , NR2A, ctc 1
MrcJriinirm f o r crcco<
1IrI~tednessof I he fundrrmcntnl srihtinrt NR 1 'nun- N M D A ' s 11 h unit pwcs
-
10
(FPP
N R 2 series- and
08-05-04: NR 1 coding scqucnccst sharc only 18% scqucncc hrmolr,~:yt with thr 'potcnt!atlng' NR2-tvpc suhunits. NR2A-2C sharc S5-JQTr svqiicncr itlcntitvi with uacli othcr".H. Typical hnmologics between thc NR 1 suhunit If'.1 5 and subunits cncoding AMPA- and kainatc-sclectivc rC;luR arc -22-2.7'% (for C.IuII-A-GluK-13),-2.3-24'Xx [for Gl~iR6and GluRJI a n d -22% (for KA-21. ncspitc thcsc rcrativcly low ~ c q i 1 t . nhomohgies ~~ thu NR1 i w l a t u show< ovcrall v / r w t u r r i J similaritics with AMPA and kainatu rcccptors ( ~ c r EL,[; CAT I ; I . I J hMPAIKAIN. v n t r y 07, rind Ilomnln (I r r ( i n a y ~ ' miu ,n OH-2 71.
,%y ucn ci> viirui n t s within popiilrt i ions 08-05-05 Minor drfhcnccs in scqucncc hctwcen the NR2A-D suhunits, poqqihly due to polymorphic variatinnt hctwccn diffcrcnt strains {if rat havc hucn rcprwtctl nnd d i w i w d H .
1
Subtype classificatinns
OH-06-01:For a whtypc chsific;ition hascd on similarity nf NMDAR protcin pnimry I ccqucnccs, v w ~;enra~cirnilv,O H - O . ~
Table 1. Distinguishing features of genes encoding NMDA receptor-channels (From 08-Qti-02) Genelsu bun1t (equivalent nomenclature in brackets)"
Description"
NR1 = NMDARl Homolope:
NMDA 'fundamental' or 'key' subunit. Forms homomerid charnels; significantly potentiated+ by co-expression with anv NR2 subunit.Different NR1/NR2 series cornhinationshave different p r ~ p e r t i e (see s ~ ~other fields)
= zeta 1
(
NR2A = NMDAR2A
Homolope: = epsilon 1 ( E 1)
NRZB = NMDAR2B Homolope: = epsilon2 ( ~ 2 )
NMDA 'potentiating' or 'modulatory' Does not possess homornelict channel-forming activity
Encoding'
938 aa
Transcript size
Predicted mol. wt (non-glycosylated)
Two adjacent
hybridizing bands 4.2 and 4.4 kb'" or 4.5 and 4.8 kh (human")
-1464 aa 1445 aa, mature
mRNA
-
12 kh
mRNA
-
15
Sib: 19 aa
NMDA 'potentiating' or ?noddatory' Does not possess homomerict channel-forming activity
1482 aa
kb
-- 105 kDa' I16 kDa hy SDS-PAGE+
-
1RO kDa [glycosylatedt] I65 kDa de-glycosylated see rZE2 and Protein I T I Q k C U h Y weight (purified),08-22
-
NRZC = NMDAR2C Hnmnlogue: = epsilnn 3 \,-.IF
NMDA 'potentiating' or 'modulatory' s u h u n ~ t ~Does . ~ . not posscss hornornerlcT channel-forming activity
I W 9 aa
mRNA
- 6 kb
NR2D = NMDAR2D Homologue: = epsiIon4 1.41
NMDA pot en ti at in^' or 'modulatopr' , suhuntt",'. Does not possess homnmerlci channel-forming acaivrtv
1.323 aa
mRNA
-
See
See Dntnhnse l i ~ t ~08-53 n ~ ~ .
Other N M D A R See Datahnsc lretlnps. 08-53 as~ociatedsubunits
Dntnhase
7
kb
Ser
Dutahase
l i s t l n g q . 08-5.7
11stln.y~.
08-53 "For discussron of convcntional nomenclatures, see Channel deognatlon. 08-03. "FOT sequence retrieval via accession numbers, see Datahn5e listings, 08-53, "Encodang' data show thc number of ammo acid residues In thc specified channel suhunit, with signal peptidc restdues denoted hy the prefix 'Sig:' Thc isvmbal for the NMDA rcccptoa suhunit cloned in Nakanishi's l a b o r a r ~ r ~is' part ~ of the Greek letter nemenclature used hy Mishina and colleagues for rnousc lGluR subunits'" (we Gene f a m ~ l yunder ELG CAT GLU AMPA!KAih7. 07-0.51. 'Radlatlon inactivation analysis' prcviouslv ~ u g g e s t e d 'that ~ the NMDA/glycine-brnding site of NMDA rcccptors was -.110 kDa, in reasonable agreement with this prcdicted ~non-glvcn~vlatedil M,. L(
c
Multiple nRonist activation of single receptors 08-06-02: Purification and characterization of two Xenopus CNS excitatory amino acid ionotropic receptors show that they display unitav receptor1 activity (i.e. they have more than one class of excitatory amino acid agonist speci Ficity within nne protein oligorner). A subunit-exchangr? h ypnt 11csi s hns hccn proposed to acctmnt for thc known multiplicity of excitatory amino acid rcccptor typesf6.
Pu 1 n 1 ive pre-synnpt ic ionotropic Glu R s OR-Oh-03: A cDNA clonc cncnding R 33 kDa protein (GR33Jhas hcen nhtaincd h y scrccning a lihrary with a n antibody gcncmtcd apinst ~lutarnatcbinding protcins”. The scqucnce of GR33 is identical to that of thc previously reportcd prc-synoptic protcin syntaxin. When GR33 i s exprcsscd in Xennpw oocytes, it forms glutamate-activatedirin channels that arc pharmacolapcally similar to NMDAR hut have distinct clcctmphysialngical properties. In vim, the GLM may hc a pre-syntiptrc glutamate receptor".
1
Trivial names 08-07-01: Thc NMDA rcccptor; the NMDA receptnr-channcl; thc Ca2’-
pcrmcahlc glutamate rcccptorxhanncl; thc NMDAR; the NMDAR-channcl.
Cell-type expression index For detnfls ol selective NMDAR siihiinit gene exprcssion, S E C other ficlrls, prtjciihdy mRNA disrrihution. 08- 13, nnd Protein di.~trihrtion,08-15.
Uhiqrrity of jGluR expression in the CNS 08-08-01: Receptors for ionotropict NMDA rcccptor-channcls (iGluRs) arc uxprcsscd on virtually every neurnnal cell, hut are notshly ~ i h e n !from glial cells (ct. Cell-type expression rnikx tinder ELG CAT G L I J AMPAIKAIN, 07O X ) . Functional divcrsity of NMDAR may arisc thrnugh co-expression of mdtiple types of swhunit genes within single cells, which is a cnmmnn occurwnce”. Tahlc 2 givcs cxamplus for pattcrns of t‘xprcssion, couxprcssion and absence of expression fnr NMDAR suhunits tlurivcd from
irnmunochernicnl and RT-PCRI studies in a range of neurnnal cell prcpsrations. In gcncral, regional variations in NMDAR properties arc cffcctcd hy differential exprcssiont nf NR2 subunit gcncs (see m R N A diTtrihsitron, 08-12, nnd Dommin functions. 08-29). Specific cxamplcs of wcllcharacterized prepantinns for studying NMDAR arc listcd in othcr ficlds.
Restrictiw exprcwion of NMDAR 08-08-02: Although the ncurotransmittcr glutainntc cxcrts cxcitatoryf effects in hoth CNS ncuroneq and gIiaI CCIIS, thu Iattvr dii not cxprcss N M D A R ~ ~ . T h u typc 2 nstrocytc (one typc of rnacmglial ccll] from rat ccrchcllum posscssm AMPA- and kainatc-sclcctivc GluRs cvcln thniigh it lacks rcccptors sclcctivc for NMDA2’.
Tahfe 2. I'ntterni ol cxprc.~$jr)n for NMDAII s ~ l h t l r ~In ~ todlllr s rr1l ~lrurt~nrrl cr,IE r v p U cI~tr~r'rl (In r~vrl~lr~lllr* 'strhnnrt-.rprc~/lc'prtlhc..; (From O X - O X - 0 1 ) Subunit gunc
I
CAI C A,? Dcntato Ccrchcllar Purkinrc pyr;~rnirlal pvminidnl g r n n ~ ~ l cgranule cclls ccll~ cclls cclls culls
Spin;11 cord motor nuuroncs
Channel density N M D A R densities ~nudnlatedby develnpmcnr a1 stjmuli 08-09-01: Transient increased densities nf NMDA-hinding sitcs have hccn rcpnrtcd rn t h d~c v c l c l p ~ nrat ~ hippocamyus22. 1ncruast.s In NMDA aRonlst sitt. dcnsrty can hc rup~~lnrctl by thc ovarian horlnonc oestradfnlZ7 ( ~ e mlro r Ilr,vcfoprnentxll rr~grrlntlon.08- 1 1 )
Cloning resource Ex])rcssio~~-clor~ir~~ 08-10-01: T h c principal subunit of the rat NYDA rcccptor-channcl INRI J was f ~ r s t1st11ntt.d hy both expressinn cloning1 and suhscqzrcntly hv Iowstringency' crt~rs-h{~inolr)gy " Tcrccnlng of hmin cDNA Iihraricq. ~x~rcssir,n-cloninKt proto~o1s for ~ O R O ~ T O ~NMDAR I C ~ wcrc IJascd on :I scnrch for NMIIA-tvokcd currcnts mcasurutl in ~ ~ " - f r cIwffcr c in thu prcrcncr (1f gl ycinu (\rip Ivollrtrtln p r o l ~ c ,IIX- 12. (lnd Rrpurl~torrrayonrs!7, 08-50)
'"
Ccll line cnurccs lor NMDAII m l I N A 08-16)-02:Thc :IstrocytoInn ccll Elnc K-I 1 I has hccn usctl as a stnlrcc nf mRNA cnccidln~acctylcholinu and glutnnintc ruccptclrs supporting the cxprcssfon of :I sinall nunihur c ~ NMI3A f rcccptors whun injcctcd tntn X r n o ~ w ?n o ~ ~ t c s ~ ~ .
Exprcsslon of ionotropic glvtamntv recuptor gcncs has also hccn reported in p19 cmhryollal cnrclnoma ccllsZ5. Thc tcrminallv d~ffcrcntiated postmitotic human ncuronal ccIl linc hNT has hecn ruportcd to cxprcss functional NMDA- nnd non-NMI3A rccuptor-channcl2'.
Developmental regulation N M D A l l conrrol of gpne-ncrivnrion event,?in hippocnmpnl cellx OR-11-01: rnflux of C:a'+ t h r n u ~ hthc NMDAR and L-type ~ a "channcls of hippc~can~palncunmus has hccn shown to rcgulatu gene transcriptionalt events In thu nucluus2". Activation nf snultifunctiona2 ~ n " / c a l n l r l d u l ~ n tlcpcndcnt protcin kinasu ( C A Mk~nnsc)is cvokctl by stimulntinn of c ~ t h c r
NMDA rcceptors or L-type Ca'+ channcls [hut appcar 'critical' only for propagating the L-type Ca" channel simal to the nuclcus). Thc NMDAR and L-typc Ca"' channcl pathways activate transcription hy mcans of differcnt cis-acting regulatory elementst in the promotcrf of the immediate-earlyt proto-nncngenet ~ - f n s ~(see ~ ' below). For rolcs of NMDAR activation in modulation of synaptic plasticity (resetting of synaptic strcngth), see Phenotyprc expression, 08-14, Thc rclationships uf ncuronat activity t o gcnc cxprcssion h a w hccn rcvicwcd".
NMDAR control of cell migration ('growth cone movement']in developing c e r e h e k m 08-11-02 NMDARs appear to play an early role in the regulation of Ca2'dependent cell migration before neuxones rcach their targets and form
synaptic contacts2Y.Rlnckade of NMDA receptors by specific antagonists curtails granule cell migration in dcvcloping momc ccrchcllum2Y. Furthermtirc, enhanccment of NMDAR activity [hy the removal of ME'+ or Rlycine application] increases the rate of cell movement. Increase of endogenous extracellular glutamate (by inhihition of its uptake) also accelcrates cell-migration rates".
N M D A channel activation is required for continued growth ~f c e r e b e l h c d s in culture 08-1 1-03: Cerebellar granule cells in culture develop survival requirements which can he offset either by chronic memhrane depolarization /25 mM extracellular K', see Iielnw) or by stimulation of ionotropict cxcitatory amino acid receptors3'. This trophic effectt is mediated via Ca" influx, cithcr through dihydropyridine-sensitive, voltagc-dependent calcium channels (activatcd directly by high K' or indirectly by kainatc) or through NMDAR-channels. Calmidazolium la calmodulin inhibitor] counteracts the trophic effcctt of elevated K ' with high potency (IC5n-0.3 pu], indicating that thc trnphicf effects involve a Ca7'/calmodulin-dependent protein kimsc 11 activity'".
interrelations hetwcen neurotrophic responses nnd synaptic activity 08-1 1-04: NMDA neurotraphict tesponses in rat cerchcllar granule cells arc
modified hy chrnnic depolarization in culturc3' Cclls culturctl in high ( 2 5 m ~ 3K' conditions only respond with a rise n cytosolic free calcium concentration when external Mg'+ is removed. When granule cells arc grown in low (5 m ~ K'), NMDA exerts a neurotrophict effect. At the critical time for this effect, NMDA elicits a ICa2'1, rise in 5 m M K' cultures even in the presence of Mg2+. Growth in 2 5 m ~K' induces the rapid appearance of M$' block (see Blockers, 08-43). Thus, rises in lCa2+1],arc associated with the neurotrophfc effect of NMDA. Note: Ncumtrophic factor production (which in turn influences subsequent cel I-developmental lincagetj may itscIf require synaptic activity (see DeveIoprnenf rqulntron undcr ELG CAT GLU AMPAIKAIN, 07-1 7).
Pharmncnlogical induction of immediate-early gene expression vin N M D R R octivntion
EQS and Junt form a nan-covalent nuclcoprotcint complex that hinds to the conscnsus rccognition scqucncc 08-1 1-05: The protn-oncogenest
(if thc AP-I tmnscriptinn factor+. FOS, Fun and othcr immediate-earlyt gcnc products have hccn dcscrihcd as ‘third rnewengersi ’ which arc rcmilatctl by sccond rncsscngcrsf such ;IS intracellular calci~irn~’~. In thc hrain, c-fast and c-junirnay hc inrluccrl bv cluvntvd ncuronal activity such a s occurs during prntylcnetctriznlc (PTZ) scizurcr. NMI3AH-gatctl Cn”-inf!ux plays a rolc in thc rnductirm of c-(ris cxprcssion in PTZ wzurcst“.
Tmns-crrprzonal nullvation cosiplrd
to
N M D A R stimulation
OR-1 1 - 4 N NMDA appltcatinn has bccn shown to tlircctly stimulatc rapid c-lm mRNA :iccumulation in dcntatu ~ y n i snuuroncs” (’stimulus-transcription coupling’”4). In thcsc cclls, Cn” sc‘rvcs as n w x i n d rncsscngcrt coupling thc CluKs t o transcriptional activation1 of c - h ~mRNA. Thu rnutc nf Ca’‘ entry into dcntntc ncuroncs, howcwr, rlcpcnds on thc uxcitatory amino acid rcccptor subtype stirnulatcd. Won-NMUA receptor activation results In thc indirect cnhanccmcnt (if Ca”-influx via vnltaRe-sensitive calcium channcls, whereas NMDA rccuptor activation results in Ca” influx drrcctly throuRh thc NMDA channc! itsclf ’ I . Noic: rnhihitian af c-Fns synthesis foil7 to protect aRainst ~Iiitarnatc-induced cell d c a t P (we Phenotypic
Modulfltmn of N M D A R gene expression d u r i q Irrnin devehpmeni 08-1 1-07: Transicnt increascs in NMDA rcccptor expression nccur during dcvulopmunt, snd application of antagonists can hlnck experienccdependent plastitityi in thc adult (rrvzrwrd in rrC.”‘*).A t early dcvcl~qmental St;igc<, N M D A rcccptors arc ’ywntancously’ activated hy cndngcnrms ncurcitransmittcr”, hut thcsc rcwptors arc normally lost by an activity-dcpcndcnt Ahscncc of sensory input dclays the Ins? of thcsc functions””. Notably, inc.rrri.r;rd lcvcls o f RNA transcripts I for CaZ’Jcdmodnlin protein kinasct, GAP43j a n d glutarnic acid dccarhnxylasel hnvc hccn rcpcirtud m d c r thcsc conditirins o f scnwry tlupr~vnt~cin.”’,suKRcstinK their ~ c n u sarc rliffcrcntially rcgril:itcrlt hy calcium Influx.
lncrcoscs in N M I l A K exprcxsfon /‘olhwiFr,qtrenttnpiit with nestrodiol 08-1 1-08: Incrcascs in NMDA agnnist sitcs h a w hccn r>hscrvcd follciwmg treatment of C A I hippocampal ncuroncs with thc ovarian hormonc oestradia12’. Nr~sr,.Oestradinl has hccn .shown t o affuct cognitive fonction a n d Iowcrs thc thrcshold for seizurest.
Control of NMDAR properties during development 08-1 1-08: In gcncral, thc molccular propcrtics nf NMDA receptors arc developmentally replated and may cnntrol thc ability of synapses 20 changc in carly Iifu. Thc physiologicnl and pathophysiological ralcs af excitatory amino ncids (and their rcccpznrs) during dcvclnpment have hcen rc.V!CWCd“.4‘.
Develnpmentd r w i r r t i m ~in e x t m c e ! I u h Mg2+-block (see nlso Rlnckers, 08-43] 08-1 1-10: Hezcrologoust NMDAR-channel cxprcssmn studies
have revcalcd
developrnrntal variations rn Mg2+-hlack qenqitivity amongst diffcrcnt
receptor s~thtypcs(for signifjclincc. x C e Phenotypic cxprcssion, 08- 14, and Blockers. 08-43): Rctcasc from Mg%lnck aftcn allows nr ‘facilitatcs’ thc occiirrcncc of long-term potentiation1 (LTP). Within thc irnmaturc visual cortex (which is more susccptihk to LTP than adult visual cortex) synaptically activatcd NMDAR haw varying hut clcarly rcduccd scnsitivitics tn Mg2’ blockd2.This variability is not ahscrvcd in thc adult hrain, and it has Iiccn pmposcd that ’initially cxprcsscd, latcr-climinatcd’ NMDAR cxhihiting ‘rcduccd’ MR”-hlock phcnotypcs may undcrlic thc grcatcr susccptiliility to plasticity+ in thc irnmaturc ncocortcx4’.
Other developmentnl chnCyesin N M D R R charocteristics OR-1 1-11: NMDA rcccptors in rat hippocampus appcar Icss voltage-scnsitivcq' and lcss ~ g ” - s c n s i t i v c at ~ ~cady stagcs of dcvclapmcnt, allowing grcatcr influx nf Ca” than in the adult (wc ~rlsnRlncker?, OX-43). Dcvchpmenza! i ncrcases in gl ycioe-binding sites associatcd with thc NMDA r ~ c c p r r ? ~ . ~ ~ can aIsn enhance ca’+-inflvx in mature cclls. NeurnnaI connectivity patterns arc altcrcd by selcctivc blockade of post-synaptic NMDA rcceptoas in dcvelnping visual pathways4‘. ( . ~ c enlso Devehpmcntal rc
N M D A R nctivii y nffccting phenor ypic dcvelnpment 008-11-13: Thcrc arc scvcral rlocumcntcd developmental phenotypic cunscqucnccs nf altcring NMUA-gatcd chnnncl function, For cxaniplc, ph:irmacological hlockadc of the NMDA rcccptor in dcvclnping rctinnrcctal ’ ~ ‘ ’ rcndcrs proicctirins altcrs rctinal g u q l i o n ccll arhnrt s t r u c t ~ r ~ ~ and striatc ccirtt‘x rcsistant to the cffccts of mrmicular dcprivaticm”‘.
Phormncolqqical
~ 0 d l 1 h t t O t 1of
neuronnl development
08-1 1-14: NMDA agonists can restore plasticityt of X c n o p u ~hinncular maps Iicyond a critical period in dcveIopmcnt”.
Envirnnmentnl ‘cues’ for control of N M D A R expresxion 08-11-15: Activit y-tlependcnt, campetitivc synaptic interactions, which stabilize some axon hranches and ricndritcs and remove nthcrs, involvc glutamate rcccptor exprcssion [see ELG ~ c fncrs. y entry 04.nnd rcview.P5*I. Dark-rearing delays ~ F F C loss d NMDA rcccptnr function in visual cnrtcx3‘
(,we hclnw). N M D A rcceplor reqponses underlying developmentnl rcdirctions in
synoptic plasticity
08-1 1-1 6: NMDA rcccptora iirc crucial for experience-depcndcnt synaptic
modifications that occur in thc dcvclnpinR visual cortex. NMIjA-mctliirtcd cxcitatciry post-synaptic currcntst [EI’SCsl in laycr 1V ncuroncs of thc
vlsz~nlcortex h a ~h ~~ t' ' ~11own i~ to 'last I o n ~ c r ~n ' y o i i n ~ratq thnn In atli~ltrats. Fi~rthcrnir)rc,rlurntlcln'i of the EIRSL'\ I~ccomcprt~xrcqs~vcly shorter, In parallcl with thc rlcvclr~prncntalr c d i ~ ~ t ~In o isynsptlc ~s p l ; ~ s t i ~ iTt h ~ tl ~I ~ O~ C T O ~ Sin U NMIIA rcccytor-~nur!i;~tcllEI'SC tlur.lt1c111 I $ tlcl~ycrlwhen thc nnlmnls nrr rcarcrl in thc d;~rk,;I c(>!(JW ( ~ t u !F(y. 1 )
N M I I A H-chnnnrl ~ clvr~t t ion
cort rx
ill
rlr'vcfopr??cnt oC lhc sc~tn(if nscnsory
08-11-17: Chnnxcs In NMLIA chnnncl activlty may oxpla~nthu transient plasticity oh~crvcrlin laycr IY tlurlng early pmt-natal devclopmcnt. NMDA ruccptor-mctll~tcdcurrunts arc pnlmlncnt In 1;lvcr IV cells of immeturc inotrsc somatownriorv cnrtex (thnl;arnt~corticalsynapses) Ixfnrc maturation of ~ n h i h ~ t i t m ~Errrlrcr ". thtsn post-natal day 9 the ~ n a i o r ~ zofy rcsponscs arc mt~no.;ynnpticnnrl pllrtly cxcltatory, with both non-NMDAR and N M D A R mcrliatcd g l ~ ~ t a l r ~ a t c r g ictimprmunts. c In c~ldrr r l l l i n ~ c r l q , disynaptic inhihitnry currents sl~rnmatc wrth the excitatory oncx nntl Iuwcr thc rrvcrsnl pcltunti;~l c ~ f thc rcsponsu to v t ~ l t a ~ c ';tt i which the NMDAR cr,ntll~ct.lncr1s lnrgvly hlockcdi6
Isolation probe Fxprr vsiclt? c l o n E n ~prof ouol 08-12-01: A function:~!cllNA clone fnr t h t N M n A rcccptor was ftrst ist~latcd hy expression claningp'". P o I ~ ( ArnRNA \~ prcparctl from thu forchmins clf 4wcuk-rhd malc r;lts w:~s s~ilycctcrl to cuntrifugntirln cln 5-25?, sucrosc tlcnslty grad~cntq(srbru H r f 5 ' ) . A f r ~ c t i o nxtvinl: 'prltcnt clcctrt~phydolo-l rcspon.;csl t o l ( l O , r ~ NMDA (i~~casurcrl In ~ ~ " - f r cimctliz~m c suppluil~tntctl with i U / l r n glyclncl followlnp inject~oninrtj X t . r m j i ~ r \ oocytcr was u r d for constr~tction4 d a dircutlr)n;il ,clINA 1lhr:iryi in n p h n ~ clambda cloning vcctorl. TJ R N A p o ! y ~ ~ ~ c r a sCRNA-runofft ci from DNAs prcpnrurl from ~ntlividu;~l suh-prlots of this I~hrarywas used t c ~~ n l c c tinrlivirl~lnlXcnopr~s oocytcq. I ~ r ~ l a t i oofn a slnglc NMIJA rcccptor chlnc (lnmhda vcctor jNhl)/ pl;lqm~d vector pNhO) wns ncl~icvctl hy stcpwrw fr~ct~onaticln(lf u 'rc.;ponsc-cvok~ng'clSNA rnixturu"'.
1
I
Ilr~rnrrrrf rJ IJC1l pmhal; for isolnt rng rolr~tcd,cone ff~mily rnenrhcrs
OR-12-02: An RT-T'CK~str;ltcgy was ump1clyc.d to ~ s o l a t cprnhcs for t l ~ c NMI)AII sithtypc.; N ~ I ~ A - N B I This ~ I I ~mcthorl rclicd o n 3 cnnscrved aminn acid scqucncc hctwccn NRT and nthcr irlnrjtrc~plc GBiiKs (YTANLAAF in tlic vicjnity elf the pl~tntivctransmcmhranc M,3 wgmunt rlf 1 thew rcccptorq. ~ e ~ e n c r a t cPCR l primers synthcslzct! I~ctwccn this sltc .inil a T7 KNA pr,lvmcr;~scf prc~inc~tca+.;cr~llcnccin the Iihrary cl(ln1nr: vcctort prrmlttcd rutricv;ll rh IJNA fraxrncnts w h ~ c hwcrc urctl nF; NR2A- N [{?,I ) - S I ) C ' E ~ ~ I C~ T O ~ ~ C S .
entry OR
Imrnunoprohes for detection of ,ylutamate-hinding proteins 08-12-03:k cDNA clonc encoding a 33 kDa protein (613333with similar pharrnacolrigical prnpcrties to NMDAR has been obtained by scrccning a library with an antihody generated aRainst glutamate-binding pmteins" (see Sulitype clnsdicntrons, 08-06).
mRNA distribution See the mcthoddngicnl notc m m R N A drstriliution under ELG CAT GLU A M PA JKAIN, 0 J - I.?.
'Neor-universnl' expression of the 'fundamentnl' NMDAR subunit in the CNS 08-13-01: In situ hybridization to adult rat brain sections shows that the
NRl mRNA is distributcd in almost all neurrrnal cells throughout thc brain. Thc spatial pattern observed with NRl mRNA is 1argc:cly consistent with autoradiographic studies using several radiolabelled ligandsf nf the NMDA receptor. expression of NR1 mRNA is ohscrvcd in thc cerebellum, hippocampus, olfactory huh and hypothalamus. In the latter, large signals arc ohserved in granule cclls and CA4 cclls of the dentate gyrus and in pyramidal cclls throughout the CAI-CA? reginns'"". Sec irlso Tahk 3. 08-13-02 'Prominent'
N M D A R siihiinii m R N A dis!rihution by single-cell RT-PCR 08-3 3-03; Glutarnatc rcccptor subunit mRNA cnrnpnsitinn has hccn mcasurud using RT-PCRt in scvcral individual ncurmes within rat hippocampal CAI ~ l i c c s ' ~Gcncrally, . cach CA 1 ncurone contains varying amounts of most glutamatc rcccptor mRNAs. These single-cell expression studies h a w rcvcalcd novcl, alturnativcly spliccd forms of the NRI ( s w Gene or$y~nfzfitlnn.08-20] and kainatc receptor typc 2 subunitsfn. SurprisinREy, cnmparcd to other GluR suhunit message, NMDAR typc 1 mRNA is of rclativcly low abundance at the smgle-ccll lcvcl (see helow). Furthcrmorc, RNA cditingt WAS shnwn nnt to he a Renernl cellular
mcchanism for GluR divcrsity (cornpnrc Gene ar,qonization under ELG CAT G1-U AMPAIKAIN. 07-20).
Differential gene expression of mR NAs encoding 'potentiating'
snhunits
08-13-04: In c m t m t to the wide distribution of the zcta 1 ( N R l )and epsilon I (NR2AJsubunit messenger RNAs in thc brain, thu epsilon 2 (NR2B) subunit mRNA is cxprcssed only in thc forebrain and thc epsilon 3 (NR2CJsubunit mRNA is found prcdominsntly in thc cerebellum5Y. 0 t h than thc ubiquitous NRI, NMDA-selective GluR subunit gcncs display differential
spatial expression"
[SEC
Tflble 3).
Surnmnry of regional m R N A expression potterns for N M D A R rn brain
OR-13-05: Individual mRNAs for the NR2 suhunit series (which providc a molccular basis for the 'functional diversity' of the NMDA rcccptor)
entry O X
Table 3. nlRNA clisfrih~ltion:slln?n?rIr\. of rc.porfrd fissrlc 1iistril)trtions NMI>A rrcc.ptor f ( ~ n ? i lmHNAs \~ (Frorn OX- I,?-041
NRl NR2A
NR2R
NR2C
NR2D
o{
Expressed ubiquitously (i.c. almost all ncuronal cells in all hrain regionsl'". T h c hypothalamus contains only thc NRI transcript, suggesting the existence of atlrlitional NMI>A receptor subunits" Wirlcly cxprcsscd in many hrain rcgions. Ahi~nrlantin ccrehral cortcx anrl hippocampus, internal granulc layer of the olfactory ht~lh,nntcrior olfactory nuclci, olfactory tuhcrclc, certain thalamic nuclci, infcrior c o l l i c u l u s , p n t i n c nuclci, inferior oliv;iry nuclci and ccrchcllar cortcx . NR2A mRNA distrihution hears the closest rcscmhlancc tcl that of NRI anrl is prcscnt in both forchrain and ccrchcllum". T h e amyjidaloid nuclci cxprcss mRNAs encoding NR2A and NR2R hut not NR2C" Widely cxprcsscrl in many hrain rcgions, hut morc restricted than NMDR2A. Ahundant in cerebral cortcx and hippocampus, tclcncephalic and thalamic rcgions, hut lowly cxprcsscd in the hypothalamus, lower hrainstem anti ccrchcllumx. NK2H is cxprcsscd in forchrain, and has a complcmcntary distribution to NliZC mRNA". T h e amygdaloid nuclci cxprcss lnRNAs cncorlin~ NR2A and NR2R hut not N R ~ C " ,NR2R-specific mRNA is cxprcsscd mainly in granulc cclls" Shows localized mRNA distrihutic)n in comparison to NMDR2A :ind NMDRTR. Prominently cxprcsscd in the ccrchcllar granular 1:iycr. Motlcratc cxprcssion of this mKNA is sccn in the gl(~mcrul;~r and mitral crll layers of thc main olfactory hulh, Also ohscrvcd in some of tho pontinc, thalamic and vcstihular nuclein. NR2C is cxprcsscd at highest levels in thc ccrchcllum, and has a complen~cntaryrlistrihi~tionto NK2R and mRNA". The ;~mygcl:~loid nuclei cxprcss mRNAs encoding NR2A and NR2R I ~ u tIIO!NII2C:. Siniil;~rly,in thc caudate-putaincn, thcrc is no NR2C: mRNA, hut motlcrntc signals arc dctcctctl with NR2A and NR2R l~rohcs".NR2C-specific I ~ I R N Ais cxprcsscd mainly in tilftctl ;~ntlniitml cclls" Highly cxprcsscd in the dicnccphalic and lowcr hrainstcm (suhcortical) rcgions. High in sift1 hybridization signals arc ohscrvcd in the glomcrular laycr of the main olfactory hulh, the ventral pallidurn, thc majority of the thalamic nuclci, the hypothalamus, superior colliculus, suhstantia nigra, vcstihular nuclci, pontine nuclci, and deep ccrcbcllar nuclci. Low cxprcssic~n is ohserved in the central cortical rcgions and the gmnular laycr of the cerchcllumn
Notc: T h e spatial tlistrihution of channel suhunit-specific mRNAs presumahlv reflects a 'functional specialization' in particular cell types. Mapping of cxprcssion patterns is a complex task anti has to take Inany variahlcs into :iccoiint, such a s in s i t t ~localization, dcvclopmcntal regulation, suhunit stoicliiomctry, ;lntl factors regulating overlapping or co-expression. For notes on the integration of computer-l>;iscd information resources ahle to crossreference thcsc diverse fiictors, sot* Fr.ctll,clck cu) (7SN c.ircr.ss, cntrJr12, mnrj A17[lrsrltli.u / - .%,c~rc.llc.ritvric1 0 1 C S N clc*\,cplopn~cnt,rmtr!r 0.5.
overlap in some hrain rcgions hut also show localized expression pattern^'.^. Anatomical distrihuticins which havc hccn dcscrihcd in thc litcraturc arc givcn in Tahlc 3 (sre ihc ori,pinrtl in situ hvhridizntion s t r r r i i e . ~- rr~i.~"*~*'*. ond ~ h (oornotc c tn Tohlr 3 ) .
''
Phenotypic expression NMDA receptors contrihrrte to n lnrgc number of neurophysiolopicol phenoiypes 08-14-01: Thc demonstrated involvcmcnt or 'association' (if NMDA rcccptors t n several neumphysiological and pathophysiological proccsscs has given grcat impctus for thc dcvclopment of sclcctivc pharmacological modulators of thc NMDAR (scr Kcccptor an!osoni.~ts,08-51) and tcir dcfinitinn of thcsc phcnotypcs at the molecular and cellular Icvcl'"."' (Trthlr 4). A morc extcnsivc list of chmnic neurodegenerative diseases in which iGIuR dysfunction or 'ovcrstimulatinn' mnv play 3 role is included in ~ c f . ' ~ .
Table 4. Fllnctionnl nlki1.v o f N M D A R-chnnncls (Frot?~OX- 14-01) Physiological tunction/pnthaphysicilogic~1phcnntypc involving NMDAR-channels
Sclcctcd rcfs
Developmental synaptic p~asticityt (src hcl(~w and I'rnlr-in intrSrric.tir~ns, OH-31) Learning and memory Trans-synaptic gene expression controlt Normal information processing Srr cllso CollingritIgc, IYHV, rrrldcr Rclrttcd sn1lrcr.v csl rrvirws, OX-56 Sensory ncurotransmission including amplification of auditory responses nnrl ainpliticatinn of excitatory and inhihitory visual signals Anxiogenesis 1 ncvclopmcnt and maintcnance of epileptic+ states Dcvcloprnent of olivopon toccrchcllar atrophyl Dcvclopmcnt of motor neurone disorders ~ ~ ~ o ~ l ~ c a cepisodes rnict Post-ischacmic hmin dnmagc: pathophysiology and ccll death C'nrdic~vascularprcssurc alntrol (haror~cc~tiont ant! control of vasomotor tonet] CcntrnE scnsitiz;~tionduring nocicrptiont (pain reception) (SCP r11.w I,clnrv) Activation and maintcnancc of motor rhythms [ c . ~in . rcspiratory control NMDAR hlockadc cnuscs prolonged inspiration)
u t , SO. 5.7..54.6.1-72 .5.1.6<,.
7.3.74
2s. .?4 75
.TI(.
7.6~2
~3
84-85 ~b
~ 7 . 1 ~
RB 4R'M9.3
Y~-V(,
' ' 9 ~
Kt~r~ir~rvc~d in
r c k .'':If''
C o n c e n t r a t ion- and t i m e - d c ~ p r n c l l ~ nglutrlrncltr t to.uicity 08-14-02: Excess net ca2+-influxthrclugh NMDA receptor-channels is a key
step in triggering the nel~ronaldcath intluccd hv hrlcf, intense glutamatc cxposurcfn'. T h c amtilint of " ' ~ a ' ' accurnul:~tion correlates closely with the 'tlcgrcc of ncuronnl' cicath 24 h I:~tcr'"' over ;~pplicdgliltatnntc conccntr:ltions ranging hctwccn 1(1 and I O O 0 p h ~ ;ind d i ~ r a t ~ o nof s exposure not(': Rcl:ltivclv little ~ ; ~ . " - e n t(as r~ hctwecn 0 anti IOniin. Cornprirtrtir~~ measured hy ' " ~ n ? ' acctimulatic~nlhas Iwcn ohscrvctl in sinlilarlv treated gli;~lcells.
A lr;r,yc nrinihrr o f . s i g n r ~ I l i nc:cort7ponents ~ r?tr~,vc o n t r i l ~ u t ct o ,yI~itr;nzr~ic toxicity 08-14-03: As summ;~rizctiin Tnhlc 7 (sr,v I'rotcin intclnlc-tions. OX-,311 and reviewed in r ~ f . " ' ~ ,several caz+-activated enzymes may contrihutc to excitatory arpin~,acid toxicity, inclutling prntcin kinase ~ t ' ~ ~ ' - ' " ' phns, pholipase Azifn'pf'"',phospholipasc CI "", Y ; I ~ ~ ( I I I Scndonucleasest"', ca2*/ calmodolin-dependent protein kinase II"*, nitric nxidc ~ y n t h a s e ~ ' . ~ ' ' " calpain 1 [i-nicrt,mtrl;~r (23"-sensitive protcolysisl and calpain 111 (mill~molar~ a " - s e n s i t i v e prt~tuolysisl""12n. Since regillation of c;I."-influx f expression, dysrcappcilrs tilndamcntal to the 'orrlcrcd progression' c ~ Kcnc p l a t i o n of ~ a "hnmcostasis is nlso likely to have longer term effects on ccll phcnotypc or the initi;itiirn of apnptnsisj. Kc1c~vr;nccof ~ J u t o m n ! et o x i c i t y to n ~ ~ i r o ~ l ejm~ ~ ~nO C~ CrS ~S Ct S 08-14-04: A ni~nihcr of pathological/neurndcgenerative cnnditions can hc cxpl;lincti hv glutamate toxicity :IS a central mcchanistn 1c.g. when prolongc~l r c c r l ~ t c ~ r - n ~ c i l ~ : ~dcpol;~rization tcd results in 'irrcvcrsihlc 'j2.*"' 121-'f5 1. T h e correct disturh:~nccs' in ~ c ic~ n hr ,l~~ct~st;~sis*"."~. functiclning of Na+/K+pumps is ; ~ l s oimportant for l>rcvcnticin of glutam;~tc toxicity ( ~ r v I'rotr,irr . intrrrrr.iions, O X - ~ ? l ) . Notit: Alth(lugI1 NMDAK nlny hc the p r r ~ r i o r ~ ~ iroiltc ~~rr~ for~ l~ a " - e n t r y a t toxic Icvrls, other routes nlav hc involveti. o t h e r c:~nrlicl;ltc pathwa y s f ( ~ neurotoxic r ~a"-influx includc Land N-type vnltagc-gated Ca" channcls ( s r , r p VLC; (:(I, ctnrrtr 421, nnnN M D A i(;luRs ( t h r r ~ ~ i gAMPA-sclcctivc h iGluR I(rc.kins GluR-1%subunits . s ~ ( E~ l . ( ; ( : A T ( ; I , / , AMl'A/KAlN, rntry 071, influx channels intlircctly opened hy metahotrnpict glutamate receptor stimulation, ~ a * / ~ a " exchangcrs, and non-spccific rncmhrane leak'". Some of these routes arc illustr;ltcd in Fjcy, 4 rrnrlcr I'mtrin intc,rrlrtions, OH-,?I. N c u m p a t l ~ o l o g i u o cl o n d i t i o n s ru.sociatcd with ' o v c r s t i m n l n t i o n ' o f ionot ropic , y l u t r ~ m r ~rteec e p t o r s 08-14-05: The involvc~ncntof iGluR arc wcll-established in the initiation and and in the t ~ ~ n s s i vneuronal c cell deatht that propaga tion of scizurcst "&* follows pcrir~ds of strnkct-inducetl isrhaemial and hypnglycaemiatpR'" (S(Y Iwloiv). ~ I V S ~ I I I I Cof~ g~II~~tiaInI ~ i n c r ~ ipathways c has llccn implicatcd Ithough not sl~r.c.i/ir*r~lIyasst~ciatctllwith the pathogenesis of a numher of nentoclcgcnerativc diseases (sr,r h c k ) ~ rfull , references listed in Alzheimer's diseaset (tentativrl, Thcsc. tiisortlcrs incl utlc cpilcpsyt'", Huntington's discasel, AIIIS encephalnpathy~/dementia complex, amyotrophic lateral stlcrnsisi and lathyris~n .
'"
entry 08
Time-dependent changes i n synaptic transmission failure in models of 'in vitro ischaemia' 08-14-0(,3 Fivc minutes of oxygen and glucosc deprivation ('in vitro ischaemia') causes long-term synaptic transmission failure (LTF) in the CAI region of the rat hippocampal slice. In huffer containing 2.4 mM ~ a " , thc extent of LTF largely depends on thc average levcl of 'exchangeable' cell c a L ' in CAI during this proccdurc',7". In this model, unidirectional ca2+influx during the 'first 24 min' of ischacmia depenris entircly on NMDARchannels; howcvcr the NMDAR antagonist MK-801 has no effect during the 'second 2f min', prnhahly as a result of NMDAR dcphosphorylation. ~schaemia-inducedca2'-influx during the second 21 min of ischacmia can he partially attenuated Ily -25% hy the voltage-gated channel hlockcr nifedipine ( f i o i l ~ and ] hy an adtlitionnl 35% hy the ~ a ' l ~ a exchange " Thc AMPA/kainatc antagonist DNQX inhihitor benzamil ( 1 0 0 ~ 1 ~Note: ). has nn effect on the ca7-'-influx in this modcl'"".
Total cell ca2+ prior t o in vitro ischaemia is adequate to cause complete LTF 08-14-07: In the hippocampal C A I slicc model of in vitrn ischaemia (previous purogruph), pharmacological hlockadc of enhanccd cal'-entry during ischaemia in 2.4 mM Ca?' has no effect on the LTF phenotype'"". Howcver, thc NMDAR antagonist MK-801 strongly protects against LTF when the buffer contains caZ' at concentrations closcr to physiological lcvcls (1.2 m ~ )A. cornhination of hlockcrs for NMDARxhanncls (MK-801) and AMPA receptor-channels (DNQX) prcvunts LTF in huffcr containing 1.2 mM Ca7'. Thus, AMPAikainate receptor ~ctivation makcs some contrihution to ischaemic damage, although this ApDCArS independent of enhanced ~ a ~ + - c n t r y ~ . " .
L o n ~ c rterm NMDAR-associated neurotoxicity 08-14-08: Slow incrense.~in N M D A channel I:,,, may also provide an excitotoxicf mcchanisrn in that ca2*-influxcan increasc markedly in cells suhject to prolonged depolarization'"' (see below nnd Current-voltage rclntion. 0835).
Extended neuron01 depolnrizntion following removal o f glutamate agonist
u
08-14-09: 'Physiological rcsponscs' of hippocampal pyramidal ncuroncs in primary culture to prolonged (-10 min) exposure to 5 0 0 i 1 ~glutamate show that the ncuroncs remain depoladzed (a20 mV from rest) following 'washout'. This depofarization is accompanied hy a -57.8% increase in mcmhrane conductance, initinllv hy NMDA channel opening and suhscquently through othcr ca2'-influx channels. Since depolarization can he maintained for long periods 1.30 min to < 4 hJ, the phcnomcnon has hcen dcscrihcd as extended neuranal depolarization (ENDJ"'. During END, cells retain 170th the ability to fire action potentials and the ability to respond to glutamate, and can cxcludc vital dyest'?,'.
lndircct neurotoxic effects 08-14-10: In retinal ncurnncs. metabolic inhibition results in the loss of
voltage-dependent ~ g ? hlockadc ' of the NMDA receptor-channels, leading to an incrcascd potency of glutamate agonists"" ((or rlgnil~roncr,svr
Hlock~rs.08-4.31. 'Protection mechnnixn~s' for cxcitotoxic phcnotypc.~ 08-14-11: Extracellular 2n2+dccrcascs NMDA receptor-mediated toxicity1"".
This effect is in contrast to its potentiating effect on AMPA reccptormctliatcrl ncurotoxicity (srpe IJhcnotypic expression under ELG CAT GLIJ AMi'AIKAlN. 07-141. Nitric oxide may havc a role in protection of NMDAmciliatcd cxcitotoxicity (for tlctnils, see Chunnrl modulrition. 08-44). ~ n t i s e n s e oligonucleotides ~ to the NRI sequence havc hccn shown to 'protect' cortic;~lncuroncs from 'cxcitotoxic' reactions hy reducing (i) NRI gene expression and (iil the volume of experimentally induced focal ischaemic infarctionsn4.
I'revcntion o f 'cell death phenotypes' in cells expressing NX IINRZA hetrrotncric NMDAH 08-14-12: Co-cxpression of NRI and NR-2A subunits in HEK-29.1 cells rcsults
in cell clc;~tht, hut viahility can he maintained hy including the NMDAR antagonist 11~-2-arnino-5-phosplionopentanoic acid (AP5) in the culture tncdium post-transfcction" ( s c ~I
NMDA R modificr~t ion in mnintenance o f cpilcptic stntcs 08-14-13: Chronic epilepsy induced hy kindlingt can he considered as an NMDA receptor-dcpcndcnt form of activity-dependent ncuronal induced in rrirro, which results in Lirtin,y n~odrfictrtion.cin the function of singlc NMDARS'.~'. In control ncuroncs, the amplitude of whole-cell NMDA currcnts is not sensitive to the presence of an intraccllular ATP regcner;ltion systcm'l, whereas NMDA currcnts in k i n d l e d cells show a xrcat vari;~hility, with larger ;unplitudcs ccmsistcntly rccordetl in the prcscncc ot intraccllular 'high-energy' phosphates.
NMDA chnnncl propertics nffclctcld
kindling
1 7 ~ 7
08-14-14: Kindling'-induced epilepsy predominantly affects the mean opcn
timci, bursti, ilncl clusteri duration of NMDAR-channels, their sensitivity to intraccllular 'high-energy' ph!~sphates, and their hlock hy M , hut not rntcs of receptor dcscnsitizationi or single-channel contluctnncc 'values'"". Such alterations may reflect a change in the molecular structure of NMDA channcls ant1 may underlie the rnriir~tcncir~cc~ of the cpilcptic state.
r'
I'uI.si~tilc glutmmrlte re1c.osc~ following N M D A R-channel mctivntion 08-14-15: Periodic inwarrl currcnts have hccn shown to he gencratcd in
nconatal ncuroncs hy n synchronous, persistent, pulsatile glutamate release fmm prc-synaptic nerve terminals (secondary to NMIJA receptor stimulation and oscillations in intracellular c n l c i u m ) ' " ~ ~ s eFix. r 4 11nrlr.r I'rott-in intr~mc.tions.OX-,? 1 ) .
NMDAR-channel frinc'tion In centrtll wn
entry 08
-
fibre inputs to the spinal cord and hrainsteml"' and, in addition to semtonergict, peptidergict and noradrenergict mechanisms, may mediatc pain receptive input in thc dorsal horn (Note: C-fihrc inputs wcrc first asaociatcd with pain reception pathways over 60 years ago1"*). NMDARchannel activation mediates tllrcc rclatcd phenomena associated with pain rcccption in dorsal horn neurones (revicwcd in ref.'"): (i)the formalin response, a model of pain rcccption by suhcutancous injection of formalin, (ii)amplification or 'wind-up' of dorsal horn ncuronal response, and (iii) modulation of the flexion reflex, initiated by pinching of the cutaneous surface of thc foot (the response can hc incrcascd hy 'prcstimulation' of C-fibre inputs). Notcs: 1. Thc 'wind-up' response has also hccn charactcrizcd as hcing mediated by L-ty c ~ a "channels in turtle dorsal horn ncuroncsl.'" and can be potentiated hy protcin kinasc C in isolated mouse trigcminal ncuroncs (see Channel mndnlotion. 08-44). 2. All three responses (ohove) can he hlockcd by systemic administration of NMDAR antagonists in dcccrehrate animals. 3 . Antagonism at the glycine site on thc NMDAR reduces spinal nociceptiont in thc ratPR.
-r"
Role o f NMDA receptors in non-vesicular endogenous release o f C:A RA 08-14-17: The glutamatc-induced endogenous release of thc ncurotransmittcr gamma-aminnhutyric acid (GARA) is niainly mediated hy NMDA receptors, and consists of a single, sustained p h a s ~ ~ 'This ~ . phase is insensitive to nocodazole, partly inhihitcd hy verapamil and can hc blocked hy c o 2 + ions + hccn attributed to a block of as well as SKF 89976A. Thc action c ~ ~f o ' has NMDA-associated ion channcls. Note: The majority of CARA rclcase occurs from vesicles (whosc rclcasc can hc stimulated experimentally hy K'-induced dcpolarization) whcrcas the glutamate-dependent release is nonvesicular14n. Scc rrlso SYN (ve.~iculor), entry 40.
NMDAR-channels ns 'hinsensors' for release o f neurotransmitters 08-14-18: Outside-out mcmhranc patches cxciscd from rat hippocampal ncuroncs have heen used to dctcct rclcasc of cndngcnous excitatory amino acids (EAAs) from synaptic terminals of isolatetl turtlc photor~ccptors'~'. Electrical stimulation or application of lanthanum chloride to photoreceptors induces an incrcasc in the opcning frequency of 50 pS NMDA rcccptorc h a n n ~ l s ' ~ ' .Spontaneous channcl activity is also ohscrvcd near synaptic terminalst. Note: Exocytotic release of cndogcnous EAAs is an important criterion for clilssilication as an authentic transmitter. Involvement o f N M D A receptor-channels in synoptic plasticitJrt special note
J
08-14-19: Within this cntry, thc descriptions of processes affecting synaptic plasticityt havc hccn limited to phenotypic aspects dependent upon the molecular characteristics' of thc NMDA receptor ("see OH- 14-2h).Thc subject of neuronal phenotype rnndulation is a complex one, ant1 is difficult to clcscrihe outsidc of a neurnphysiological and neurnanatnmical context. For comprchensivc accounts of thosc sspccts, inclurlin~thc molecular pcrspectivc,
scc the sclcc~edrcfcrcncra listed in ~ c l r ~ t sot~rces cd d revicws, 08-50,
-
Cicncrml ~111ctiotypi~c,hrrngcs in long-tcrnl potcnt irrtion o f sytirrpt ic t rrrnsmission 08-14-20: T h e phenomenon of long-term potentiationt (LTP), a long-lasting
increase in the strength of synaptic trans~nission11uc to brief, repetitive activation of excitatory afferenti fibres, is a striking example of synaptic plasticity~'J'. Rricf, high-truqi~cncytrtanic stimlllation c ~ faffcrcnt fihrcs in h~ppocampals l ~ c c sinduces an LTI' of synaptic transmissioni, which causcs an 1ncrc;isc in the size of the synaptic response clicitcti hy low-frcclucncy stimulation of the same synapse. LTI' persists for scvcr;~lhours in rri!ro ant1 111' to scvcral wccks in virw, ;in11 is ;it present the most extensively studied rrvicrvs, form of activity-dcpcn~lcntsynaptic rlasticityt (for cr~ri~pn.~hr~risir~(~ g l , l , r r , f y 5 J 67. . 143-148
r!r1(!
1 < 1 ~ 1 ( 1 ! 1 Y 1,Yollt(.cs(I*) t(>lficB\\t,v,
(l8..50/,
Expcrit??cr?trJlinrluctiot? mnd phr~scso f LTI' 08-14-21: LTP processes can he induced hy ilclivcry of a tetanust [typically
50-100 stimuli at I00 Hz or greater) to the pathway of interest. Alternatively, LTP can he inducctl by ( i )theta hurst ~timulation'~" (typicnlly hy multiplc hursts of four shocks ;at I00 H z dclivcrcd s t 200 ms intervals) or (iil primed burst stimulationl"~typicnlly by delivery of n 'priming stimulus' followed hy n single hurst of four 100 Hz shocks LOO m s later). Thcsc cxpcrimcntal proccdurcs appear to simulate synchronized firing patterns that occur at similar frcqucncics in the hippoc;~mpustlt~ring1cnrning1",
Mr1thotlolo4yicolnotrJ - r??c,rr.~lrrr'n?rnt o f s!7nrrl~ticpotentintion phcnorncna 08-14-22: L T P ~p l ~ ~ n o t y p carc s commonly dctcrminctl hy plot tin^ ;I graph of the slope of the field EPSPi versus time in experiments where stimuli of
.spc,c.i(i(~cl frcqi~cncy;Ire dcl~vcreil(i.c. low-frequency, -1-10 Hz, ranging up to tct;tnici stimuli ( s r ~ rFi'y. ~ 11.1. EPSP slope parameters (mV/ms or "A) c;ln hc ilctcrmincil in the prcscncc ot ph;~rmncologic:iI:~gonists/nntngonistsot the NMDAR, or ;~ctiv;ltors/inllil>itorsof ;~ssoci;iteil sign:~lling components. St~stainedenhancement oi synaptic tr;lnstnission (i.c. LTPl is indicate11 hy ;I 'non-decremental response' ( s c ~ cFi,q. ~ 1 / 7 1 whcrcns short-term potentiation (i.c. STPl I S chnmctcrizcil Ily ;I 'decremental response' (sr,rB l~c-lorv rlr~rlk'i,y. I l l l i i ) ) .
N o t n r n c l r ~ t u r ~for ~ srrltrrnntivr fort??.^
oI . s y n ~ p t i pc o t ~ n t i ~ t i o n
08-14-23: T h e use of the phrase 'long-term potentiation' has hccn non-
sy.srrn7rrtic.rrll\, applied t o any form of synaptic enhancement1 lasting more than a few minutes. Howcvcr, scvcral cxpcrimcntnl manipulations can result in potentiationf of synaptic transmissioni that declines over -540 rnin (dccrcmcntnl synaptic tmnsmission or short-term potentiationt, STP). STP is likely to hc a prcrequisitc for stable LTI' [i.e. non-dccrcmcntnl svniiptic cnhanccmcnt). For further tlctails on thc relationships hctwccn thcsc multiple forms of potcnti:ltion, s r Fi,q. ~ Itl cjnd rr.h5J.6'.-'.
LTP rrnd LTD 08-14-24: T h c rcl;~tionshipof LTP to long-term depression (LTDI of excitatory .
syn:~ptic tr;unsmission is discussed in rcf.I5'. Thcrc is cvidcnce for the involvcmcnt of NMDA rcccptors in the intll~ctionof homosynaptic L T D ~ ,
entry 08
whcrc post-synaptic dcpolarization and increases in Ca2' resemhle those for LTP induction (as described below], howcvcr LTP requires a markedly stronger post-synaptic depoIarization (see ref.*"' for further tlctails). Development of LTD phenotypes depends on functional metabotropict glutamate receptors (SEC Tahlc 7).
Dependence o f LTP on NMDAR activation and depolarization 08-14-25: In the CAI region of the hippocampus, the induction of LTP
requires activation of NMDAR hy synaptically released glutamate with concomitant depnlarization c ~ f the post-synaptic mcml>rane'J2~'"". This rclicvcs thc vnltngc-dcpcndcnt mawcsium hlock of the NMDA rcceptorchanncl, allowing calcium to flow into the dendritic spine. A synaptic model of memoryt involving LTP formation in the hippocampus has hccn proposed (reviewed in ref.").
A n overview o f experimenlol models used i n the study of hippocampol long-term potentiation (LTP) 08-14-26: Within the scope dcscrihcd above (pawgraph 08-14-19), Fig. 1 attempts to summarize aspects of NMDAR LTP phenotypest that
ultimately depend on molecular characteristicst of the rcccptor-channels. Supporting information on scveral subtopics can he fnund under relevant fieltlnames of this cntry and the ELC; CAT GLiJ AMPAIKAIN cntry. ( A hrood illustrrrtion of !he 1ype.s of prc- nnd post-synnptic signalljng proteins internctin,~with N M D A receptor-channels is also shown under Protcin interactions. 08-31 . )
Direct experimental evidence for NMDAR-medinred ca2+-influxi n synaptic plusticity 08-14-27: Optical mcasurcments of synaptically induccd c a 2 ' transients
through NMDA rcccptclrs have been ohscrvcd undcr conditions which eliminate activation of dcndritic voltage-sensitive ~ a ? 'channcls in pyramidal cell dcndritcs within hippocampal sliccs (i.c. steady post-synaptic dcpolarization to the synaptic reversal potential)'". ~ a " - i n f l u x through synaptically activated NMDA rcccptclrs arc ciircctly implicated in thc induction of L T P ~ , since the magnitude of LTP is diminislied when induced with the post-synaptic mernhranc hcltl at progrcssively more positive pcltcntials (with cnmplctc suppression at pcltcntials near $100 ~vI'"~ Note: . Induction of LTP can he blockcti hy injection of intracellular ca2+chelatorst - c.g. microinjcction of EGTA into post-synaptic ncuroncs blocks the induction of LTP'"" and furthcr implicates a dircct rolc for c a L ' signalling in the induction proccss.
'Auqmentntion' o f NMDAR-associated calcium transients and other Caz'' sources 08-14-28: The ~ a * + - ~ e r m e a h i l of i t ~NMDAR-channels t (sec Selectivity. 0840) has Icd to a common nssumption that NMDA receptors play a dircct rolc in potentiation of synaptic rcsponscs. However, vnltage-gated ca2+entry (src VLC: Cn. entry 42) anil ~a?-'-relcascmediatctl hy neuronal ryanodine receptor-channels (scc ILG Ca Cn RyR-Cof, entry 17) and neuronal InsPl receptor-channels (sec ILG CN lnsl'?, entry 19) appear to
OVERVIEW OF LTP INDUCTION
a
I
PERSISTENT ('long-term') INCREASE ('potentiation') of SYNAPTIC STRENGTH
Signal transduction
post-synaptic NMDAR
I mechanisms 1)
I
b
I
EXPERIMENTAL MODELS
Brlel trelns of high-frequency stlmuletlon
I
(i) Typical 'non-decremental' response (LTP)
(ii) Typical 'decremental' response (STP)
Excitatory connections (e.g. perlorent path appllceble to other hippocampal excltstory pathways)
text)
granule cell
synaptlc transmission" see B~ISS, (1973) J Phys101292:331-356
............ .....,,...,,....,,........
Time, hours
c
TIME COURSE OF LTP-INDUCTION AND PERSISTENCE
induction phase: In vivo and In vitro generally
[lor d.scrlptlon of experlmentel meeeuramants, see text; for further dellnsatlon of 'decrementel responses: see Malenka and NIcoN (1993). Trends Nauroacl, 16: 521-5271.
Persistent phase: In vitro (hippocampal slice)
In vivo (non-anaesthetlsed)
several hours
several days
In vivo (anaestheala) several hours
Figure 1. Experimental models for synaptic LTP in the hippocampus. (a) Overview of LTP induction. (b) Experimental models. (c) Time course of LTP induction and persistence. (d) Classification of potentiated synaptic responses. (e) Absolute dependence o f synaptic potentiation o n 'sufficient' post-synaptic depolarization. ( f ) Molecular mechanism for NMDAR-dependent induction o f LTP. (From 08-14-26)
CLASSlFlCATlON OF POTENTIA TED SYNAPTIC RESPONSES
d
I
I I
-
- - - - -- - -
(i)
NMDAR-dependent responses - I hour 'Short-term potentiation' 8ppNcatlon of NMDA alone (wlthout depolarizatlon / ~ ~ + x p u l s b- n sea penel E, b l o w ) S I insufficient to induce LTP, but I t can induce STP.
-
sea (e.g. MK-801 Blockers field);
(
Receptor antagonieta f/eld).l
I I
I I
t I(ii)
STP con be converted into LTP b y manipulations that Increase influx of calcium into the postsynaptic cell See Malenka, (1991) Neuron 6 53-60
NMDAR-independent responses
-
I
1
I
I
I
General potentiatlng responses s d d l t l v e to LTP I
PAIRED PULSE FACILITATION Max~mum duratron - several mln
.
I
!
; > I hour or longer 'Long-term potentiation' Ssnsitlve to protein kinase lnhlbltors, nduclng persistence to 30-60 mln sansitlvo to decraarlng number of stlmuN In tetanus Sensltlva to the degree of post-synaptic NMDAR actlvetion Sensitive to the magnltude and tlmlng of post-synaptlc depolarization
.
/ drstmgu~sh~ng features Subd~v~slons
Durat~on 1
POST-TETANIC POTENTIATION (PTP) Saturation of PTP prevents lnductton of chemically-~nduced potentlatlon' - see note 4
I
NMDARindependent I LTP components i n area C A I e.g. 'Mossy fibre LTP' see note 5 and I mGluRl under Prote~n 1 rnteracbons, below.
.-
1 Note 4: Agents characterlsed as el~c~tors of chernlcaliy-lnduced
YES (note 2)
Note 1: LTP duratlon 1s several days In non-anaesthetlsedanlrnais Note 2: Proteln synthes~s lnhlbltors affect 'exlstlng mRNAs Note 3: lnd~cattnga requlrement for transcnptlonal acllvatlon
I
I potentlatlon tnclude arach~donlcac~d,metabotroplcglutamate I 1 receptor agonlsts, the potasslum channel blocker TEA, calctum Ions I and G protean activators (e g sod~urnfluor~deI alummlum chloride) I Note 5: Sens~t~ve to calc~urnchannel antagonists requlres stronger II I tetanlc st~mulat~on than NMDAR-dependentLTP
NO (note 2)
LTP-2
YES (note 3)
I
I II
LTP-1
Other
NMDAdependent forms
*
"Non-Hebbian" Long-term potentiation (see references In Related sources and reviews)
and
e.p.s.p.-spike (E-S) form of activity-dependent potentiation (see references In Related sources and revlews)
e
ABSOLUTE DEPENDENCE OF SYNAPTIC POTENTIATION ON 'SUFFICIENT' POST-SYNAPTIC DEPOLARIZATION
Typical experimental arrangement:
Experimental condition
(' )
,
Hz
Schematic synapse on to a 'target' dendrite\
,
Post-synaptic 'Target' locus of post-synaptic NMDARdendr~te channels ,*'
-, Pre-synaptic -'stimulating electrode (depolarizing shocks)
Post-synaptic stimulating 8 recording electrode
LTP phenotype
+ no depolarization of
pre-synaptic stimulation
& LTP induction.
Analogous l o weak i n vivo stimuli (i.a. those activating only a few input fibres) not belng able to approach a 'threshold' of post-synaptic depolarization.
post-synaptic dendrites h h
+ (*)
pre-synaptic -
(3)
Robust LTP-induction; simultaneous depolarlzation (even at low frequency) facilitates LTP (the NMDAR-channel acts as a 'molecular co-incidence detector').
'paired' depolarization of post-synaptic dendrites
-
pre-synaptic stimulation
I
Robust LTP-induction; analogous to 'synaptically-coupled' neurons i n vivo (see sections on temoorel summation of svnaotic inputs by the NMDAR under Activation, be1owj.l
I & LTP induction.
There Is an absolute dependence for 'sufficient' post-synaphc depolarization for LTP induction lor underlying molecular mechanisms, see below.
-
stimulation
experimentally during tetanus
1
f
MOLECULAR MECHANISM FOR NMDAR-DEPENDENT INDUCTION OF LTP
'Sufflclently dcpolarlzed' (Strong tetanic stimuli inducing Mg2+a ~ p u l s i o n and agonlst blnding)
Under 'resting' conditions (or weak stlmull or those produced by synaptic inhibition) ( H y pcrpolarizcd)
(sss Panel
'Inaulilclmnt' glutamale/
Extracellular Ilgands (agonirts) bound with concomitant post-synaptic depolarizing 'threshold' reached
F, above)
"':.
-* N~ LTP-~ *duction
-_..
For further detatls see thts field (Phenotyprcexpresston) and the ffelds Protern tnlenctrons Cumnr-vofiage retatton. InaCbVahOn. Blockers, Receptorhnsducer mteractfons. Receptor agonists and Receptor antagonists. See also the entries ELG CAT GLU AMPAMAIN and VLG Ca
v
[ LTP-induction I
.: NMDAR-channel {unoctuplrd b y mgonlnla)
Ilmgnerlum ion
ion
block rlh (wlthln the pore1
GtUtmm*qm (ag0nl.t)
IGlyeine (co.agonlat)
-
augment t h c ~ a "transient associated with the synaptic activation of NMDAR. Thcsc results, togcthcr with ohscrvationson the inhihitinn of LTPhy dantrolene (SVC l'h~riotj,pic r x p r ~ s ~ i olin(Ier n ILG CIICOK,vK-C(lf,17-141 and the ~ a store " ~ a " - u p t a k e pump inhihitor thapsigargin (sec I'l~cnotypic.(.xl)r(*,ssion ttndcr II,G C11 CSI
Norrcl NMLIA-indc>pcndcnihippoc(imp(i1LTP h y K' c17mnnc~ll>lockors i~nhnncin,q,ylutnrnrrt[j rrlctrsc OR-14-31: In hippocampal CAI, ir transient block of I<:,IM and the dclaycrl rcctificr llvl hv the K ' ch;~nnclhlockcr tetracthvlammonium ~ r o d u c c sa
ca2*-dcpcndcnt,NMDA-indepcndcnt LTP, rcfcrrcd to as L T P K ' ~ .This novel form of LTP is induced hy a transient enhanced glutamate release which generates a dcpolarizatinn via non-NMDA receptors and the consequent actlvatlon c ~ f voltage-dependent ~ a ? 'channels1"". Compr~rr~tivcnote: NMDAR-dependent post-synaptic mechanisms of LTP induction can also lead to sustained enhanccmcnt of prc-synaptlc transmitter rclcascl"'.
Externr~lM$+-block of NMDAR 'masks' ohservotion o f LTI) 08-14-32: Long-term pcltcntiatic~nin the striatum (a hrain rcgion associated with acquisitic~nof memory motor skills] is 'unmasked' hy removing thc voltage-tlcpcndcnt ~ ~ " - h l o c kof NMDA receptor-~hannclsl"~ (sce Hlocltcrs. 08-43). Under control conditions, NMDA rcccptor-channcls arc inactivatcdt hy the vrdtagc-dcpcndunt ~ g " ' - h l o c k and rcpctitivc cortical stimulation induccs long-term dcprcssiont (LTD) which also docs not rcquire activation of NMDA channels in this preparation. Removal of external M ~ ' + removes voltage-dependent hlock and rcvcals a component of the E P S P ~which is potentiated hy rcpetitivc (tetanici) activation1"'. For an + during LTP illustration of the rnle of voltagedependent M ~ *'expulsion' induction, sei' Fig. Ic.
Synnptic activation o f N M D A R cnn induce ‘short-term' potentiation in mt striatrrm 08-14-33: Maintained activation of NMDAR hy synaptically releascdglutamate in the corpus callosum can pmducc a sustained enhancement of the E P S P ~
which coul(l contrihutc to basal ganglia-rclatcd motor function1"'. ~ g " - f r c c ccrchmspinal fluid (artificial CSF) increases thc duration of prc-tctanus E P S P ~ together with incrcascs in amplitude and duration of the direct rcsponsc to tctanict stimulation. past-tetanict potentiationi in normal artificial CSF (containing ~ g " )is followed hy a long-lasting tiepression of thc EPSP. Thus ~ g - " - f r e eartificial CSF cnahlcs the expression of a short-term potentiation o f thc EPSP amplitude and duration1"'.
LTP enl~ancementb y antihody 'a,qonists' nctin,p nt thc glycine recognil ion site 08-14-34: A monoclonal antihody (R(iR21) which (i) Jisplaces [ , ' ~ I - g l ~ c i n c
hound spccifically t o hippocampal NMDA rcccptors and (ii) cnhanccs the opening of the NMDA integral cation channel in a 'glycinc-like' fashion can he competitively antagonized hy 7-chlorokynurenic acid"" ((see Receptor antrr~pr~is!s, 08-51]. Antibody B6R21 also enhances LTP in hippocampal slices1'". Intraventricular infusions of R(iR21 significantly cnhanccs acquisition ratcs in hippocampus-dcpcndcnt trace cyc hlink conditioning in rahhits, halving thc numhcr of trials required to rcach a ts criterion of 80% conditionctl rcsponscs (see m1.w effect o f pnrticll ~ ~ o n i s~t the flycine site under Rccept or rrgnnists. 08-50).
Hctcrr~~yeneity of synoptic firin!: pat terns associr~tcdwith NMDAli in different nellronnl loci 08-14-35: ~onntropictNMDA rcccptors expressed in rat arca C A I pyramidal cells (PC) and interncurones (IN] d~ffcrin their modc of f~ringanrl pos.icss
entry O K
different kinct ic propcrtics when dcpnlarizcd by a prolonged current pulse"f: Whcrcas PCs firc n single action potential, most INS rcspnntl with nonaccommodating high-frequency spiku firing. No!c: NMDA EPSCS~in thc majority nf channels located in the oriens layer and alveus display unusually slow rise times"'.
Intrmcclln~nrmodltlnrion o f N M O A chr~nnclnotivntion 08-14-36: A cc~nsiticrationof how cach of the distinguishing propcrtics of the NMDA receptor 1i.c. filycinc co-agonism, voltage-Jepcntfent ~ g "hlock, relatively long open stntcs nnd ~a?'-pcrnlcal~ility)contrihutc to the modulation of synaptic strcngth and firing synchrnnization in computational netlral circtlits has hccn made1*'. "*. (SCL' (11.~0V o l t r ~ ~ ~ ? sr.n.s~rivit!~, 08-42, I~loclrcrs,OX-43, nnrl Chrrnncl modl~l~~tion, OH-44).
PnrolleI 'down-modulntion ' o f inhihit ory s~gnollin~y nssociat cd with induction o f LTP 08-14-37: During intluction of LTP, GARAh inhihition decreases, causing the NMUAR-mediated excitation t c ~incrcasc, and induce LTP fscr Fi,e. 4. ON-.?I). During 5 Hz stimulation, ZOH-Saclofen (a GARAR-receptorantagonist) has been shown to prevent (il reduction ot ~nhihition,[ i i ) incrcasc of cxcitation, and (iii) induction of LTP".'. Notr: CARAIt receptor modulation of synaptic plasticityi ('disinhihition'l can occur at -5 Hz which is within the frequency range of the thcta rhythm+ endogenous to the hippocampus and which has hccn shown to rnoclul;~tcLTP in rri~,o.
~ vN' M D A rccept or stimulniion Rc,yulat ion o f intr(lcclltil(~rM ~ I ~ 08-14-38: T h e rcgulaticln of int rrlrc~llulr~r M ~ ion ~ concentration ' 1.ry neurotransmitters such as gli~tamate may also hc important in controlling ncuronal rxcitahility: NMDAK activation hy glutamate plus glycinc has hccn shown to raise intrnccllular ~ g "from 1 t c ~ more than 1 1 mM (compnrcd with the rcstinx cclnccntraticln of 0.5 MI"^. T h c maior component of this glutnmatc-induced intraccllulnr ~ g ? 'incrcasc is dependent on cxtraccllular ~ a hut " is indrper~dcntof cxtraccllular ~ g ? 'A. sccond (minor) cclmponcnt of the incrcnsc is independent of cxtmccllular c;I?', hut rcquircs cxtraccllular M ~ ? anti ' can he n~nplificdhy cxtraccllular Na* rcnic~val'~'. Con~parrltivc no!?: Scc also the effects of intraccllular ~ g ? on ' inwardly rectifying potassium channel gat in^ within the INN K srrics (cn!rics .10 t o 33). Antr!foni.srn o f ntctahotroPic-t aqlntcrrncrtereceptors rcdnct: the durorion o f LTP 08-14-39: T h e [relatively non-sclcctivcl mGluR antagonists 2-amino-4-phosphonohutanoate [AP4) and 2-amino-3-phosphonopropionate (AP3) reduce thc duration of LTP' ". Convcrscl y, sclcctivc mClluR m,yonists such as lS,3R-aminocyclopentane dicarhoxylate (ACPDI can ~ u ~ m c ntctsnust induced potentiation and can induce NMDA-dependent LTP with ' s i ~ h t h r c s h o l t l ' ~or ' ~ low-frcqucncv stimulif77. Notr: ACPD is known to augment rcsponscs of hippocampal ncuroncs to NMDA"" and inducc a 'slow-onset' NMDAR-indcpcndcnt m t ~ d c of LTP'.~" (see rr1,vo Kct.rptor/ trtrns(lltc~rir1tclrr1c.rions. OX-49).
entry OR
Comparative note: Role of metahotropic glutamate receptors i n parallel release of ~ a "from InsP3-sensitive stores
08-14-40:Synthesis of InsPq following the activation of rnGluRs (see ILG Cn InsP?, entry 19, and Resource A - G protein-linked receptorp, entry 56) can elevate [caZ+],from I n ~ P ~ s c n s i t i vstores e (see ILG Ca InsP.,, entry 19) in add~tion to caz+-induced c a b rclcasc initiatcd hy ca2+-influx thmugh NMDAR-channels (.FCC ILG Ca Ca RyR-Caf, entry 17) Actrvation of mGluRs can induce LTP by a thapsigargin-sensitive mechanism even in the presence of NMDAR antagonists isoc I'hcnotypic exprewion undpr [LC: Ca CSRC, 18-14, nnd Channel rnodtrifitlnn under ILG Cn I ~ T P . ? , 19-44)'". These results suggest that glutamate-induced release of ca2' from stores can substitute for ~a"-influx following activation o f ionotropict NMDAR. Note: A more general role for ca2+-store-mediatcd release In nmplrfying localized transient signals through NMDAR-~hannelsis suggcstcd by the 'insufficiency' of { i ) slow depletinn of ca2+ or [iiJCa'+ currcnts'"~' to induce LTP.
Alternative forms o f synaptic plasticity dependent upon the degree o f post -synoptic depoIntizatron 08-14-41: Intracellular recordings from granule cells of the hippocampal dentate gyms maintained i n vrtro havc shown isolated NMDA receptormcdlatcd synaptic responses to express hoth LTP and L T D ' ~ ~ Additionally, . the level of post-synaptic depolanzation can determine which of the two forms of synaptic plasticity is expressed in response to an identical input. These studies havc also shown that post-synaptic caz'-influx as essential not only for induction of LTP hut also for induction of CTD of NMDA receptor-channel function"'.
Protein distribution See the rnethodolo~icnlnote in mRNA distribution under ELC: CAT GLU AMPAIKAIN, 07-13,
NRI protein distribution 08-15-01:The 'fundamental' NMDAR suhunit NRl is generally confined t o the ccntral nervous system. Following suhcellular fractionation of the cerehral cortex, NRI protein 'co-enriches' with synaptic membranes. Prominent selective immunostaining for NRI protcln occurs In several layers of the cerebral cortex, in the hnppncampus and clcntatc gyrus, as well as in the ccrchcllumSR.
Di{fereni ial NMDA R distributions as determined by displacement of a,ponists 08-15-02: Under conditions of differcnt~al receptor activation, regional diffcrenccs in NMDA rcceptor pharmacology can he detected in the CNS using hinding and quantitative autoradioRraphy1'". The compounds CPP and 7-C1-Kyn (see Receptor nnto,pnni~tc,08-51 ) havc dist~ngu~shed at least thrcc populations of nativct NMDA receptors by displaccmcnt of ['HI-MK-801"".
Functioncil c~ssa,vsfor NMDA protein distribution 08-15-03: Ry use of sclcctivc antagonists for NMDA rcccptors, it has hcen shown that NMDA rcccptors contribute only a small and variahlc amount to the EI'SPS~in hippocampus, cortcx, spinal cord ant1 ncostriatum.
Suhficltls 'devoid' o f NMDAIl in hippocl~mpus 08-15-04: Mossy fibres1 which terminate in the stratum lucidum of area CA.7
arc dcvoitl of NMI)AI< and LTP is not hlockcd hy NMDAR antagonists in this suhficldl. Mossy fibre LTP thus fornis one class of NMDAR-independent synaptic potcntiat~on in the hippocampus (srr. c.lassific.rltion in ~ c ( . F ~ ~ ~ * " " crnd tlic c./tr,s,sliic~cllior7 o f ~ v r i i ~ p tNi c~ S ~ O I ~ SinC SI'I?~~io!?~pjc c ~ p r ~ ~ 08-14). ~I'o~.
Subcellular locations 08-16-01: NMDA
rcccptors arc generally located nn post-synaptic membranes, closcly apposed to transmitter rclcasc sites across thc synaptic cleft [ 2 pm). NMDAR arc generally rrssrrm~d t o hc located on dendritic spines; a dcndritic location of NMDAR proteins may nct to localize c;' s~gnalsas spines can restrict the diffusion of ~ a " ' ~ ' .A comprehcnsivc di~cussionof prc- and post-synaptic factors affcctin~LTP phenotypes has appc:~rcd5'.
'Cll~strrin~y' and 'mobility' nf cnnrrntokin-C:-sen.~itive NMDAR 08-16-02: The Conus ~co~qrtrphus venom pcptidc c0nantokin-G (CntkC) has
hccn rcportcrl as a 'rcliahlc' prohc for dctcrmination oi NMDAR protcin d i s t r i l ~ u t i o n ' ~Thcsc ~. studies have shown NMDAR t o he clustcrcd and immobilized on dendrites of l i v i n ~cortical ncuroncs. In hippocampal slices, the C A I dendritic suhfield is strongly lal~cllcdby CntkG, whcrcas the CA.3 mossy fihrc region is not Iahcllcrl. On CAI hippocampal ncuroncs in culture, dcndritic CntkG-sensitive NMDAR arc clustered at sites of synaptic contacts, whcrcas somatic NMDAR arc distrihutod diffusely and in p ; i t c h ~ s ' ~Not;ihly, ~. NMIIAR distrihution differs from the distribution of voltage-dcpcndcnt calcium ch;inncls. A significant fraction o f labelled NMDAIi on somata and dendrites has hccn found to he highly mahile. Rates of mohility arc consistent with rapid recruitment of NMDAR to specific synaptic Iocation~'~'.
Methodological note 08-16-03: Ncuroncs rcccive many excitatory synapses (lfiz-10' pcr ccll) which arc located nn slender dcndritic clcmcnts generally far away from thc somatic
recording site, making in sit11 studies of iliscrcte (localized)synaptic events difficultfH7.Typiciilly, whcn pr~pul~tions of synapses are activated, NMDA receptor-mcdiatcd synaptic potentials appear as slowly rising, long-lasting waves supcrimposcrl on faster, non-NMDA-receptor potential^'^^. Excit;~toryautaptir 1 currents (identical to thnsc found in vivo) havc hccn shown t o I>c mcdiatcd hy NMDA rcccptors in isnlatcd hippncampal ncuronrs m;iintaincrl in coll culturefHR.
Co-localization and co-activation o f NMDAR find non-NMDAR channels at single synapses 08-16-04: Monosynaptic excitatory post-synaptic potentials (EPSPsJevoked hctween pairs of cultured neurones from either mouse hippocampus or spinal cord have dcmonstrated that two functionally distinct excitatory amino acid receptor-channels can he simultaneously activated hy transmitter release from a single prc-synaptic ncuroncrx.'. Thc co-localization of NMDA and non-NMDA rcccptor-channcls at singlc synapses is important for the tlcvclopmcnt of L T P ~ , which may hc diffcrcntislly cxprcsscd s t cach synapse according to the mix of receptor suhtypcs at that synapsefHJ(see Protein interactions, 08-31). Note: As summarized hy Daw et al.'" NMDA agonists multiply (amplify) synaptic rcsponscs (increasing the slope of the rcsponsc curve), whereas non-NMDA agonists add to them (moving thc rcsponsc c u ~ u c wards). Thc rolcs of postsynaptic calcium in thc induction of LTP have hycn reviewed, e.g. rcfs"4, I", 186 . For cxpcrimcntal protocols of LTP' induction, see Phenotypic expression, 08-14.
P
Transcript size 08-17-01: Sec Tullle 1 under Gene fomily, 08-05,
Note: The symbol [PDTM]denotes nn illustrnted fentore on the channel protein domain topography model (Fig. 2).
n
U
Chromosomal location 08-18-01: The human gcnc cncoding thc NRI suhunit (NR1, zeta I ) has hccn mnppcd to chmmosomc 9q84.,1"-'HY with gcncs encoding potentiating suhunits epsilon 1 and epsilon 3 hcing localizcd to chromosomcs I6pl3 and 1 7q,3S rcspcctivcly lHv.
Encoding 08-19-01: For open reading framet lengths of reported cDNAs, see Datnllc~se
listings, ON-.5,7.
Gene organization Alternative splice vnriants o f the filndamental suhunit 08-20-01: Alternative splicingt generates functionally distinct NMDA r c ~ c p t o r s ' ~ ' ~ Analysis ?. of thc gene structure cncotliny: NR1 rcvcalcd eight splice variants arising from (iJdifferent comhinations of a singlc 5'terminal exon insertion and (iil three different 3'-terminal exon deletion^'^.' (for further details. sce Domain filnctionx, 08-29).
A n t ~ l v s i sof N R I Rcnr ' U ~ ? S ~ Y C ( I sI ~~I ~ ~ ~ c T I c ( ~ s ' 08-20-02: Cloning and scqucncc analysisl"'of a 3.8 kh EroRI fragment of the rat NRI gene incli!dcd .? kh of promoteri and enhancert region, cxonl 1 ant1 a portion of introni 1. NRI posscsscs scqucncc motifs characteristic of a housekeeping genet rcgulatcd hy immediate-early' gene products. Two major transcriptional start sitest were idcntificd at 2 7 6 and -238 from the first nucleot~cicin codonr onc. One C;SC; and two SPI motifs, hut no TATA hoxt or CAAT hoxi cxists in the rcgicln proximal to the transcriptional start sitcsi"".
1
Homologous isoforms
08-21-01: Thc human NMDA receptor cDNA hNRl shares high scqi~cncc homology (--c)YY/, with the rat hrain NMDAI and the mouse zeta I ~ u h u n i t ' " ~ ~The . rodent and human h o m o l o ~ i c s diverge near thc Cterminus, suggesting that they represent alternatively splicedl messages of the same gcnc (scc (;rnc or,ri~nizc~tion, 08-20. and I>otol~itsc1istin.y~.OX-53). Of the 7 of Y,?H amino acids which nrc tliffcrcnt hctwccn the rodent and h u m m scqucnccs, three occur in the region of the signal peptide+ and thc othcrs in the cxtraccllular (N-tcrmin;lll dom;~inpreceding the four putative tr:lnsmcmhrnnc scKmcntsl' ( s c ~ irlso irllrrnnrivi, (tivc t r r ~ n s m r ~ m l ~ n ~ n c (/OIZI(II~I) rrioil~l, v l i o ~ 111 ~ ~ /I'DTM/, r~ Fi,y. 2).
Protein molecular weight (purified)
C;lyco,~ylr~rc~d m o n o m e r i c and o l i < y o m y r ip r o t c i n s 08-22-01: Roth native and hctcrologouslyi exprcsscrl rat hrain NRI suhunit have an appnrcnt molecular mass of 1 I6 k n a dctcrmincd hy SDS- PAGE^"'. Chemical cross-linking of nativc synaptic mcmhranc protcins shows that the Nlil protcin is p:irt of n receptor protein complex with a molecular mass c ~ f7.30 k ~ I 1 ~ 'The . NRI rcccptor protein is heavily g ~ y c o s ~ ~ a t (sc7r rdt l)r,lor4.l.
N-C:lyc~o.<~rInt ion c o n r ~ c c o u n tfor differr~nccsh c l t w c c n pretlictcd a n d purified M, o f NMDAI< 08-22-02: An antihody raised to an positions 14.35-1 445 o f NR2A recognizes four inimunorc:~ctivcspecies with M, of 180 kDa, 122 kDa, 97 kDa and 54 kDa in rat hrain, hut only a s i n ~ l chand of M, of 180 kDa in HEK-29.3 cclls transiently expressing N R ~ A ' " .Dc-N-glycosylation elf HEK cell mcmhrnncs yields a 165 kDa immunorcactivc spccies, which agrees with an M, prcdictctl from thc open reading frame1 length of the cDNA scclucncc for the maturc NR2A suhunit (scoi,rl1.vr) Si~clrrc-nee nlotifs. 08-24),
Sequence motifs Mult iplr N-,ylycosvlation s i t ~ s 08-24-01: The amino acid seclucncc of the NRl c l ~ n c ' ~ . predicts .~" 10 possihlc N-glycosylation sites at the cxtraccllular domain. In the isoforms NR2A to NRID, (under the assumcd mcmhranc topography dcscrihcd in Amino m i d
cnrnpo~ition,08-25) there arc 6, 6, 5 and 6 canonicalt Asn-X-SerlTha sequences rcspcctivcly for potential N-glycosylation in thc cxtracctlular Ntcrrninal regions8. A large number of pmsihlc N-glycosylation sitcs arc also present in the cxtraccllular C-terminal regions of NR2A (12 sitcs) and NR2R { l o sites) but only one in NR2C ant! none in NR2D. At the Cterminal of these polypeptides, all but NR2D-1 share a common amino acid sequence of undetermined function: ScrlPro-Scr-Lcu/llc-Glu-Scr-GlulAspVal. The cloned NMDAR suhunits also posscss many phosphorylation moti fst (see Protein pho.sphorylntion, 08-32).
Note: The svmbol [PDTM]denotes nn illustrnted f ~ n t u r em the chonnel protein domnin topogrnph y model (Fi8. 2). In-press updates (we critera rrnder Introduction el lnyorrt o f enrries, entry 02): .Tee notc ahove field 07-26, for important new informntion relotins to ionotropic glutamatc receptor strucmres.
Amino acid composition Aminn acid n.rxipnments of N - and C-terminol residr~es 08-26-01: T h e averall structure of NRl suhunits is similar to thnt prcdictcd
for the AMPAtkainatc family of receptor-channels, i.c. a large N-terminal extracellular domain followed by four putativc transmcmhranc scgmcnts (TMI-TMIV or M1-M4 rcprescnting the 'core region' (see Dornoin canservdtlon. 08-28)'" ~ ~ d r o ~ h o b i c ianalysis t~t of the NRI receptor predicts a hydrophobic N-tcrm~nalsiWalt pcptidc and four hydmphobict transmembrane domains (hi see Dnmoin arran
Domain arrangement 08-27-01: Nntivc NMDA receptors arc composcd of a fundamental clr key suhun~ t (NRI ) and its pntentiating suhunits (NR2A-NR213). Variants of NMDAR-2 suhunits potcnt~ateand functtonally d~hhcrcntiatcnativc NMDA rccrptors by forln~ngdlffcrcnt hctcmrncriet configurations w ~ t hNRI (for further clcro~ls,sce Ilomnm funct~ons,08-29),
Alretnntive domain models predict five trnnsmem hrnne segments 08-27-02: T h c suhunits N R ~ A - N R ~ D 'also pnsscss large hydrophilict damsins a t hnth N- and C-terminal sides of the fnur putativc transmcmhranc
entry OH
1
segments, though NR2 suhunits clearly display five hydrophobic segments consisting of 20 uncharged amino acid residues. NR2 suhunits also show a large C-terminal extension following the M4 segment which is not sccn in othcr cloned ligand-gatcrl ion channels (cf. >4?0 aa residues in NR2 versus 'five trans50-100 residues typical of othcr ionotropicT ~ l u ~ s l ' The . membrane domain' model (c.g. see rcf."l is represented in (PDTM], Fig. 2, with dotted lines representing the 'extra' transmemhrane domain.
Domain conservation Determinnnts o f ~ , y ~ + - s e n s i t i vand ity ~ a ~ + - s c l e c t i v i t ~ 08-28-01: NMDA ant1 AMPA receptor-channels contain common structural motifs' in their transmembrane M2 segments that arc rcsponsihle for some of their ion selectivityi and conductanceT properties. The high ~ a " pcrmcahility and extracellular ~g"-sensitivity of the NMDAR are imparted by asparagine residues in a putative channel-forming segment of the protein, transmcmhranc domain 2 (M21. In the NRI suhunit, replacement of this asparagine by a glutamine residue decreases (la2'perrneabilityt of the channel and slightly reduces ~ g * ' - b l o c k ~ (see ~~ I)ommin junctions. 08-29). The same suhstitution in NR2 subunits strongly rcduccs magnesium hlock and thus increases the ~ ~ ~ ' - ~ c r m c a h ihut lity docs not significantly affect ~a"-pcrmeahility. These asparagincs arc in a position homologous t o the site in the M2 region (Q/R site) of AMPA receptors1"" (cf. Ilornnin functions undcr ELG CAT GLU AMIJAIKAIN, 0729, (2nd srr Domain functions, 08-29).
Variation o f secluence homology in different structural domains o f the N M D A R family 08-28-02: Levels of scqucncc homology arc variahlc across thc structural domains of NMDAR. Homology is extremely high within the four transmembrane segments (-80-90%,, the 'core' region1 hut only moderate in the N-terminal portions (--45-60%) and 'vory low' in the C-terminal portions (-20-.3094,)n. Cln structural grounds, the NR2A ant1 NR2R sequences closely rescmhle each othcr, as do the NR2C and 2D subtypes. Thus, they may he classified into two suhgroupsH.Subunits N R ~ A - N R ~ Darc ' only ahout 15% identical with thc key suhunit of the NMDA receptor ( N R l )hut arc highly - homologous (approximately 50% homology) with one another.
Domain functions (predicted) Agonist clnd co-ngonist hinding site - signal transduction via electron trrlnsport 08-29-01: In the NRl subunit, the agonist-binding signal may he carried from Y456 to W590 through an electron transport chain, ~ncludingW48O which is a candidate locus for the glycine modulatory site. NMDA channel opening may arise from repulsion of negatively charged Trp590s, analogous to Trp4,15s of the Shakcr K ' ~ h a n n e l ' " ~ .
'"*3
--
Ilalched rcpiaa lhorv hrmolngv to glulrrnlrr pcmrrn nf f..dr - m Nakanlthi. N f u r m 11900)5 56V.XI.
(b) Psntameric arrangement(putative)
Y 3 ~ 2
-__
(a) Monomeric
M
- -.
I ' M 2 M 3 ,.-'I3
domains
*----
-M4
I
1
--
. V . C . CrlHwi M l I W scfds found m Yt (THIJ domain cf homologous porlrlons In t h . PDTM under ELG CAT GLU AMPA KAIN
. I ' ?lornologous r#sldues t o the C , ~ I I C ~ I O-R sns (present rn aubunns NR1. NRzA to NR2D). wg MK-eoibloek (N -4 mutations elso decrease cslclum permeebrllty)
-
-
_--
-
-- --
--
- -
~
Y4
PutMlva ollgomerlc structure (cee Doman arrsngemenl undo1 fhe ~ELGCATGLUAMPAUAfNI
coon -
~
Cornpart rcIattve l e n ~ t hOJ~ C-ttmttnn~dnmorns: NU1 ( - 1 I l l aa, a\ hcrc) \E.\ (43a ~ l\R2R 1-641 an). \R2C ( 4 1 ad). URZD-I I - 4 V l aal. VH2I)-I ( - 4 M l aa) Re tLdrl~P lcnqrhr harrd <,nubmnmc*nrt tn lrhrr I r ,/I l W l r r l 9) -
%--_
lntracsllular
Z
--
\----,
1
;
w a Y M4 1 u2. M J u3 M i
Y ' ~ r ,
-
Extracellular
y1ni4
m1 u i
-
--
-
Approxlmmtm loc.llona o f 'CO~..I)SUS' phosphoryletian SINS tor CaMUN end p h l n klnsse C (See Appondlx G. consensus slfes) For further deferlr. phosphoryfef,
AddltloMl (hypoth.tlc*I) dommln inlroduced In #om. topology models to n l l n consislency with structure :luncllon data I.. Seeburg (TBPJI Trends Phannecol Compare l o Scl 14: 297-303 aHern*tlv* model. wlth Isrge (M3-M4) '?tr.cellular loop and a r t r u c e l l u l ~ rCOOH tall (8.9 ELC CAT 5-HT3)
-
-
KEY
Y-
\ Note
ma
s1rvnur.s
Typlcrl numhr mnd clu*hrlng ol oonwnlu* w - l w s r t n t m sbt.l rsw rim turd %worn maws
-Po.nbmn e l mawma117 esnu.*sd In l l R l snd 1 1 1 nF.~ubunl1..q-s (sea th* fgeld I d m J o n pmW. llpun dear mi emphsdr* Uiftemnc~sbstwnn ma primsry of 8ubunlls NU1 mnd NRZA-0 ur4.a Rae D1tab8se Ilrtlnpr)
NOTE: AN mktfvo posltlons of motifs, dOm8hl ahepes a n d ~ 1 - s are dl8gr81nmatlC and a m subJect to re-interprel8tlon.
Channel symbol
ELG
-
Figure 2. Monomeric protein domain topography model (PDTMI exemplified for NMDA-selective ionotropic glutamate receptor (iGluR) NRI subunits. In press updutes: (see criteria under Introduction d Iavout o f entries, entry 02): A modified 3-transmembrane domain model based on N-glycosylation site tagging data has appeared i n press. For brief details, see note above field 07-26. For further details, see entry update pages via the CSN. (From 08-27-02)
entry 08 ~
-
.-
Amino (!(.idsrr:g~~lcrtiri
NR I splicr v ( ~ r i n n tf i f n c t i o l ~ ~ 08-29-04: Propcrtics o f scvcn isoforms
of the NMIIA receptor guncrateti by altcrn:~tivcsplicingi h:~vcI>ccn d e ~ c r i h c d ' " ~Co~nl,inatorial . RNA splicingt h3s hccn shown t o ; ~ l t c rt h e surface charge o n the N M D A rcccptor'"'. SpIicc vari;~nts of NKI rncotlcrl hy m t ventral niidhmin c D N A diffcr in tllcir functional properties (for tltc Ioc.orion ol NMIIAII protrin r c'ions ~. ( ; l i ( ~ t i ,!>J!- ~ l t ( ~ r ni\uB ( 1 t .spli(~lt~,y (*\vbnt,x. , s ( ~/l)/)TA4]F'i$y.2). T h e structi~ral variation in some splicc v:~ri:~ntsis shown in T'tl~lc.5.
'
Prcsuncc o f N R 2 scrirls 'potcnrirtting' suhunirs in h e t e m m e r i c c o m p l ~ ~ x rc~protluoc~ cs nat ivc N M D A R p r o p ~ r ics t 08-29-05: ~ o r n o r n u l t i m c r s t formed from cxprcssion of N R I J n (zeta sul>units exhibit scvcr:~l fc:~tiircsof nativc N M D A receptors. Si~nificantly, thcsc i n c l ~ ~ dpronoilnccrl c ~ a " - ~ c r r n e a h i l i t(~~ r , cSr~1r.c-tivitj,. ~ 08-40), a rnotli~lntory ; ~ c t i o no f glvcine ( s c ~(:iir~nrirpl iiiorlr~lrrtion. OH-441, and n ncg;~tivc slopc cond~tct;lncc of currents in t h e prcscncc of M R ~ +(src C l ~ r r r ~ r i t - i ~ o lrc,Irr/ion. / ( ~ ~ v OX-.?5). Sithitnits NR?A, NIi213 ant1 N10C yield prominent, typical glutnmatc- and NM11A-:lctivatc(l currcnts clnlv when thcv ;Ire in hctcromcrici configurations with N R I . NRl JNK2A nntl N R l / NR2C channels diffcr in Ratingt behavinut and magnesium sensitivity. FJctcronicric N M D A receptor suhtvnes nrohahlv exist in nativc ncuroncs.
I
entry 08
Figure 3. N M D A R n~utntionsriffecting M,F.'' block. ~d'-pcrmcnhi1ityand sensitivity to ~ n ' ' und open-chnnnel blockers in hctcromcric comljinntions ol NMDAR. The figure cornpnrcs phenotypes for (n) thc wild-typet subunit cornhination r2/CI; (h) the rnlttant snhunit comhinntion r2/Cl-N598Q: (c) the mutant srihunit comhinntion 62-NL589Q/<1 und ( d ) r2-N589Q/C1N598Q. Tlte hetarornerict epsilon Plzetn I N M D A receptor-chnnncl with the mutation on 170th sslhunita - shown in panel (d) - displojrs grently
reduced sensitivitv to dizocilpinc (MK-801) but is st ill susceptildc to inhibition hy ~ n " The corre.~pond~ng m u totion o f thr cpsilon 2 subunit /I[I.F n similnr effect to pnncl (d). Thr fi
entry 08
Table 5. RNA splice rrarinnts o f NMDA receptor-channel (From 08-29-04) Splice variant
Stmctural variation
NRla and NRlb
Thc NRlh splice variantt differs from N R l a hy the presence of a 21 aa insert near the amino end of the N-terminal domain and hy an alternate C-terminal domain in which the last 7 5 amino acids are replaced hy an unrelated sequence of 22 amino acids2"'. Otherwise, NRl h is virtually identical to N R l a in the remainder of the N- and C-terminal domains, at the 5' and 3' non-coding ends, and within the predicted transmemhrane domains and cxtracellular and cytoplasmic loops
NRlc
The N R l c splice variant has been shown to he identical to N R l h in its C-terminus hut lacks the N-terminal insert2"'
(see olso Protein phosphorylntion. 08-32, and Channel morltrlution, 08-441 Note: Features and differences of alternative splice variants designated NMDARI-la, NMDARI-1 h, NMDARI-2a, NMDARI-2h, NMDAR1-3a, NMDARI-3h, NMDARI-4a, N M D A R l 4 h and others can he found in the original references shown in the Database listings. 08-53 since NRl messenger RNA is synthesized throughout the mature rat hrain, while NR2 messenger RNA show a differential ciistrihution" (for summnrj: scc rnRNA distril7ution, 08-13). Further 'potentiating' properties of NR2 series suhunits in hctcromeric NMDAR complexes are listed in Tahle h. (Scc also Domain functions under ELG CAT CLU AMPAIKAIN, 07-291.
Subunit-specific patterns o f modtilation for recombinant homomerzc NMDARs 08-29-06: ~ o m o m c r i c iNMDA channels of the splice variant N R l h possess clectrophysiological properties distinct from those of N R l a homomeric channels2" (see Genc orp~nizotion,08-20).N R l h channels cxhihit a lowcr apparent affinity for NMDA and for glutamate. Furthermore, N R l h channels exhihit a lower affinity for n-2-amino-5-phosphonovaleric acid (APV] and a higher affinity for ~ n ? ' The . two receptor variants show 'nearly identical' affinities] for glycine, ~ g * + and , phencyclidine. Spermine potentiation of NMDA resl~oiiscs,prominent in oocvtes iniccted with rat tnrehrain mRNA, is also prominent for NRla receptors, hut is greatly reduced or absent for N R l h receptors2"'. For mechanisms of spcrminc potentiation, FCC Ch(lnne1 modr~lntlon,08-44
n Predicted protein topography 08-30-01: 'True' transmemhrane topography has not yet been direct1,v dcterrn~nedfor any ionotropict glutamate receptor-channel. For pr~dicted arrangements, see Amino acld compos~tion,08-20. In-press updates: See note a b o ~ vfield 07-26,
Table 6. Functional properties and molecular features contributed to NMDAR complexes by NR2 series subunits (From 08-29-05)
Property/feature
Description
NR2 subunits alone do not form functional channels
When expressed individually in Xenopus oocytes, NR2A and NR2C show no electrophysiological response to agonists. However, when NR2A and NR2C are co-expressed with the uhiquitous NRI, complexes show marked potentiation7 of NR1 activity and produce functional variabilities in the affinity of agonists, the effectiveness of antagonists, and the sensitivity to M ~ blockade ~ '
'Full reproduction' of native NMDAR-channel properties in NRl/NR2 heteromultlmers
Heteromeric NRlINR2A and NRl/NR2C combinations display all of the hasic properties characteristic of the native NMDA receptor, including ~a'*-permeabi~ityt, glycine modulation, voltage-dependent ~ g ' +hlock and selective inhibition by competitive and non-competitive antagonists and open-channel blockcrs. Note: In contrast to its role as a co-agonist with glutamate, glycine alone can activate epsilon 1/ zeta 1 suhunit heteromultimers when co-expressed in oocytes
The cpsilon 4 (NMDAR4, NR4) subunit forms heteromultimers with distinct properties
The epsilon 4 (NMDAR-Dlsuhunit is distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 suhunits, and contributes further diversity to the NMDA receptor-channel. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists when expressed in Xenopus oocytes
Refs
R
7
""
1
Variations in regional expression in patterns of diverse NR2 series suhunits
The molecular diversity and different spatial distribution patterns of the NR2 (epsilon\ subunit family underlies the functional heterogeneity of the NMDA receptor-channcl (see m R N A di.strilwtion. 08-131. For example, the heteromerici epsilon 1/zeta 1, epsilon ?./zeta 1 and epsilon .?/zeta 1 NMDA rcccptor-channels exhibit distinct functional properties in affinities for a ~ o n i s t and s sensitivities to competitive antagonists and ~ g ' + block
Consen~cdresidues in NRZ. subunits
All NR2-type 'potentiating' suhunits contain a positively charged lysine residue within M2. The corresponding residue of N R l subunits (Thr6OZ.lcontrols ion permeation at homologous positions in the nicotinic ncctycholine receptor (see ELG CAT nAChR. rntry 001and may explain its conservation in NR2A to -2D suhunits
The 'asparagine ring' predicted for NR1 h o m o m u l t ~ m e r sis conserved within heteromultimers
A corrcsponding asparagine residue forming the putative NR1 'asparagine ring' /see ilbor.et is conserved in NR2-type suhunits, indicating the rlng may torm and contrnl ca2+-pcrmcahilityand channel conductance properties in the pore region within hctcromerici INR1/NR2\ receptors
Variations in NR2 series extracellular domains
Structural variability in the extracellular domains of the NR2 subunits is responsible for governing different affinities of agonists and antagonists that act at the glutamatcbinding site and the glycinc-modulatory site. A gating mechanism for NMDAR channels has been proposed which shares features with Shaker-type K' channels
Relative length of intracellular domains
NR? suhunits possess relatively long C-termini [typical size -. 530 aal which generally exceeds that of cxtraccllular domain preceding M1 (typical length 500 aa in all iGluRsl
--
''
c C 3
-f 7
VI
"'
entry 08
-
Protein interactions Co-localization of N M D A and non-NMDA receptor-channels 08-31-01: NMDA and thc non-NMDA (AMPAIkainate) channel subtypes are often co-localized at individual excitatory synapses1R4.This assumption is critical for modcls of Ionpterm potentiationt (see Phenotypic expression. 08-14). The NMDA class, by virtue of its voltage-depcndcnt channel hlock by magnesium and calcium permeahilityt, provides the ' t r i ~ e r ' for thc induction of long-term pntentiationi, whereas the actual enhancement of synaptic efficacy has been proposed to be contributed by the non-NMDA class of GluR channels.
Frequencies of NMDARlnon-NMDAR subtype co-localizations 08-31-02: As described under Domain arrangement. 08-27, and nornoin f~~nctions, 08-29), interaction with N R l is necessary for the functional
expression of all other cloned NMDA receptor subunits. Therefore, the NRl suhunit is likely t o he a central component of all known NMDA receptors in brain (see re["). Measurement of rniniaturet synaptic currents in cultured hippocampal neurones has shown -70'14, of excitatory synapses to possess hoth the NMDA and non-NMDA classes of receptor, although to differing extents. Of the remaining excitatory synapses, -20% contain only the nnnNMDA subtype and the remainder possess only NMDA receptors1u4. The depolarizing post-synaptic potentialt (DPSP)in cerebellar granule cells has heen determined to he part-mediated hy NMDA, GARA* and AMPA receptor proteins (see Protein interactions under ELG Cl GARA*, 10-31).
Operation of extracellular M P - b l o c k depends on function01 Na'IK' A Tl'ase 08-31-03: Central to the prevention of 'neurotoxic' influx of calcium through
the NMDAR is the operation of voltage-dependent block by extracellular ~ g (described " under Rlockers, 08-43). Failurc of the Na+/K'-ATPases (for example under conditions of ischaemiat] can directly affect NMDAR function in two main ways. First, if pre-synaptic Na'/K' pumps fail, the clcvated intracellular sndium concentration can lead to failure or reversal of 'glutamate (uptake) transporters' (see Fix. 41 thcrchy increasing glutamatc concentration within tlac synaptic clcftt. Morcovcr, if post-synaptic Na'/K' pumps fail, the neuronal mcmhrane will depolarize, and extracellular ~ g ' + hlock will not operate (see Fig. l e and I3lockcrs. 08-43). Thus 'ambient' levels of glutamatc will open the NMDAR channel^'"^. This typc of glutamate toxicity has hecn demonstrated for NMDAR in rctinal ganglion cells maintained in vitroZR3.
u
'Functionnl clusterin,p' o f other pre- and post -svnclptic sjgnal trunsdtlction proteins 08-31-04: Co-localization of a large numhcr of signal transduction protein5 (.see Fiiy. 4 and Tahle 7) within single synapses permits rapid, local cross. modulation through multiple diffusible factors. Figure 4 illustrates a sclcction of these protcins and their intcractions. Note: The arrangcmcnt
shown is intcnded to sunnlerncnt the text of scvcral firlds
~ n t dotw i not
entry 08
-
take into account the important roles of potassium and chloride channels which can r c p ~ l a t erates of neuronal firing. Likewise, the contrihution of G protein-linked receptor signalling to modulation of ion channels is largely imored. Rricf descriptions of functional processes dependent upon interactions of synaptic protcins (such as long-term potcntiationfl are included under Phenotypic expression. 08-14, though thc complexity of potential interactions at individual synapses is difficult to gencralizc into a single model (see ndditional references listed under Related sources el
reviews, 08-5h). Properties of other signnlling components associnted with N M D A Rmediated phenotypes 08-31-05: Neurons co-ordinate the expression of a large number of signalling proteins which contrihutc to phenotypes and functions associatcd with synaptic transrnissiont [as dcscrihcd in thc previous paragraph and partly illustrated In Fig. 41. Table 7 summarizcs important features of somc of thcsc molecules, where they are likely to involve interactions ~ ! t hthe N M D A R . For descriptions and classifications of synaptic phenotypesi (such as long-term potentiation, LTP) as mentioned in thc tahlc, see I'henotj~pic expression, OX- 14.
-
Protein phosphorylation Sever01 other pre- and pnst-synoptic signallins proteins contributing to or undergoing 'phosphnmodulr~tion'are also described in Tahle 7 under Protein intcmctions. 08-3 J . Conservntion o f kinase-modulntory sites in cloned NMDAR subunits 08-32-01: Cloncd N M D A receptor subunits display many potential phosprotein kinase type 11 and phorylation sitest for ~a~+/calmodulin-dependent protein kinase C. (For the Incatic~nsof thcsc sites, see re/erences given undcr Dnrl~hascli.~rinjis,08-53). Ry analogy with othcr 'ELG superfamily' channels, kinase sitcs located on intraccllular domains (such as the putative M3-M4 intracellular Inop, see IPDTMI. Fi,q. 21 can act as Important modulators of channel function'".
Exnmples o f homorneric and hetcromeric N M D A R current potentiation h y PKC activators 08-32-02: The N M D A zcta 1 homomerict channel activity is positively modulated hy treatment with the protein kinase C activator phorhol 12myristate Id-acetatc (TPA)'? ~ c t c r o r n c r i c tepsilon l/zcta 1 and epsilon 21 zcta 1 channels [hut not the epsilon .?/zeta 1 channel), are activated hy treatment with TPA when expressed in Xenopus o o ~ ~ t c s " ~ . Relative 'susceptibility' of NR In and NR 1b t o PKC activators 08-32-03: Treatment with TPA (see above) potentiates NMDA responses in oocytes injected with mRNA encoding the splice variantT N R l b hy ahout 20-fold compared to -4-fold potentiation in splice variant NRla-injected o o ~ ~ t c(.see s ~ Genr? ~ ' nrgnnization, 08-20),
entry 08
0.
Key: @ ,vtimtr/rrior~.
rtrhrhrnon: scr olro Ahhm*inrionr und Indtrr ro Comp)rrnd~und Prott.in.r . - .
+-t+
Pre-synaptic N a+sodium voltage-gated channels rc*v VIX; ,A'N
Glutamate carrier reversal I inhibition
K+L-glutamale
*d
carrier molecules ('rrpmhc
+,.. Y L-Glu <
~ ~ s i c lr rel c
L-GIu
Nitric oxide - z guanylate cyclase activation -> c(;\ll' formation
Acti~.alion of innotropic and gl~rtamatr rrreptorr
7
:
I*
I I I
/
I
I
I I I i \,
J
Arachidnnic acid
I
f-
ca2+
Pre-cynaptic t.nltage-gated calcrrrm channels (%, I' O - r ~ [ l r \ ) 5rv I'\ (, ( 0
I' ! t 1 1
;
Pre-synaptic C protern-coupled metahotroprr ~lrrtamarr receptors
\ I \I \ '\
.'.
\-
--. ......
11,
I
----
,llnfe: Pofasrium channclr (not chorn) have important netrronal excrrahrlrtv
-
Diffusion from port-wnapric neurone 'refrogradu mescen~ers' we Rv t~l)ror-rron~vdlrrr.r rrr~r~rortmnv
---- ---__
Figure 4a. Overview of pre-synaptic GluR-linked signal transduction proteins. stimulation; -, inhibition. (From 08-31-04)
+,
I
DAG
('nlri~lrn
ca2+
/'ft~I-~~~r#{lllr d~pft/ar~:neon-nnr~nI~d r.nlrrrtm r h n ~ r ~ r r l * $r( \ I ( ,
( , I
L-GIu
receptor-
{this entry)
Glycine (to-agonist)
depolarization
AMI'A /
kclirtnt~ receptor-
+
Na r
-
chnrt tlels \<*t*
1I(,
(
\ I (,I I
1 ifl' l / A l I V
r
--
-"-,
---------
1 Nitric o\icIt# \ ! ~ ~ t h a wi \~ r : ~ c h i f i r ~ n a tIv -> \ i t r ~ c r ~ x i ~ l t - 1 1 5 t / I ! ( t A \ \ 1 I 1, , , , * --,,, Jc -J
---,-,---
I
d
0
Figure 4b. Overview of post-synaptic GluR-linked signal transduction proteins. stimulation; , inhibition. (From 08-31-04)
+,
cntry 08
-
Table 7. Summary of NMDAR-ossociatcd signal transduction components (From 08-31-05) Class and suhtypc of protein
Key rolcs/intcraction
Regulatory functions/notes
Adenylyl cyclases calmodulinsensitive
Increased levels of CAMP have been demonstrated following tctanict stimulation of the Schaffcr collateral pathway in the CAI regionm4
Calmodulin-sensitive adcnylyl cyclasc in this preparation depends on both activation of the NMDAR and incrcascs in IC~?-']~*" (SCC111.so 'CRIUI~I~II channels. I~igll-voltagc activat cd', this
tahk) ca2'/ calmodulindependent prntein kinase (C~MKII)"'
Targeted disruption of the gene cncnding rrCaMKII (i.c. gene-knockoutt) markcdly reduces (hut docs not climinatc) induction of LTP in brain sliceszn5 ~ C ~ M K I T - ~mice U I Iare ~ dcficicnt in both L T P ~ induction and spatial learning hut show no Rross morpholo~ical changes in the hrain
rrCaMKII i s highly exprcsscd in post-synaptic densities. Primary scqucnccs of cloned NMDAR cxhihit conscnsus~ phasphorylstion motifs for CaMKII (see /I'DTM/. Fig 2 nnd Protein phosphorylntion, 0832)
rlCaMKII is also likely to 'positively-modulate' AMPA/kainatc iGluR receptor-channels (see 'Non- NMDA-type. ionotropia glutmn~otil rcccptors', this t a l ~ l rnnd ELG CAT GLU AMPA/ KAIN, cntry 07) Calcium channels, highvoltage activated (scc V1,G Cn, entry
Calcium influx through both NMDA and non-NMDA receptor-channels in c u l t ~ ~ r crat d hypothalamic ncurones activates a calmodulin-dependent inhibition of the high voltage-activated (HVA)~ a " currcnt2""
Compr~rr~tivc notc: NMDA rcccptor activation has hccn shown to incrcasc CAMP lcvcls and thcrchy the fractional open timct of high-threshold ~ a ? + channels in CAI pyramidal 'ccl
cntry 08
n
Table 7. Conrinurd Class and suhtype of protcin Calcium channels, voltage-gated, type, N presynaptic (we VLG Cu, Pntry 42)
Key rolcs/intcmction
NMDA receptor agonists have hcen reported to sclectivcly and effectively depress N-type ca" channels which modulate neurotransmitter release frnm prc-synaptic sites2"' (see VLC: Cn, entry 42). The inhihitory effect is eliminated hv the competitive NMDA antagonist n-2-amino-5phosphonovalcratc (APV) and tlocs not require c a 2 ' cntry intn the cell
Implies a 'negative feedhack' hetween liberation of excitatory transmitter and cntry of c a 2 ' into the cell, modulating prc-synaptic inhibition and regulating synaptic plasticityt '"'
Calcium-store release channels: Rvanodine receptors lsrr 1LC: Cil Co RvR-Cal, cntry 17) ond InsPl receptors (sec 1f.C: Cn InyP,. pntry 19)
Pcrfusion of dantrolene on to In ccrcbcllar gmnulc cclls a major component groups of cclls during thc sustained plateau phase of of hoth K ' - and NMDAinduced elevation of ~ a " the Ica"], response to K' or involves release from NMDA reduces the response intracellular stores2oR. to hoth agents in a concentration-dcpcndcnt The ~ a " - s t o r e dcplctors thapsigargin (which marine?"'. Note: Dantrolcne is uscd as a clinical antidote blocks thc action of the ~ 3 ' 'ATPascl and for ryanodine rcccptorryanodine (src !LC: CN med~atedmalignant R,vR-CoI, cntry 1 7 ) hyperthermia (see [LC: Co display partial additivity Cn R,vK-Cilf, cntry 171 to K t -and NMDAind~iccdresponses, showing that thcsc aRcnts affcct two overlapping hut nonidentical ~ a "pools
Cytoskeletal proteins
A protein interaction hctwcen actin and NMDA channel regulatory proteins can affect the 'nindown' phenotype of NMDAR in native cclls
Scc Rundown. 08-39
-
Table 7. Contin~red Class and suhtypc of protein
Key roles/intcraction
Regulatory functions/notcs
Endonu~leases'~'
Scveral ca2+-activatcd enzymes may contrihute to excitatory amino acid toxicity, which may includc the activation of various cndonucleasest
Regulation of ca2+-influx appcars fundamental to thc 'ordered progression' of gcnc expression; dysrcgulation of ~ a "homeostasis is also likely to have longcr term effects on cell phcnotype or the initiation of apoptosist [see also 'co2+-scnsitivc pro tease.^', this tnhle)
G proteinFor role of GARAR receptors coupled GABA in low-frequency (-. 5 Hz) receptors, induction of LTP (see (SARAR Reccptor/tran.sducer subtypes interactions, 08-49) G proteincoupled glutamate receptors (mctahotropict glutamatc rcccptors, mGluRl
mCluR which activatc protein kinasc C appear to lowcr thc threshold of induction for LTP (see rer~icw,ref.",? and 'lJrotcin kinnsc C', this tahlc). Targctcd disruption of the gene encoding rnCluR, in micc having sevcre dcficits in motor cn-ordination and spatial learning2""
G proteincoupled muscarinic receptors (MI
Muscarinic receptors which See Appendix A - Index of G activatc protcin kinasc C protein-linkcd rcccptors. appear to lower the threshold entry 56 of induction for LTP (seu rcview14-' and 'Protein kinasc C', this tol?lo)
G protein-
14-opioid rcccptor agonistst potcntiatcr NMDARactivatcd currcnts in trigcminal ncurones of rat medullary slices, probably via activation of protcin kinasc C (scc tliis tahlr nnd ref.'10)
coupled opioid receptors, 11subtypes
~ G I ~ R - n u micc l l t have no gross anatomical or hasic clcctrophysiological ahnormalitics in cithcr the cerchcllum or hippocampus, hut show impairct! cerchcllar long-term depressiont and hippocampal mossy fihrc long-term potentiationzop (see Fi,p. Id). See also Rcceptor/tmnsd~rcer intemution.~.08-49
1,-opioid potcntiationt may hc a feature of synaptic plasticity1 ohscrvcd in central gain reception pathways21o (.?PC I'hcnntj~pic oxpressinn, 08-14, mnd Channel modulation, 08-44)
Class and suhtypc of prcltcln
Kcy rolcs/intcraction
GARA* (inhihitoryl receptorchannels (,s(.r, E l . ( : CI
In most cortical ncuroncs t h e nctiv;ltion of t h e NMUARs (and hcncc the induction of LTP1 - srv,
R c ~ u l a t n r yfunctions/notcc
Morph(~logicaldamage and psychotominicticr effects indilccd hy NMDA-active d r u ~ can s he prcventcd hy I'l?r,nr,tj,picc,xy)nb.scion.OH- 14) other drugs which act a t t h e ( ; A H A A .~'rltr!, rcquircs :I concotnit;~nt gnmm:i-;iminohutyric acid 101 rcctuction of ~ ~ 1 3 h c r ~ i c t (C7ARAA)receptor-channel inhihition hy low tloscs of t h e complcx2", indicating a CAIIA* ;tnt:igoni.;t functionnl inter;~ction h i c ~ ~ c n l l i n eThis ~ ~ ' .inriicatcs hrtwccn these channel types in viva Isrr. Rrcrptor th:~tin the ncocortox t h e activation threshold' c ~ the f r~nrr~y Monosynaptically evoked inhibitory processes inhillitory post-synaptic For intcr:~ct,ionsof currents in hippocampal GARAergici, chnli,nergict pyramidal slices diminish in and g l ~ ~ t a m i n r r ~ i c i t h e prcscncc of CNQX (an pnthw;~ysin t h c control of AMI'A receptor-sclectivc NM1)AII-nicdiatct! a n t ; ~ ~ o n i sand t l APV (.sr*(, glutamate tnxicity I, l I r ~ . i ~ p t (rntrr,qor~icts. or 08-5 1 ) .+('r, Fi,y. 7 following ;I t r ; ~ i nof action potentials. However, rcsponscs t o GARA applied hy iontophorcsist do not change signitic:~ntlyIcf. CI?onncl E l . ( ; C1 rr?orltrl(i!ion~rnrl(*r
,,
c ;A [ { A , 10-44,
Guanylate
cyclase
Sustained activation of ~ u a n y l a r ccyclasc and accumulation of cvclic C M P has I,ccn ohscrvcd following NMDAR activation in prlrnary cultures of ccrcl~cllargmnulc cells2'"
c G M P formation can occur via ~ a ? ' - d c p c n d c n t stimulation of nitric oxide synthasr and lipnxygenase metabolism of arachidonate (rclcascd hv phnspholipase A21. For further d c t , ~ ~ol ts t h e apparent neuro-protective role of c C M P a n d n ~ t r i oxldc, c \',(, r r f < l l 3.214.215 rind C / ~ r r r ~ nrnorltrlrlt cl ion, OX-44
entry 08
-
Table 7. Continued Class and suhtypc of protein
Key rolcs/interaction
Regulatory functions/notes
Nitric oxide Inhihitors of NOS appear to synthase (ca2'- hlock induction of L T P ~ tlcpcndcnt) (NOS\ll.%ll"
See nlso ' C ~ ~ a n y l acjrclr~se' te this table, and Channel moclulation, 08-44
non-NMDAtype ionotropic glutamate receptors (AMPA- and kainateselective glutamate receptorchannels)
In the maintenance phase of LTP, both NMLIAR ant1 nonNMDAR currents appcar to he enhanced2'", possibly as a consequence of ( i ) increased pre-synaptic glutamate r e l e a ~ c and ~ ~ (ii) ' ~ ~ ~ ~ increased post-synaptic rcsponsivcness of ~ c ~ u R ~ ~ ~ . ~ ~
The co-involvement of NMDAR and non-NMDAR in L T P ~induction is wcllcharacterized in the Schaffcr collateral-CAl pathway (see refs544.216for reviews). Generally, non-NMDA (AMPAlkainate receptors) provide 'sufficient' depolarization for removal of Mg" hlnck from NMDAR, facilitating the induction of LTP (see Fix. l e . fl
Phosphatases In cell-attached recordings of (various, CA"*- acutely dissociated adult rat sensitive) e.g. dcntatc wrus granule cells, Ca7+/ applicatic~nof okadaic acid (a calrnodulinnm-sclcctivc phosphatase depcndcnt inhibitor) prolongs NMDARphosphatase 2R channel openingsonly at a ( c a l c i n ~ u r i n ) ~concentratinnthatinhihitsthe ~'
See also ELG CAT GLU AMPAIKAIN, entry 07
Calcineurin inhihition prolongs the duration of single NMDA channel r.rpcnings, burstst, clustcrs and superclusterst
Note: Intracellular dialysis with calcincurin does not ~a~'/calmdulin-dependent induce rundownt of phosphatase 2 R (calcincurin), NMDAR in mt hippocampal and is ineffective when ~ a ? ' ncuroncs2*,' entry through NMDA See also Protein phoschannels is prevented222. In adult dcntatc gyms granule phorylation. 08-32 cells, calcineurin is activated hy calcium entry through native NMDAR channels and shortens the duration of channel openings. Simulated synaptic currents arc enhanced following phosphatase inhibition. Application of a calcineurin inhihitor (FK-5061mimics the effects of okadaic acid222
Table 7. C o n t ~ n u c r i Class and suhtype of protcin
Key rolcs/intcraction
Phospholipapes, Post-synaptic [ ~ a ' ' ] (varic~ils,C:a ' - elevation activates PLAI. scnsitivcl c . ~ . Inhibitors of PLAr block phospholi ~ s c L T P ' ' ~ * " ~ ~ A? , P L A 2 , ~ ~ ~ j ~ q nntl phosTrnnsirnt appliration of pholipasc c"" arachidonic : ~ c i d(sr:r*noxt c o l r l n ~ nt/ o hippocampal synapses induces a 'slowonset' potcntiation
Proteases [various ~ 3 ' ' sensitive) c.g. calpa~n"~
Conversion of trtrl?sicnt NMDAR-mctliatctl c a " influx into prr%istc.nt modificaticms of synaptic strcngtht in LTPi indi~ction
Rcgulatoty functions/notcs
Ser Fis. 4. PLA?, releases
arachidonic acid ( A A )from mcmhranc phospholipids which may serve a s n 'rutrogradc mcssengcr' (i.c. diffusing from post-synaptic t o prc-synaptic loci) ( f o r derails, scrJ 1iat:eprorl trnnaduccr intpmcfions, 0849). A A stimulates phns-
phoinnsitidc (PI1turnover. PI tiirnovcr has heen ohsorvcd prc-synnptically during LTP induction"". AA may stimulate prc-synaptic protcin kinasc C (sce Fig. 4 ) ~ a " - s e n s i t i v e protcnscs inclutlc calpain 1 1 ( , r ~~ a " scnsitivc prcltcolys)~)and c a l p d n I1 ( m Ca~ '-scnsi tivc p r o t ~ t ~ l y s i s l " " ' ~(scr l ' m t ~ i n ~ ? h r ) s ~ ~ / t o n ~ l rON-.32) itinn.
Protein kinase C
(c;I?'/~~o.;-
pholipidtlcpcndcnt protein kinnsc; I'KC,l"%lllS
NMDAR-mcdi;~tctlc;I"influx which ;~ctivntesPKC directly mcdintcs ( i l 'positive modulation' of t h e NMIIAR itscli (i.c, increased gli~tarnatcsensitivity durinx the maintcnancc phnsc of LTPI; ( i i l positive niodulntion of AMPA/kainatc receptors ( s w t h i , ~t ( ~ / l l ( ,ant1 / [iiil activation ot Ca- '1 calmodulin-dcpcndcnt protcin kinnsc I1 (this tmhlc) T h c r c is likely t o h e a multiplicity of PKC iqoforms, with post-synnptic kln;lsc (PKC--,?Iactivating transiently ( T , ? -several m i n ) foll(~wcdhy prcsyn;~ptickinasc ;lctiv;~tir)n (PKC:-.]?I with sirstained ; ~ c t ~ v i (t ryl ? . l IIJ?'~
PKC inhihitors can hlock LTP intluction afrcr t h e tetanicl stimulation phase (srarpF I X . 1 / 7 1 , PKC inh~llitors [at low doscs) I)lock intluction of long-term potentiation without affecting short-term pr~tcntiation(ser, Fiiy. Id) PKC activators 1c.g. phorhol cstors) induce synaptic potentiation2."' when inicctcd pc~st-synapticall o r applied e x t r a c c l l i ~ l a r7'l ~ ~ ~
N ( ~ t rAs : r c v i c ~ e t l ['KC ~~~, (~lrln has ~ hccn judged insufficient t o intlucc LTP I>ut tn;ly convcrt STP t o LTPl (.YCP
Fi
entry 08
Table 7. Continued Class and srlhtypc of protein
Kcy rolus/intcmction
Regulatory functions/notcs
PKC incrcascs P,,,,, and rcduccs valta~c-tlcpcndcnt ~ ~ ' ' - h l n celfk the NMDAR
~rirnar~ scqucnccs t nf clonctl NMllAR cxhihit conscnsust phosphorylation motifs for PKC (see [PDTMI. Fig 2 ( ~ n dI'rotein phosphorylntion, 08-32)
(srr r ~ i . 'c~nd ~ Rlockrrq, ON43)
Currents conducted through hctemlngously~expressed NMDAR in Xenopv~nocytcs can hc potentiated hy PKC activators59-22Y
Tyrosine kinase; TyrK
Expcrimcntal induction of LTP can be blocked hy coapplication of tyrosinc kinasc inhibitors2,"
NMDAR activation has hccn linkcd to tyrosine phosphorylation of MAP-2 kinaset",'
For furthcr information on thc rnles of NMDAR protein phnsphnrylation and G protein-mediated signalling, see Pmtein phospj~nrJ~lc~t~on, i8-;12, nnd
Role of phosphomodt~lfltiono f N M D A R in development o f LTP phenotypes 08-32-04: Direct, positive modulntion of NMDA currcnts hy protein kinase C (17KC)has hccn ohscrvcd, ant1 it has hccn suggested that PKC activity may dctcrrninc the threshold of L T P ~induction (see r e v i ~ w r, ~ r . ' ~ ,Enhanced ~). kinasc activity may underlie the ccntral rolc of thc NMDA rcccptorchanncl cnmplcx in neurnnal plasticityt (src .scctinns on LTP induction under Phcnorvpic exprc.~sion,OH- 14).
NMDAR phosphorylation m a y he nssocinted with the mointenonce of epileptic states 08-32-05: ~indlin~t-inducedepilepsy affects thc sensitivity of NMDAR channels t o ~ntraccllular high-cncrgy pho~phatcs'"~ (see Phenot,vprc cxpressron. 08-141.
Mcchnnism o f PKC potcntiotion o f N M D A R responses 08-32-06: In a numhcr of nouronal prcp;Imtions, protein kinase C has been shown to pcltcntiatc the NMDA response hy incrcns~ngtho prclbahility of channel clpcnings and hy rctlucinr: thc voltage-dependent ~ ~ ? ' - h l ( l cofk NMDA receptor-channelsY7 (srr, Chnnnr.l ~nodulrltion.08-44),
entry O K
----
Activation S l o w risc a n d ~ L Y : Ctir?i('~ I J ~ o f NMl>A-nctirmtcd c u r r c n t s 08-33-01: NMDAR-channels elicit the slowly rising, slowly decaying ( r several hundrcd r n i l l i s ~ o n d sopen state1 1 ctrmpc>ncnt of cxci tstory postsynaptic currents (EPSC:s7 l in rcsponsc to glutamate (sc'c* I t i i ~ c ~ t i ~ ~ o tOXion, 371. In comparison, A M P A receptors niedi;itc the most rapid synaptic cxcit:~tory ncurotransn~ission~ ;lnd conduct mainly Na' currents (src E1.G CA.1' ( : I , l l A h4I'A Ih'AlA'. c,ntrrr 1/71, l n ~ r i n s i cpropcrl ics o f NMIIA R-c.hrlnn(~1.s drt c r n ~ i n cd u r n 1ion of currcnt 08-33-02: Intrinsic NMIIAR channel kinetics dctcrminc the time conrse of NMDA receptor-11icdintc.d synaptic c i ~ r r c n t s " ~ ~For . ~ ~example, ~. kinetic responses of NMDA r e c e p t o r s i n cxcisctl mcml~ranc patches from hippocnmpus nnd supcrior colliculus show simil:~ritics to that of the NMIIA E I I S C ~suggesting , thc time coiirsci of the NMDA EPSCi rcflccts slow NMI)A channel properties in this prcp;ir;~tionJ". ' K c - l ~ i n t l i nof ~ ' ,q1litn1i1ritr l o ~ h Nc M l l A R fro171 thl' s,vn(lplic cleft ~ O P . S1701 OC('1lt
08-33-03: Rricf pulsrs of glut;~mntcapplied to outside-out rncmhr;lnc patches results in openings of NMIJA chnnnels that persist for I~undrcds o f milli.si~i.on(l.s, indicating that glutamate can rrrrlr~in I ~ o u n d for this pcriot12". Current rise' and decayt is mnrkcdly temperature-dependent, intlic:~tingthat changes in rates of free transmitter diffusion cannot ;ilonc ;lccoilnt tor its time
(:Iutarnotc (117ti ~l!~cirii'(1c.t iv(lliP 1 1 1 NMI>AlZ ~ (IS indcpcnrlcrll c o ogonisl s 08-33-04: Occupation of ;i separate, allnsteric 'glycine receptor site' is also an absolute r c q ~ ~ i r e m e nfor t NMDAR activation (vcc*K(;.c:ptor ( ~ ~ o n i s tOX-501. s. T h c c.onc.r,r?trl~tioriof ~ l y c i n cat the synaptic cleft1 is I>c\ocv a saturated level. In trigcminal ncuroncs, cxtcrn:il C:I" contrihutcs to unusunlly high glycine affinities for NMDA receptors (potcntiatic>nt c~ccuning when glycinc sites ;Ire unsatumtcd - scc E r ~ f ~ i ~ i I l r idi<socir~tion tlm coristnnt. 08451. This form of c a 2 + mndlllation may havc a rolc in regulating thc NMIIA receptor-ch:inncl activities during intensive or sustained ncuronal stimul;ition2'". Analysis of NMI3A clisnncl activation kinetics in outsidcout patches of cultilrrd hippocampal ncuroncs (following mpid steps into . Glul has dctcrniincd the high conccntr;~tionso! glut;~niatc,c . ~ -20011~ ;~ctivnt/ontime c o ~ l r s c to ' he concentration-inclcpcnilcnt and limited hv transitions' hctwccn the shut (hut 'fullv-ligantlctl'l state nil the open s t a t c l . Kinetic motlrls which c:ln :Iccount for activation kinetics following glutam;itc concentration jumps havc hccn derived for NMDAR in hippocam pal n c u r o n c ~ ~(vcc, - ' ~ F I X . 51.
'Temporal summntion ' of synaptic currents relieves ~ p - b l ~ hy c k depolarization 08-33-05:Single, brief applications of glutamate arc sufficient to producc an extended activated state of sever01 hundred millisecond^, whosc transition 10 pM Glutamate 2 -
#
MIC A
MIC
of bindins sites
5 pM Glycine
10 pM Glutamate + 10 WM Glycine
I
Figure 5. Mode1.s nccounting for N M D A R nctivot ion kinetics following low agonist concentrations ( e . ~ . ngonist concentrrttion jumps. At rc21r~tively -2-If) I'M ~ltltnmnte)a two glutamate-binding site model can flccollnt for nctivntion kinetics followins glutcrmntr~ conucntrntion itimps (upper panel)2~'7.A two ~lycinc-bindingsite model cnn also he fitted for channel nctivntion in the continuous presence o f glutnmnte (middle pan~1)2". Agonist and co-n~onist l~indin,p kinetics nre better described l7v nn independent co-agonist binding model, rr~therthnn n scqtrentiol hindins niodrl (lowcr pnncll"". Datn points shown represent npproximntelv thr first 30 nin following mgonist nddition, with fitted model predictions indicrrted hv arrows. MIC denotes rtddition o f 2.5 mn4 methoxyindole carboxylic acid used here to ellminnte spurious grltlng rit~e to glyc.ine contr~minr~tion from solutions (see Receptor nnto~onists,08-fil). Note: Thesc studies predict the NMIIAR to he nt lenst tctrorneric, contninin,y four liwnd-binding subunits, oasum in!: a sin,ylc binding site prr sul~trnit"~.(Ilr~procluccriwith prrmission from Clrmrnts mncl Wcsthrook (199 I ) Neuron 7: 605-18.) (From 08-3.3-041
entry OX
-
t o the closed state is independent of the c o n d u c t i ~ gstate of the channelz3'. This modc c ~ gating f cwlscs temporal summatian1 of synaptic inputst (and conscqucntinl: rlcpolarizatirlnt). Notr.: Summation can cxplnin why 'singlc shock' sti~nulationof nffcrcnts is not ns cffcctivc ns natur;~](rcpctitivc] stinii~latioii,since sin& pulses do not 'sufficiently depnlarizc' ncuroncs t o rclicvc t he M ~ ' ' hlockH' ({or siqniiicrlnt.r, srr F ~ R . I ru rirltl I{lor.Kcrs. OX-43).
Nr~rnhc~rs o f N M D A c:hrjnnt.I opcnirtgs rc!cplirrti to ,ycnc>rrrtc
(In
EI'SC~
08-33-06: Rricf applications of ~ c l u t a m a t cto outside-outt patches from
hippclcampal ncuroncs in the prcscncr and ahscncc of the opcn-channel hlockcr MK-801 havc shown that about .3B'Y0 of the L-glutamatc-hound channels arc open at the peak of the ~urrcnt'"~.Tho high prohahility of opening for NMDA rcccptor-channels following stimulation by LRl~~tamntc2"H sumcsts that rolativcly few channels arc required to 'guarantee' a large, Iocnlizerl post-synaptic c;~lciumtransient.
A~onistsprohnhly remain hound d u r i n ~'supc.rc/ustering' 08-33-07: N M D A receptors havc an unusual propcrty of binding ccrtnin
agcmists (inclutiing gliitarnatcl h)r a long period c ~ ftimc. This property may ] a partly explain why I~ricf(-1 ms) ;~pplicntionsof glutamate [ l m ~prtiducc slowry decrying current, the major component of which has a timc constant of -200 ms. ' ~ u ~ c r c l u s t c r i n ~ hchaviour 't ohserved at low i ~ n ~ ON-41) l may correspond glutamate conccntr;~tions(scr S i n ~ y l ~ - r . l ~ i rclilr(!. t o ;I singlc pcrioti during which one or tnnrc mc~lccralcsof glutanintc arc tlound tt, the rcccptc~i4".
-
Current-val tage relation E f f ~ c tosf difkrcnt cxtrclccllr~l~r inns on 1-V relllrinnships closc to E,,., 08-35-01: T h e response nctivatcd hy NMDA sgonistst cxhihits n voltage-
dependent cxtraccllular ~ g "hlock. Ca"-influx is restricted unricr resting cc~ntlitions, hut post-syniiptic membrane depolarization can rcmovc the hl{lck. Furthcriiiorc, thc largcr thc driving forcei tor M ~ ' + to pcnctrntc tho , Illr~ckcrs.08-4.7). nicmhranc, the largcr the t~lock( f o r frlrthc~rr f ~ t r ~ i l ssrc However, in tight-scnlt, wholc-ccllt r c c o r d i n ~ sof cultured spinal cord and hippocnmpal ncuroncs, high concentrations [ 2 0 m ~ of ) ~ n " ant1 Ca'' display linear I-V relationshipst [within +IS mV of the rcversal potcntintl althouxh thry d(i reduce slope contluctanccs. Ry contrast, extracellulnr ~ n ' ' ions produces a strong, voltagc-dcpcntlent block o! responses to NMDA, such that cvcn close to the rcvcrsal potentiali, the NMDA currcnt-vt~ltagc rclationshi is high1y nt~n-lincaP4'.
Tlir. slow r~oltr~~c-depcndc~nce of I', , ,
in ~,y*'-freesolr~tions
08-35-02: Studies of non-lincart wholc-cellt I-V curvest in free solutions hnvc shown that NMDh channels in cxciscd pntchcs rcvrrsihly shift their I),,,,.,, in n vriltaxc-tlcpcnrlcnt ~ n a n n c r(i.c. they cxhihit -,I- to 4folrl Arc;Itcr I),,,,,.,,at positive pc~tcntialsthan at rcstl'?'. Changes in I),,,,,,, nrc ~n:linly ;ittrihutahlc to shifts in openinp: frequencyt: I:,,,,.,, changes over a s c~l>scsvcd - 'vcrv slr)wJ time crlursc 1-2-15 niinl which ~ ~ n i l r r l i c the
entry 08
hysteresisf of whole-cell current-voltagc curvcs ohtnincd undcr noncquilihrium 1i.e. non-steady-statct ) conditions. Thc slow increase in P,,,,, provides a potential cxcitatoxic mechanism in that CA"-influx can increase markedly in cells dcpolarizctl for prt~longctlperiods of time1.".
Quontitotive N M D A R nciivniion models precIiciin,q E,., nnd cap*infltlx n t different v o I t a ~ c s~ n Ic~'"],, d 08-35-03: In accordance with a quantitative model dcvcloped for NMDARchanncls cxprcsscrl in cu!turccl! hippocampal ncurt)ncs2'*, increasing [ ~ a " l , , markedly shifts the reversal potential to positive values and simrrltmneortsiy dccrcascs thc single-channel conductance at potcntials ncgativc to thc rcvcrsnl potential. Using the model, rclativcly s i ~ p l c quantitative descriptions of calcium pcrmcationt anrl channcl hlackl hy calcium ions can account for ohscrvcd channcl hchaviour and accurately predict rcversnl potcntials and mngnitudcs of calcium influx over a widc rnngc of condit~ons'~~.
Dose-response Thrcshold.~for nctivation of ,qlutamatc receptor-chonnel subtypes in mixed populations 08-36-01: In dorsal horn ncuroncs, activation of ~a"'-pcrmcahlc NMDA receptors cvokcs intraccllular c a 2 ' transients that arc largc 1-780 nA], rise at a morlcratc ratc, and maximize amplitudct at NMDA conccntrations of - , 3 0 0 / 1 ~ . Whcrc mixed subtypes of glutamate scccptclrs arc cxprcsscd, glutamate rcsponscs at conccntrations less than 3 1 1 ~arc d ~ l crxclusivrlv to NMDA receptor activation2*". At higher glutamatc conccntrations, intraccllular rcsponscs arc mcdiatcrl hy I~r~thNMDA ant1 non-NMDA rcccpt~rs~~~'.
Inactivation Inactivation parameters shfipe decay times o/ N M D A receptors 08-37-01: T h c characteristically slow decay of NMDA-mediatcd EPSCS~
appcar to he duc t o (i)persistence of bound glutamate and ( i i ) the long open state of the channels. In hippocampal ncuroncs, repetitive stimulation ot glutamate receptors clicits increasingly smaller ionic currents. For example, in thc prcscncc of 2 . 2 n - 1 ~Ica2'],,, repetitive glutamatc applications ( 1 5 episodes of 4 slmin) elicit progressively smaller currents which stahilizc at -45% of their initial peak This 'interepisode inactivation' is cxacerhatcd hy elcvating extracellular ~ a "to I 1 mM, and is attenuated hy reducing extracell~llar ca2' to 0 . 2 2 m ~ . Current decay shown during individual stimuli ('intra-episodeinactivation') is ciepcndcnt on cxtraccllulnr ~ a ? yet ' remains stat~lcduring rcpctitivc stimuhtion. Thus, inter- and intra-cpisodc inactivations of NMDAR currents result from two distinct proccsscs triggered by ~ a " . Thcsc 'modalities' of inactivation may arise from ~ a - "hindiny: cithcr to the - reccptor or to closely associatcil rcgi~latclrv
I
cntry OX
Distinctions hct~vccnNMDA rcccptor-cl~(~nr~c.l rlcscnsitizrltion nnd intlctivntion 08-37-02: T w o distinct mechanisms have hccn s u p ~ y s t c dfor modulation of N M D A receptors by intracellular ~a"'"'. I)c~~c~nsitizc~tir,n nf N M D A rcccptors is inducetl when I>oth ~ntr;lccllul;~r C;I" is increased ant1 NMIJA receptors activated hy ;igonist. Note: T w o types of steady-state desensi-
tization for t h e N M D A i~gonistsaspartntc and glycine 1i;lve hccn shown in isolated rat hippocamp;ll n e i ~ r o n e s ~ ~I n" .c ~ c ~ t i r ~ r ~oft i oNr ~M D A receptors is produced hy increased levels of intracellular C;I" hut docs not require NMLJA rrccptor activation for intli~ction'~' /scsc. l~c~lrnc,).
'IIo~~~-rc:qulr~tior~ o f jlost - s y t ~ t ~ j ~~ (t1i.c" -c1t1trj7 08-37-03: Studies of calcium-dependent inactivation of N M D A channcls in
cultured rat hippocampal ncuroncs h;ivc suggcstctl a mechanism for downregulation of post-svnaptic c a l c i i ~ n ~cntry during sustained synaptic a c t ~ v i t ~ ' ~In~ .norlnal [ ~ a ? ' ] , (, 1 - 2 m ~ land lO/rhr glycinc, macrnscopic currents evoked hy 15 s applications of N M D A (10/rh~linactivate slowly following a n initi;~lpe;lk. At SO rnV in cells huffcred t o I c ~ ' ] , < 10 M with 10 mh.1 EGTA, t h e in;~ctiv;~tion tirnc constant r,,,,,,., is -5 s. Inactivation does r ~ o t occilr ; ~ t~ n c ~ n h r ; ~potenti;lls ~ic ot -140 mV and is ahscnt at IC;~' ' ] ,, 0.2 mhi, s u ~ e s t i n ,
Trtlt~sirt~l ~ ( 1 " ' -itlduc.el! itlrlc.1 ivrll iorl
Ic;~' ' ] , following cell tlepolariz:ltiont also resrllt in inactivation of N M D A channels without altering t h e single-channel contli~ctancc.~ n - " cntry through local voltagegated ~ a "channels may suhstitutc for (or aitl) this inactivation process, ;ilthough C:I-"-entrv through N M D A channels is more efficient. [ ~ a " ] , tr:lnsicnts m:ly inclilcc NMIIA channcl inactivation hy hinding t o either the channcl or ;In 'nssoci:ltcdr rcgul;~toryprotein t o ; ~ l t c rchanncl 08-37-04: In ccll-;ltt;lchcdi patches, transient increases in
M i ~ i ~ h o n of i~n NAIllIA ~ rrpcivj~tor rractitrr~tionfollowing innctiv(1tion 08-37-05: Untlcr conditions of I~ricf( 1 m s ) glutamate application ( 1 - 1 0 mhr),
ncuroncs in r:it visual cortex prcldilcc ;I rcsponsc th:lt mimicks t h e time coilrsc of miniature EPSCS~/mEPSCs). T h c ratc of nnsct of desensitization is m u c h slower th;in t h e decay ratc of t h e response to a hricf application of glutam;ltc, implying that t h e tlccav of mEPSCs reflects channel closure into a statc rcndily avall;~hlc for rea~tivation"~.Furthermore, a t steatly statc, MK-801 complctcly hlocks suhscqi~cnt responses to NMLJA, suggesting trl1or~i.l c;ln re-open a t ste;~dystatc. Inacthat 'in;1ctiv;ltctlJ ch;lnncls tivation is fully rcvcrsihlc in t h e presence of A'T'I' hut is not hlockcd hy inhibiting p1iosph;ltascs o r proteascs. For a description of the complex closed-time tlistrihi~tionsof t h e NMDAR srr .?ir~,ylr~-c.hannrl dnto. 08-41, (
~
c
~
p
Trt~ns~clnt Cl currrnt5 rrc.t~v~ltctl h\~Cc~'+-~nflux thmu,qh NA4DA r1.c-cptori r ~ . ~ p r i ~ ~ rI d I ~s Xcnopus ooc.\rtci 08-37-06: NMI)A receptor\ cxprc\\cd In ,Yenopus oocvtes ~nlcctctlw ~ t hrat
brain RNA elicit a rapid inward current on NMDA application that dccays in scvcral seconds to a relatively stahle level ('apparent desensitization'). Howcvcr, thc early ~ a ? + - d e p e n d e ntransient t component can hc cvokcd morc than oncc during singlc applications of NMDA, sumcsting that thc rcccptor does not d ~ s c n s i t i z c ~ ~ A" .variety of chloride channcl hlockcrs 'almost eliminate' the transient component and, in addition, inhihit the plateau current. Thus it has hccn proposcd for oocyte expression systems that a significant portion of thc NMDA current rccorded is carried hy a transient inward calcium-activated chloride current (see ILG C1 Ca. entry 25).
Rundown Relotionship o f cytoskeletal element function to 'rundown' 08-39-01: T h e ATP- and calcium-dependent rundownt of NMDA channels
can hc prevented when actin depalymerization is hlocked hy phalloidin2"". Comparative note: Rundown of AMPA/kainatc receptors is unaffcctctl hy phalloidin. Application of cytochalasins (which enhance actin-ATP hydrolysis) inducc NMDA channel rundown, whereas taxol or colchicine (which stahilizc or disrupt microtuhule assembly) havc no cffcct. Protease inhihitors also have n o cffcct. Calcium and ATP can thercforc influence NMI)A channel activity by altering thc statc of actin polymerization. T o cxplnin thcsc findings, a model has heen proposed in which actin filaments 'compartmentalize' an NMDAR channcl regulatory protein2s".
Sen.~itivitvo f rundown to intracellular cn7' 08-39-02:Increases in intracellular calcium lead to NMDA channcl runtlownt [luring whole-cellt recording of rat hippocampal ncuroncs hy rcducing thc open probability of thc NMDA channcl".'.
ATP regeneration retards rundown 08-39-03: Although high concentrations of ATP (or thc inclusion of 'ATP-
regeneration' systems') in thc patch p i p c t t ~ ~can ~ ' prcvcnt or rctard runtlownt, their action has hccn s u a c s t c d not to hc a direct result of rcccptor p h o ~ p h o r ~ l a t i o n ~ ~ . ' .
Selectivity Activntion o f N M D A receptors mediates colcium influx 08-40-01: Among the ionotropict glutamatc receptors (iGluR), NMDA
rcccptors arc thought to mediate thcir physiological response mainly through thc influx of extracellular calcium - cf. AMPA- and kainatcsclcctivc iGluR which (with some cxccptions) mainly mcdiatcs Nai-influx (SCT s p e c i ~ lC ~ C Sin Selectivity under ELG C A T GLIl AMPAIKAIN, 0740). High ~ a ~ + - ~ e r m e a h i of l i trecombinant ~t NMDAR has hccn directly demonstratedf'.
The struct~rralhasis of cap+-selectivityin i(:lrllZ-chnnncls 08-40-02: NMDA receptor-channcls display sclcctivc pcrmcahilityt for ~ a ? '
-
over ~ g ions " dcpcndcnt on the placement of an asparagine (Nl residue at the cquivalcnt locus to the glutaminelarginine (Q/RI site within the M2 domain of 'nnn-NMDA' channels. For further details of structurc/function at this sitc, see Domain functions. 08-29, and Gene organizntion, 07-20. Domoin functions. 07-29, Current-voltage relntion, 07-35. Selcctivit~r.0740. and Rlockcrs, 07-43, rrnder ELG CAT GLU AMI'AIKAIN.
Direct compnrisons of cn2+-influxmediated h y NMDA- versus nonNMDA receptor-channels 08-40-03: The CaZ+fraction of the ion currcnt flowing through glutamatergic NMDA and AMPAikainatc reccptor-channcls has been directly compared in forebrain neurones of the medial septum2"'. At negative membrane potentials (extraccllular frcc ca'' concentration of 1.6 r n ~ the 1 ~ a " fraction ' of the current thmugh thc NMDA receptor-channcls was only 6.8%. Compflrr~tivcnote: A high fractional ~ a "current of 1.4% has been dctcrmincd for thc lincarly conducting AMPA/kainatc rcccptor-channcls found in thcsc n e u r o n c ~ ~ ~ ' .
Influence ot'/cn2+J,, nnd NMDAK
on the condzlctnncc mechanism o f the
08-40-04: In tight-seal, whole-ccll recordings of spinal cord and hippocampal neuroncs in cell culture, raising the extracellular calcium concentration shifts the reversal potcntialt of rcsponscs to NMDA in the depolarizing direction (calculated p(-il/fN, 10.6, with cxtracellular Na' hcld constant at 105 nlhl). Thcrc is at1 apparcnt incrcasc in Pc,/fN,, on lowcring cxtraccllular Na', which may rcsult from interaction of permeanti ions within the - channc12" (src nI.~oCtrrrent-voltrl~erelation, 08-35),
-
Single-channel data superc cluster in^' properties o f N M D A channel openings at low ~ 1tomate u conccntrn tions 08-41-01: Single-channel rccardings of NMDA receptors in adult rat h~ppocampus{ C A I )at low glutamate concentrations (20-100 nM glutamate with 1 ~IM-glycincrvithout ~.xtracullulardivalcnt cations] show NMDAR activations cnnsist of clustersT of channel opcnings in this prcparation2.tv. Snmplc tiistributions of thc length of thcsc clusters havc mcan time constants of 8811s (4504, of cipcnings), 3.4 ms (259/n]and 32 ms (,30°/,1. Long clusters contain short-, intcrmcdiatc- and long-duration openings as well as subcnnductancet openings, with the average open probabilityt within clustcrs averaging -0.62. Thrcu cornponcnts are evidcnt in distributions of the number of openings per cluster, having mcan values of 1.22, 3.2 and 11 openings per clustcF3".
Complexity o f NMDA R closed trme distrihutrons 08-41-02: S~ngle-channcl rccordings of NMDA rcccptors in adult rat
hippocampus ( C A I ) at low glutamate conccntratlons (20-100 nM glutamate wlth 1 )l~-glycinewithout extracellular divalcnt cations1 display complex closed time distributions, requiring fltting of flvc exponential cornponcnts
for adequate dcs~ription"~.Of thcsc five components, at least thrce, with time constants of 6811s, 0.72 m s and 7.6 m s (relative areas of 38, 12 and 17%) rcprcscnt gaps within single activations of the r e c e p t o ~ ~ ~ .
Sublevel transitions reflect NMDAR subunit composition 08-41-03: Single-channel propertics fnr NMDAR have hecn dcscrihcd in a numher of preparations217'R7~2"2-2h0 . follow in^ cxprcssion of cloncd
NMDAR, single-channel conductances and characteristic patterns of suhlcvel transitions can hc uscd as diagnostic criteria for suhunit composition'. Tahle 8 lists some single-channel charactcristics resulting from dcfincd subunit combinations co~nparcdto those of native cells.
Voltage sensitivity Voltage-dependent extracellular M<$+-block o f the NMDAR-channel 08-42-01: Thc hlock of NMDA receptor-channels by extracellular M ~ ions ~ +
cxhihit strong voltage-dependencet, and allows the NMDAR t o function as a molecular 'coincidence detector' (for further detnil.7, see Fig. 1e.f and Hlockers. 08-43). In M,?-free solutions, slow voltagc-dependent changes in channel open-statc prohability underlie hysteresist of NMDA responscsl"' (see Current-volta,yc relation, 08-35).
Voltage-sensitive block b y arcnine 08-42-02: The volto~e-dependenthinding sitc for thc polyaminc antagonist arcaine (see Channel modulation, OX-44) is distinct from either the phcn-
cyclidinc/MK-801 sitc or the voltage-dependent channel site far r n a g n e s i ~ m ' ~ ~Thc . voltage-sensitivity of arcaine block indicates the hinding site is locatcd in a rcgion of the NMDA rcccptor ionophore complcx capable of sensing transmemhrane potential2"'. Arcainc has also hecn shown to act as an NMDAR open-channel hlockert, an action that is independent of thc polvamine site (see Reccpfnr antn,yonist.~.OH-.51). For modcls of voltagc- and usc-dcpendcnt NMDAR blockade by thc dissociative anaesthetics kctarnine, phcncyclidinc (PCP)and dizocilpinc, see Rlorkcrs, 0843. For thc voltage-dcpcndcncc of polyamine modnlation, see Channel - modulation. 08-44.
Blockers Under resting (hyperpolarized) conditions, NMDAR-chnnnels are blocked b y extracellular M$' 08-43-01: At negative mcmhranc potentials, ~ g ~ + - b l ocurtails ck transmittercvokcd ion conductance through NMDAR*"~~'~'.Thus glutamatc rcleasc from pre-synaptic sites is unahlc to activate thc chsnncl unless thc postsynaptic membrane is sufficiently depolarized t o remnve the hlock (for an illustmtion o f this principle, see Fig. I r 7 ,I). Thc larger the driving farcet for Mg?+to penetrate the memhranc, the l a r ~ c rthe hlock. Variant strengths of
Tahlc 8.
S(.l(pctc~l\rn,y1c~-t11crririvlchorcrcfc~rr\flc.\ rrpsulflr~,y{ ~ o mrfcfrncd
h'hll)AI< \rr17rrnrt c~or?i/~rr?crfron\ c~)r?iptrrc~l fo 111o
Refs
NRI-NRZA ant1 NRI-NRLR comhinations hoth have 5 0 pS openings, hricf 40 pS suhlcvcls (in 1 mM external c;I."), with s~mil:lrmc;in lifetimes and frcqucncics. NR I -NR?A ;lnd NRILNII?I< comhinations also show close clu;~ntit;~tivc rcsc~nhlanccto the channcls of hippocampal CAI and d e n t ; ~ t cgyrus cclls ant1 of ccrehell;~rgranule cclls, except that t h e NRI-NR2A coml7in;ltion has ;I lower glvcinc sensitivity than t h e n;rtivc ch:lnncls T h e NRI-NRZC comhin;~tionpr(xluces a channel with (36 pS ;1nc1 19 ITScondl~ctanccsof similar (hricf) tlur;~tion.NRILNII2C channcls closely rcsemhlc t h e .Is-I H pS channels that have hccn ohserved (together with SO pS channels, scr 1)clowl in large cerehell;~r ncuroncs in c i ~ l t i ~ r c NRI ; ~ l o n c
"
rn Low current ;~mplitudcsohscrvcd following expression of sulwnit NR I in X(,nopus oocytcs predict that 'n;lturall NMDA receptors occur in hctcrooligomeric configurations
Single-channel recordings of native N M D A rcccptors Nativc NMUAR, rat in : ~ d u l trat hippocalnpus ( C A I ) at low glutamatc h i p p o c ; ~ m p t ~ s concentrations 120-100 nh.1 glutamate w ~ t h1 I r M glycinc w i t h o i ~ tcxtraccllul;~rdivalcnt cations) show t w o main conductance levels of 5 0 pS ant1 4 0 pS (extr;~ccllularCn" at I m ~ 1 Approx. . %Onitof openings arc t o t h e 50 pS contluct;lncc level. Single-channel conductances increase as cxtr;~celIular~ a ? is' rcdt~cctl.Single-channel opcn time distributions can he tlcscrihcd hv three exponential components of 87 Its, 0.91 n1s and 4.72 m s (relative arcas of 51 X,,3 1 "o ;lnd 18"Ll with t h e majority of long opcnings hcing t o t h e I:lrgc conduct;lncc level Nativc NMDAR, ~g."-free solutions
"
In ~ g ? ' - f r c csolutions, increases in tcmpcrature hctwccn 14 and 24 C, incrcascd t h c NMDARchannel contiuctance with a Q l o of .- 1.6 while t h c nlcan opcn times decrcascd with a V l l l of .- 22.5'
'.''
257
entry OH
-
Mg2'-blockade of recombinant NMDARs has been demonstrated directly6. Open-channel hlockt of the NMDAR by Mi'' may explain its ohscrved 'neuropmtective' and anti-epileptic effects. Notes: 1. Voltage-dcpcndcnt hlock of thc NMDA receptor-channel occurs at concentrations 'wcll hclow' the (millimolar] cxtraccllular concentrations found in thc CNS. 2. ~ g "is rclativcly weak as an opcn-channel hlockcr hccausc it leavcs thc channel rclativcly quickly (cf. chnnnel nccupnncy times oi mcmnn!ine c~ndMK-801, below). 3. Proper functioning of pre- and post-synaptic Na'/K'-ATPases arc crucial for operation of voltage-dcpcndcnt ~ g ~ + - h l n c k aand d e prevention of neurotoxicity (see Protein interactions, 08-,?I). For further implications of MEL+-hlock, see also Phenotypic expression, 08-14. Current-voltn~e
rclntinn, 08-35, and Chnnnel mndulntion 08-44].
Rehaviour of NMDAR in M P - f r e esolutions and with other extrocellular ions 08-43-02: In ~ ~ ~ + - fsolutions, rcc thc opening and closing of thc NMDAR-
channels leads to rectangular current pulses, the mean duration of which varies little with mcmhranc potcntia12". Following addition of ME2', thc single-channel currents rccordcd at ncgativc potentials appear in hursts of short openings separated by hricf closurcs (cornparc with fec~ttrre.~ in Table 8). While ~ o "(and to a lcsscr cxtcnt ~ n " )mimic effects of ME3' on the NMDAR-channel, ~ a " , RaL' and cdZ' do not2," (see below).
Divalent cation block versus permca hility through the N M D A Rchannel 08-43-03: NMDA receptors are distinct from othcr glutamate reccptorchannels hecause of their high ~ a ~ + - ~ e r m e a b i l i and t y t inhihition hy selcctivc cationic channel hlockcrs such as z n Z + inns (typically -5100 p ~ ) ~ " ~ . ~dizocilpine '", (MK-801, typically -1 //MI and M ~ ' ' ions (typically -1-5 mM, scScaho~cl*~? The 'hlocka~c/pcmcahility' tlistinction hctwcen Mg2+-like and ~ a " - l i k c divalent cations may cnrrcsp)nd to a difference in the speed of exchange of thc water molecules surmunding the cations in solutions. Thus, it is possihlc that pcrrneationt occurs for all thc divalcnt cations, hut is slower for those which arc slawly
Differentialsensitivity to open-channel blockers by certnin NMDAR snhunit combinntions 08-43-04: ~ c c o m h i n a n t iNMDAR suhunit comhinations
(-NRI/NR2A of rat) or
Modcllin~o f NMDAR hlockade
dissociniive anmesiheiics
1 7 ~ 7
08-43-05: Voltage- and use-dcpcndcnt hlockadc of NMDA rcccptor currents
hy the dissociativei anaesthetics ketamine, phencyclidine (PCP1 and dizocilpine (MK-801l has hccn analyscd2"". Follow~ngthe assumptions of the 'gu;irdcd rcccptor hypothesist (a model used to interpret action of thcsc anaesthetics, scr hclorz~l"'", the estimated reverse rates for anaesthetic hinding arc indcpcndcnt of hlockcr conccntration while forward rates incrcasc with conccntration. Changing the levcl of positively charged ketamine (pK, 7.5) 10-fold (hy changing pH from 6.5 to K.51 causes a corrcsponding changc in the f(~nrardratc hut has n o effect on the reverse ratc. The voltage-dependence of blockade can Iargcly he accounted for hy rctluctions of the rcvcrsc ratcs of hinding hy dcpolariz;itiont. Differences in potency can he accounted for hy diffcrcnccs in the rcvcrsc ratc constants, which incrcasc at positive potcntials2"". Important assumptions of the 'guardcd rcccptor hypothesis' arc (i) that NMDA receptors arc maximally activated at the peak of thcir rcsponsc with a IJ,,,,, approaching 1; (iil that there is no receptor desensitization and (iii) that the hlocking d n ~ gonly associates with, or dissociates fmm, rcccptor-channels which have hccn activated hy agnnist (i.e. arc open). Notc: PCP, dizocilpine and similar clrugs also affect several othcr functions and targets in the CNS - as rcvicwerl In , thcsc include inhihition q f noradrenaline and dopamine uptake, inhibition of acetylcholinesterasel, antagonism of muscarinic receptors, inhibition of activated nicotinic receptor and delayed rectifier K+ channels.
'Protecrir~c'cffects o f NMDAR 1~locker.sagainst cnlcinm ncurotoxicitv 08-43-06: PCP and ketamine 'protect' against hrain damage in neurological
rl~sorderssuch as strokei. However, these agents have psychotomimetici properties in humans and damagc ncuroncs in the ccrchral cortex of rats, ;ilthough morphological damagc can he prevented hy anticholinergic drugs or hy diazepam and barbiturates, which act at the CA13AA receptor"-'. Furthermore, dizocilpine and similar drugs show a pattern of 'huild-up' for hlockatlc, such that tollowing hinding ot antagonist molecules, they leave tlic channcl only vcry slowly ( I , , ? > 1 hJ. Notahly, open-channel hlockers which arc well-tolcmtctl clinically, such as memantine (sc,c /~c,low)leave ~. the channel promptly ( 1 ,,? > 5 s at micromolar c ~ n c c n t r a t i o n s ) ' ~C.ARAAselective drugs h;lvc Iwcn ohscrved to reduce the psychotomimctict symptoms caused hv ketamine. 08-43-07: Memantine, an adamantane, derivative related to the anti-viral dnig amantadine has hccn shown to hc an open-channelt hlockcrt for NMDA receptors. Adamantanc is well-tolcmtctl clinically unlike othcr openchannel hlockcrs like MK-801 (dizocilpine)"". Norct: The anti-viral drug amantadine is also i~scrlin the treatment of Parkinson's tiisease, hut is consirlcrahlv less potent than ~ncrnantincat clinically tolcmtcd tloscs.
Non-selcctivc. suppression o f cxcitntorj~nmino ricid receptor-channel cL1rrcni.v 08-43-08: In oocytcs uxprcssing mouse hrain mRNA, enflurane at an ;in;~csthcticconcentration (1.8 mhrl inhihits NMIIA-, AMI'A- and kainatc-
induccd currcnts by 29-40"/0, 3 6 3 3 % and 20-27%, rcspcctivcly, suggesting that all three glutamate ionotropic receptors arc susceptible to suppression by inhalational anaesthetics2". Inhibition by cnflurane is inkpcndcnt of thc concentrations of the agonists (NMDA, AMPA and kainatc] or the NMDA co-agonist (glycinc), suggesting that cnfluranc inhibition docs nt~r result from a competitive interaction at glutamatc- or ~lycinc-hindingsitcs. Enflumnc also supprcsscs the oscillation and 'apparent dcscnsitization' of NMDA currcnts, suggesting an inhibition of ca2*-influx through the NMDA channc12".
Drlal srlppression o f EL(:-channel cind voltage-~atedchannel currents h y morpllinans 08-43-09: The morphinan dextromethorphan and thc related molcculc
dextrorphan hlock NMDA-inducctl currcnts ant1 voltage-operatcd inwart! currcnts in culturcd cortical ncuroncs and PC12 cells272. Dextromcthorphan is at least 100 times more potent (ICSo 0.55 I ~ M as ) a hlocker of the current induced by NMDA in cortical ncuroncs than for voltage-gated ~ a channds " in the same preparation (ICso 80 I'M). Notably, this class of hlockcrs arc well-toleratetl clinically'2v. The net~roprotectiveeffect of tlcxtromcthorphan (which occurs in a concentration rangc of 10-100 I ~ M )may thcrcforc be due to a complete blockade of the NMDA rcccptor-channcl and a partinl inhibition of voltage-dependent ~ a ?and ' Na' channels"-'.
-
-
Non-selective channel hlock h y TEA 08-43-10: The
organic cation tetraethylammonium (TEA, 1-5 m ~ ) antagonizes NMDA responses in culturcd mouse cortical ncuroncs in a conccntmtion-cicpcndcnt manner. TEA causes voltage-dependent dccrcascs in single-channel conductance and the frcqucncy of channcl cvcnts is also decreased. Thcsc effects arc not accompanied by any change in average channcl open tim~t'~.'.
Channel modulation Protein kinase C modulation o f N M D A R chnnncl currents jn pain reception 08-44-01: Phosphorylation hy protcin kinasc C (PKC] has hccn reported to enhance NMDA receptor-mediated glutamatc rcsponscs in a number of '4.3.210.22" . For example, in the trigeminal subnucleus caudalis, a centre for processing pain-sensory (nociceptive) information from the orofacial arcas, a p-opioid receptor agonist causes a sustained increase in NMDA-activated currcnts hy iictivating intraccllular PKC (see nl.w I'hcnotvpic expression. 08- 14). Protein kinasc C appears t o rcducc thc voltage-dcpcndent ~ g " - b l o c k in wind-up'tY7 (c.g. triggering of a dmmatic increase in discharge following tissuc injury clr rcpctitivc stimulation of small-diameter afferent1 fibres (we P11cnot)pic expression. 08-14). Thus protcin kinasc C potcntiatcs NMDA responses by increasing IJ,,,,,, duc t o reductions in ~g"'-block": A role for metabotropict glutamate receptors in PKC motlulation of NMDA-mediated processes has also hccn tlcscrihcti"" ( S ~ PC I t ~ n n r dcsignr~tion, l 08-03, rind I'rotcin intcrrlc.rions. OH-31).
entry OX
Modnlntion o f LTIJ proc.rlsses h,v protcpin kintlsc (I(-tivation 08-44-02: In t h e post-synaptic cell, hoth activation of calmodrllin and kinase i z~ctivity arc required for t h e grncration of LTI'"'.'-" (SPC I'lli.not,vpic cxpri,ssion, OX-141. Extrc~ccllulr~r application of protein kinase
inhihitors t o t h e hippoca~npnlslice preparation has heen shown t o hlock t h e induction o f LTP. Intrncpllrrlor injection of t h e protein kinasc inhihitor H-7 i n t o C A I pyramidal cclls also hlocks LTI', a s docs inicction C o n t i n u o i ~ sinflux of ~ a ? ' of t h e calmodulin antagonist calmidazoli~rn~~'. through t h e NMDAR c;In h c prcvcntcd hy prior treatment with t h e protein kin:lsc C inhihitor sphingosine"'5. LTI' is also hlockcd hy inicction of synthetic pcptiilcs ch;iractcrizcd :is potent calmodulin antagonists prcsumahlv hv inhil~iting CnM-KII ; u ~ t o -and suh.;tr:~tc phosphorv1;ltion
IJrotcin pho.xp/~ori,/(~ / io11,08-s32). Nc,q,~ativcrind positive modrilntion o f NMDAR r c d u c i n ~nfpn t s
17y
oxidizing rlnd
08-44-03: NMDA responses in several tliffcrcnt nci~ronalpreparations are
'suhstantinlly rlccrcasctIf following exposure t o t h e disulphidc reducingt agent dithiothreitol ( I j T T , 0.1-1 0 in^), while oxidation with 5,s-dithiohis-2nitrohcnzoic acid (IINTR, -500 p h i ) can potcnti:ltcf the magnitude ot t h e responsr~T'. 2-(, . MotIific;~tionc ~ tf h e NMIJA response hy cithcr oxidation or rctluction a t this redox modulatory sitep7' does rlot nppcar t o affect t h e pllclrriiclcoIcl,yic.c~lproperties of the rcccptor-channel complex. Since redox statei of t h e native NMIIAR varies widely among ncuroncs, rcg~llationof NMDA:~ctivatcd ftlncticlns by i,itllcr reduction or oxidation inav operate in r,ir,r,27".?" (SVI'
Ill.~O1 1 ~ 1 0 ~ ) .
M c c l ~ ( ~ n i somf N M D A rrlc*r.prorhloc.kodr1 1 7 ~ 7nitric. oxidr at the redox n?otlrllntory ,sit[' 08-44-04: Clinically iinportant nitroso comporlnds thnt gcncrntr nitric oxide (NO*, whcrc t h e tlot signifies one frrc electron) have hcrn shown t o inliihit responses mctli;itcd hy t h e NMDA-sclectivc g l u t n n ~ a t rreceptor o n rat cortic:~l nci~rtlncs in rfitro"v"77 . It has hccn proposed that free sulphydryl groups o n t h e NMIJAR rcact t o form one o r more S-nitrosothiols in the presence of nitric oxidc. For cxamplc, rcaction of NMDAR free thiol groups w i t h nitroglycerin via S-nitrosylation has hccn s h o w n t o m o d ~ ~ l ; ~protein te function in a n anal!)~ous manner t o protein phnsphorylation' o r ncylatic~nt2". If vicinall thiol groups react in this way they mil? form dis11Iphitlc hands, constitllting t h e rrdox modulatory site f of t h e r c ~ c p t o ? ~ ' . h'otr,: Fommatic~n of tlisulphitlc honds would result in reduction 01 c;I"-influx in rcsponsc t o NMtJA. Thus, rcaction with nitric oxidc appc;irs t o protect cclls from N M D A receptor-mediated ncurotoxicity277. C:onvrrscly, rctli~cingcontiitions favour the formation of f i e thiol I-SH) groups, and this condition is prcscnt following hcing nssociatrtl with hoth a net increase in C a 2 ' - ~ n f l u xthrough NMDAR c h a n n c l ~ ~ ~ "ant1 - " ~hixhcr ~ npp:ircnt n e i ~ r o t o x i c i t ~(srr ~ ~ 'rllso I'hrnotypic r~xpr~~,ssion. OX- I J ) .
entry 08
-
'Reversible' s u l p h y d r y l redox m o d u l a t i o n of NMDAR in r a t c o r t i c a l neurones 08-44-05: Reversal of the effects of sulphydrylt oxidizing agentst hy DTT (see cjhove) can occur over a rapid time course (t1/2 0.6 min). Spontaneorislv oxidized receptors can he further oxidized with DTNB and 'fully reduced' with DTT. When the redox modulatory site275 or sites arc alkylated with N-ethylmaleimide (NEM, 300-500 j i ~ )following reduction with DTT, responses hccome 'permanently potentiatedt' and largely insensitive to oxidation hy DTNR. Blocking effects of protons and potcntiatingt actions of glycinc arc unaffected by alkylationt, and Zn" and MRL' ions produce a significantly weaker hlock of the NMDA whole-cell r e s p o n ~ e ~ ~ ~ ' .
-
M o d u l a t i o n of N M D A R-channels by (endopenous) z i n c i o n s 08-44-06: Roth pro-convulsantt and depressant{ actions of z n 2 + ions have
hccn reported (sce ref.2""). Zinc ions are potent non-competitive antagonists of NMDA responses in cultured hippocampal neuroncs at relatively high (micromolar) concentrations2"". Unlike ~ g " , the cffcct of z n L ' is not voltage-sensitive hetween -40 and +60 mV, suggesting that ZnL' and Mg" act at distinct sites. z n 2 ' could also motlulate ncuronal cxcitahility hecause it is present at high concentrations in hrain, especially the synaptic vesicles of mossy fibres in the hippocampus and is rclcascd with ncuronal a c t i v i t y . Comparcjtive note: Zn" ions also antagonize responses to the inhihitory transmitter GARA in the hippocampus ( s ( ~E L G C1 GARAA. cntrv 101. N M D A R - p o t e n t i a t i n g effects o f zn2+ions 08-44-07: Zinc ions may also he positive modulators of NMDA receptors in
certain regions of the brain2?'. Analysis of ~ n "modulation of currcnts through homomerict receptors assemhled from different splice variants of NRI suhunit show that, in addition to its well-charactcrizcd inhihitory effect at high concentrations (src ahovc), z n 2 ' potentiates agonist-induced currents at suhmicromolar concentrations (EC5(,= 0.50 /(MI. Potcntiationt is ohserved only with a suhset of NRl splice variants1"". Zn" potc?tiation is mpidly rcvcrsihlc, voltage-indepcndcnt~ and non-compctitivc~ with glutamate or glycine. Zn?' potentiation is mimicked hy cd", c u " and ~ i " , hut not hy ~ n " , co2', ~ e ~~ ' n, "or ~ g ' +lY.' . Note: For a description of the molecular 'loci' affecting ~ g " - b l o c k phenotypes on the NMDAR, see Domoin functions, 08-29, A u t a c o i d m o d u l a t i o n of NMDAR 08-44-08: The ether phospholipid platelet-activating factor (PAF, an cthcr phospholipid autacoidt) induces a stahlc, concentration-clcpendcnt longterm potentiationt (LTP) in hippocampal s l i ~ c s " ~ . I'AF-induced LTP is blocked hy antagonists of the PAF ant! NMIIA receptors, hut the former antagonists do not block LTP induccd hy high-frequency stimulation. Facilitationt induced hy PAF cannot hc reversed hy PAF receptor antagonists2". For an introduction to long-term potentiation phenotypes associated with the NMDAR, see Phenotypic expression. 08-14.
Complex modul~~tion o f N M D A R-channels hy polyumines 08-44-03: Several hiochcmical and clectrophysiological studics (reviewed in ref^.*^'-^^') S U ~ C Sthat ~ a recognition s ~ t ccxists on the NMDAR for
endogenous polyamines (e.g. normal catabolic intcrmcdiates of argininel like putrescine, spermidine anti spermine). The polyamine site is distinct from binding sitcs for ~ l u t a m a t c ,glvcinc, M ~ " , ~ n " , and open-channel blockers. Polyamines increase the hinding of open-channel blockers hut can also increase NMDA-elicited currents in cultured neurones, sumesting they may play a rolc in 'excitotoxict' responses following neuronal damagc. Polyamine segments are also associated with several neurotoxic venoms from invertebrates (see Receptor nntn~onists. 08-51). Spermine and spermidine havc been descrihed as 'sgonists' at thc polyamine recognition site, which may modulate excitatory synaptic t r a n s m l s s ~ o n ~Furthcr ~~. features of polyamine modulation of NMDA receptors are descrihed In Tr~hle9.
Modulation o f N M D A R by the co-a onist glycine 08-44-10: Glyrine-evolted p t e n t i r t i o j of NMDA receptor activity is
accompanied hy reduced desensitizationt2". Dose-response analysis for the glycinc-sensitive activation of NMDA receptors at +60 mV reveals a 3-5fold increase in apparent affinity for glycine In the presence of 1 mM spermine. This increase in affinity for glycinc is accompanred by a 3.3-fold decrease in the rate of development of glycine-sensitive desensitization, and a 2.4-fold decrease in the rate of dissociation of glycine from NMDA receptors (while the rate constant for dissociation of NMDA 1s not red~ced'~1.
Alcohol-inhibition of N M D A R currents 08-44-1 1: Potency of several alcohols for inhibiting NMDA-activated currents
arc cnrrclatcd with thcir intoxicating potencyt, suggesting that rcsponscs to NMDA receptor activation may contrihute to the neural and cognitive impairments associated with NMDA-activated currents are inhibited by ethanol over a concentration range that produces i n t o x i ~ a t i o n ~ ~ Ethanol " ~ ~ ~ " . may inhibit the NMDA-activated ion current hy interaction with a hydrophohic site on the NMDA channel protein'y4. Compart~tivenntr: Ethanol effects a number of receptor-stimulated and voltage-dependent ion fluxes with differing sensitivity. For an illustration of the relative potcntiatinp, effects on CARA,-mediated C1 flux and the rclativc depressive effects on NMDA-, kainate- and voltage-gated ca2+-flux, see Fi,q 5 rlnd~rE L G Cl GARAA.entry 10.
Non-selective inhibition o f NMDAR-mediated calcium influx by dihydropvridines 08-44-12: The dihydropyridinc nitrendipine suppresses NMDAIglycinemediated calcium influx by a rapid and direct interaction with the NMDA
receptor-channel complex2". Thus nitrendipine may exhihit anticonvulsant and neuroprotectant activity via a combined ability to modulate I~ot11 NMDA-associated ion channels and L-type voltage-sensitive calcium channel^'"^ (src Hlocker~r~ndrrVLG Cn, 42-43).
Table 9. Some features of polyamine modulation of NMDA receptors (From 08-44-09) Feature 'Concentrationdependent' effects of spermine modulation
Voltagedependence of spermine potentiation Separate mechanisms of sperrnine potentiation or block
Examples of block by other polyamincs
Spermine modulation of co-agonist affinity
Description
Refs
Spermine can produce hoth potentiation or hlock of ' " NMDA responses by binding to multiple sites in a potential-sensitive manner. Spcrminc potentiates the action of NMDA at micromolar concentrations but is less effective at millimolar~oncentrations~~~. At low concentrations (1-10 /IM) spcrmine enhances NMDA receptor current in cultured cortical neurones by increasing channel opening frequency. At higher concentrations (> 10 //MI,it produces additional voltage-dependent decreases in channel amplitude and average open time which limit its 'enhancing' action Concentration-jump responses to 100 /rM NMDA 2w in the presence of I0 /rm glycine reveals potentiation by 3 r n spermine ~ at a membrane potential of +6O mV, hut depression at -120 mV 290 Potentiation results from both an increase in apparent affinity for glycinc as well as an increase in maximum amplitude of responses to NMDA rccorrlcd in the presence of a saturating conccntration of glycinc. Sperminc produces a voltage-dependent open-channel hlock of NMDA currcnts at high concentrations [> 1 0 0 / 1 ~hut ) has nocffcct on the block by the putative polyamine site competitive antagonist arcaine (see Rlockers, 08-4.7) The polyamincs diethylenetriamine and 1,lO29' diaminodecane produce vol tage-dependent hlock of responses to NMDA, with apparent equilihrium dissociation constants at 0 mV of 0.75 and 2.93 rcspectivcly. Rlock is voltage-dependent, and most likely due to binding of polyamincs to sites within the ion channel (see Voltage sensitivity, 08-42) Sperminc increases the affinity of the NMDAR 2w for glycine
'"
Comparativenote:Polyamine modulation isnot unique to NMDARchannels. As reviewed CIin ref.*"', polyamincs rnodulatc several Cl- conductances, including ~a'*-activated channcls (see ILG' C1 Co, entry 25) and GARAArcccptnr-channcls (see ELC: Cl CARA,, entry 10).Polyamine toxins have also heen shown to rnodulatc n~cotinicacctylcholinc receptor-channels (see ELC: CAT nAChK, entry 09), non-NMDA iGluRs (see ELC: CAT GLU AMPAIKAIN, entry 07) and scvcral suhtypcs of voltage-gatcd calcium channcls (see V L G C ~entrv42) I, and potassium channcls [
cntrv OH
-- --..- .
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.....
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- - --- -
Motll~lntionof NMDAH-c:hannrl frrlctional ol7cn r i m r h!~ncurostcroids 08-44-13: NMIJAR currcn ts in hippocampal ncuroncs arc approxini;~tcly doi~hleti in the prcscncc of the nennisternicl pregnenolone sulphatc ( 1 5 , 100 1 t ~ ~ 2 VThe n . dose-rcsponsc curvc of I'S :~ction shows s~gnificant potcntiationi at cclnccntrations greater than 250 nM ;lnd shows a halfmaximal cffcct nt 29 I ~ M .Mnxirnl~mpotcnti;Itiont is reached within 25 s, with the potcntiatinnt hcing fully rcvcrsctl with 60 s of washout. At saturating conccntrations of NMDA and glycinc co-a~onists,PS does no! change the affinity hctwccn the co-aqonists ;lntI the NMIJA receptor. I'S potentiates the fractional open time1 of NMIIA channels hy incrc;~scd trcqucncy of opcnings of single NMDA-activatctl channels hut docs not ;~ffcctsingle-channel ct~ntluctances"".
-
pH ri:qnlrrtion
of
N M D A c+hr~nnrpI ros/~on.scls
08-44-14: Rcspclnscs of mouse hippocampal neurones to NMDA or to low conccntr;~tionsof gli1tam;ltc {recorded in the al~senccof ~ g ? ant1 ' with glycinc in the cxtracclli~l:~r si~pcrfusionsolution1 arc antagonizctl hy :lcidic pH ;lntl potrnti;ltctll by a!kalinc cxtrarellt~larsolutions2""~~'"". I>ccrc:~sing s responses to <3.3 I 7 " ; );lnd ;in incrc;~scin pH froni 7..3 to 6.0 r c d ~ ~ c cNMIIA pF1 fro111 7..7 to 8.0 potcntiatcs i t to 141 t 6r':~2"".
Indircct 1no(hr1(1 t ion n f hippor:r~mprrlN M V A R l7!7 tolhut amidc 08-44-15: Tlic sulphonylt~rcn tnlhutamide (500 1 1 , ~ lhns hccn shown to revcrsihlv iricrc;~scpcak ; ~ m p l i t ~ ~ d;lnrl i u s the steady-statei levels of NMDAhut not k;~in;ltc-cvtikcd i(;li~R currcnts in cultured l~ipp(>c:~~iilwl n c u r o n c ~ ~ ~The " ' . cffcct is :)l,scrvcd i ~ 1t ~ 1 t low h ant1 s;~tur;~tcd concentrations of glycinc, and thc ;~ffinitvTof the NMIJAR for glycinc does not chnngc in the prcscncc of tolhutamidc. Although tolln~tamidchas hccn gcncrallv cliar:~ctcrizctl;IS a blocker of KnrF lur,c- I h ~ Kl ~A T / ' - ; , cPrltrtr301, the :~ctionof t o l l ~ ~ ~ t ; ~ mon i t l cthe NMIjA-;~ctivntctlcurrent is r~ol mctliatcd hy KATI, cli;~nnclssince thc cttcct pcrsists in the prcscncc of ~ntr;~ccllular Cs(:l at conccntr;~tionswhich intlucc total 1,lockl of ; ~ l lK' channels. Tolhutarnide may thcrrf(~rc;~dtlitic~n;~lly rnorl~~l;lte intmcellular messengers influencing NMI>Ali-cli;~nncl;~ctivityin this prcl>;~rntion(st-c, r(*l.'"'/.
Equilibrium dissociation constant S i n ~ i I ( /~' rH / - M K - H O ~ llir~dingin nrrtir~ecrlls rlnd hcrcrologoust NR I / NR2A corill?inations 08-45-01: (:ells transiently cr)-cxprcssing NRI ant! NII?A yiclrl ;I 10-foltl Incrc;rsc in the n~rrn1x-rof [ ' ~ I - ~ ~ - ~ ~ l - h sites i n d colnparcd in~ to channcls cxprcsscd froni Iiornomcric ;~sscmhl~cs. Simil;~r;~ftinitics for ['HI-MK-801 h:~vc I~ecn measured in HEK-29.3 cells tr;msicntlv expressing NRlI2A coml>in:~ticin.i;IS in native adult rorlcnt hr:~ins".
Extcrrlnl ~ n , "r.r~n!ril~u!r..s to 'r~n~r.sr~rrl/v 111,r.h'~11~c.inr~ rlffit1itic.sfor NMIIA rc1crJ17t ors 08-45-02; T h e aff~nityof the NMDAR co-;~gonistglycinc l.sr,cJ Ai.tirwtlon. 08,?.I) is scnsitivc to the c ~ t r : ~ c ~ I I i r(1;1'+ l a r conccntr;~tionin trigcminal ncuroncs
entry 08
(the apparent dissociation constant (ECS"] for glycinc dccrcases with increasing cxtcmal ~ a "cclnccntrations, increasing hy about 3.7 times in ~a"-containing solution^]^^^". Kinetic studies of glycinc binding to NMDA receptors indicate that external c a L ' causes a dccrease in the off ratct of the glycine binding, while having no effect on the on ratci2.?' (see also Activntion, 08-33),
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Ligands 08-47-01: Commercially available radioligandst includc I'HJ-AP~ (['3~I-n-2amino-5-phosphopentanoic acid), ['HI-CGS19755 (['HI-cis-4-phosphonomethyl-2-pipcriciinc carhoxylic acid), ('HI-MK-801 ([.3~]-dizocilpinc,5methyl- 10,I1-dihydro-SH-dihenza~n.d]cycloheptcn-5,10-imine),['HI-TCP (I.'H]- 1-1 -12-thicny1)cyclohexylpipcridine)and ['HI-CPP ([.'~]-<3-(2-carhoxypipcrazin-4-yl]-propyl-1-phosphonic acid], which is a competitivct inhibitor of NMDA.
Rndioli,ymd for the &cine site 08-47-02: ['HI-5,7-~ichlorok~nurenicacid (DCKA) and I . 3 ~ ] - ~ - 6 8 9 , 5arc 60 sclcctivc rattinligamls which have high affinity+ for the ~ l y c i n erccoppition domain on the NMDA receptor complex in hrain synaptic mernhrane~""~ (set. n1.w Receptor onto~onists.08-51),
Non-competitive antagonists for rc~dioligand-bindingstudies 08-47-03: High-affinity [.'HI-dextrorphan hinding in rat hrain is localized to a non-campetitivct antagonist site c ~ fthe activated NMDA rc~eptof'~,'.The iodinatcd manohydroxyl phenyl derivatives of argiotoxin-636 (see Rlockcrs, 08-43) retain NMDA-selective hinding and can scrvc as ncln-compctitivct - antagonists fnr radioligand-hinding asssys~'"". l
Receptor/transducer interactions Several nthcr components nnd mcsscnger mnlectiles involvcd in couplin'y of NMDAli-nssocioted phcnotypcs mctnl~otrnpicfrcccptors to rnodtrlr~tior~ clrc atst) descrihcri in T
Arachidonate as a diffusible 'retrograde messenger' for maintenance of glutamate release 08-49-01: Generation of post-synaptic arachidonic acid (AA) by phospholipase Azhn (under conditions of rcpctitive NMDAR stimulation) has hecn sumcsted to act as a 'retrograde messenger' (i.e. ahlc to 'diffuse hack'] t o prc-synaptic termini and potcntiateT (maintain) glutamate release by a positive-feedback mechanism (briefly reviewed i rc{s.'""."'""' ). Arachidonic ncirl is able to sensitize protein kinase C (PKC) in prc-synaptic tcrminsis, which can increasc ncuronal cxcitahility (and thcrchy gtuatamate release) by inhihiting the IKhh channel in cell hodics of Purkinje tolls"". (See also Fig. 4 nnd Reccptorsltrc~nsdttccr intcractions under [LC: - KAA, 26-49.]
entry OH
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I're-synnpt ir m ~ohot i ropic ,pint omrric reccptors con also neiivmtc IKAA 08-49-02: Pre-synapt ic, metahotropict glutamate rcccptors (mGlitR or m C l u ) have hccn shown to incrliatc activation of protein kinase C, with coupling elf
receptors to K' channel inhibition being dcpcnrlcnt on low concentrations of arachidonic acid.'"'. These 'positive fccdhack' mcchnnisnls hnvc fornlcd the hasis ot a motlcl for sustrzincd relemsc oT glutamntc sufficient to cstahlish synaptic plasticityt and inhibition of phospholipase A2 is known to I-rlock the induction of long-term potcntiation /srra II,(; K A A , m t r y 20 r~nd NOIP: T h e involvcmcnt of mCluR in the induction of short-term potentiationi (STPJ and mossy-fihrrl L T I ' ~processes arc outlinc~l ~ ~ n t l c r i'hcnr)tj,pic cxprcssion. OX- 14.
Trichvkinin-inducd potc?n[iationof NMDAH rrspon.scs in dorsal 11orn nCIIrotws 08-49-03: Substance B and neurokinin A hoth potentiate NMDAR-induced
currents in acutely isolated ncurrlncs fmm the dorsal horn ot t h e rat"'"'. However, substance P, but not ncrlrokinin A, increases [ca"li in a subpcipulntion of neuronus, due to ~ a " - i n f l u x through voltage-sensitive c a 2 * channels. Protein kinnsc C (PKC]may mediate NM1)A-sclcctivc potentiation by tachykinins in thcsc ccIls, sincc non-specific PKC activntors 1c.g phorhr~l esters) cnhnncc the cffccts of NMDA and staumsporfne (n relatively nonspecific prc~tcin kinasc C inhihitclrl inhihits thc potcntiation of NMDA ~itrrcnts""~.
~ a " .si,rnoi omplificni ion in 'r~tro~rradr siccnallinLr' 08-49-04: Arachidonic acid potentiation1 induces a transient NMDA currcnt~"" cnnhling amplifications in intraccllular calcium concentrations to he inducer1 hy glutainatc. ~'otcntiationt of the NMDA receptor current is associated with increases in channel I:,,,,t, with nti apparent change in opcn-channel currcn t.
Nitric. oxit-lr. ricvr~iiorls mrld g~nnylrzfccyclrzsc. rrct ivation 08-49-05: G1trtam:itc has hccn shown to inrl~iccthc relcasc o l nitric oxide
(NO', EDRF, endothclium-derived relaxing factor)".3. Nitric oxirlc I S gcncratctl in a ~ a " - i n f l i ~ x - r l r p c n k nmannrr, t anil its activity can account tor nbscrvcrl cGMP elevations that take placr in the UNS following NMDAR activation by gli~tarnatc (scc IJrcl!cin intrrac!ions, OX-,711. For details of functional changes in the NMDAR induced hy nitroso - compaunds~,srtgChrznn~Im o d ~ ~t ion, l r ~ 08-44,
Receptor agonists Nr>rmalslut t~rniner~qic nrurr~tran.srnissionand s l u i o n ~ t ~ ! ~ - i n d u c P d nrlzmnal dmt 11 08-50-01: T h e excitatory amino ncidt (EAAl glutamate is present in ah(iut .3!l1X1 c ~ fccntral synapses and plays
;3 ccntr:il r t ~ l cin neuronnl transmission under normal p h y s ~ o l o ~ i c a conditions. t Following prc-synaptic rcloasc following cxcitationi of ncrvc tcrminalst, quanta of glutamate diffusc across thc synaptic cleftt and transiently occupy post-synaptic rcccptors (typically for 1-1 ma) Iwfnrc enzymatic dc~ratlationantllor re-uptake"'. This
-
normal pattern of 'receptor use' has hccn contmstcrl with 'receptor abuse'".' wherc accumulation ot glutamate in the interstitial fluids during cerebral oedemai (c.g. associatctl with traumat and stroket) Icads to persistent ncuroncs within thc activation of its r c ~ c p t o r s " ~Notc: ~ ~ ~In~ ~strokct, ~. ischaernict area (oxygen-dcprivcd) die within a short time (scvcml minutcs) while in the ischaemic penumbra ncuroncs arc cxposcd to steadily increasing conccntmtions of glutalnatc and dcgcncratc more andlor sustained ~ a ~ + - i n f l u x ' " " . " ~ ( "generally ?~~ Amplifications of Ic~"], occur through rcccptor-coupled (as opposed to ~ o l t a ~ c - ~ a t c t l ~ ' ~ " ) mechanisms. Comporotivr note: In gcncral, ~ a " channcls play a significant role in pcrsistcncc of excitotoxict phrnotypcs"4.'~"5.'" (,s(Y~ I)l~enotj~pic cxprcssion, ON- 14, ond IJrofcinintcroctio~~s, OX-I1 I ) .
Endogenous metaholitcs as c~gonistsfor the NMDA R 08-50-02: L-Homocysteic acid IL-homocystcate) is presumed to he an
endogenous agonist morc selcctivc for NMDA-sensitive receptors than the other endogenous ligands glutamatc and aspartatc2'". Some cndogcnous metaholitcs of tryptophan (c.g. quinolinic acid) arc activc at similar potcncics~to glutamatc and NMDA~'~~'.~"'.
Amino ~ c i dco-ogonism - the ~ l y c i n esite 08-50-03: I3y definition, all NMDA-type ionotmpict glutamatc rcccptor
suhtypcs arc sclcctivcly 'agonized' hy N-methyl-n-aspartate. Occupaticln of a separate, allosteric 'glycine site' (CIy13, to distinguish it from the inhihitory glycinc rcccptor ilcscrihed under ELG CI GLY, entry 1 1 ) is also an absolute requirement for NMLlA rcccptor activation. This rcquircmcnt is ohscrvcd experimentally as a dramatic potcntiationt of NMDA rcsponscs hy gly~inc.''~,which therefore acts as a co-agonist. Glycinc potcntiationt in outside-out patchcs is characterized hy incrcascs in frcilucncy of NMDAR channel opcning3".
Mechanism o f ~1,vcinepotentiation 08-50-04: Scvcml experiments havcshown that mpid application of glycinc plus
PatchNMDA spccds thc rateof receptor recovery from t~csensitizationt.'~~~'~~'"~. clamp expcrimcnts sumcst the cxistcnccof two forms of dcscnsitizationt for the NMLlA receptor in outside-out patches, howcvcr only nncof tticsc is rlcpcndcnt on the concentration of glycinc. Glycine-insensitive desensitizationt increases rapidly over the first few minutcs of rccortlingand largely occludes thc glycinesensitive desensitizationt in outside-out patches. In patchcs that display no glycinc-scnsitivc desensitization, the rate of glycinc dissociation can he increased fourfold in the prcscncc of glutamatc, suggesting that the two hinding sites arc still allostcricallyt couplcd. Thus, li~antfhinding at hoth types of sitcs can affect the affinity of the other typc for its agoni~t.'~". In ~ g . " free solutions, rcsponscs to glutamatc app!icntion irnmcrliatcly follorvin!: repetitive stimulation with glutatnatc plus glycinc is incrcnsctl I>y -25-KK'!:,, returning to control levels over 10-15 min."". Enhancement of glutamateinduced currents is also seen following stimulation with solutions containing aspartate plus glycine or NMDA plus &cine. However, currents iniluced I7y aspartatc alone arc not potcntiatcd"". A retrospcctivc/rcvicw on the glycine site of the NMDAR has appcarcd,"6.
Indcpcndrnccp of N M D A R p o t c n t i o t i o n f r o m ,qlyc-inc (1,qo17i~r?i(lt ionot ropic 'qlycinc r r c r p t o r s 08-50-05: Glycinc potcntiationt of the NMDAR can he detected nt a glycinc concentration as low as 10 nbr ant1 is 110t mediated hy the inhihitory strychnine-sensitive glycinc receptor (err EL(: Cl (;I.Y, entry 1 I ) . NMDAK potentiation is associated with increases in channel I',, activated hy NMUA agonists3". Glycinc can act ;IS ;I complete agonisti at certain hctcromultimersi expressed in Xc,nopt~soocytes 1e.g. cpsilon I/zcta 1 suhunit comhinations17. (Scc trlvo Act ir.rrtiori. OX-33,trntl I(epc.c,pror ~lntrl,qonixts.08-5 I). I m p l i c a t i o n s for g l v c i n e co-clgonism f r o m h e h n v i o n r n l s t u d i c s 08-50-06: Enhancctl activation ot the glycinc co-agonist sitc on the NMDAR appears to facilitate one forni of associative learning ;~ntlm;lv he used in other learning tasks. Pcriphcr:~linjections of 11-cycloserine (;I p;~rtial;~gonistof the glycinc sitc alllc to cross the blood-brain harrier1 1 tloul>lcs the 1c;lrning rate in rahhit n>odclsf7' (,see. crlso cplicpc.t.\ rnonoc.loric11 nriril,otlic.s ~.clhic.lltli.splrlcr ~ l y c i n cfrom thc NMDA rrc.cptor ~rntlcrI'hrnol!f[~ic- cpx[~rr.ssion,OX- 14). S r n s i t i v i t , ~of r c c o i n h i n n n t NMl3ARs t o a g o n i s t s - cxomp1r.s 08-50-07: ~ o m o m e r i c lNMI)A receptors (NR I ) expression-cloned in Xcnol~us oocytcs from cIJNA in plaslnitl vcctor pN60 (sc~c~ Iso/(rtion pro/?cp.08-12) tlisplay the following ;~gonistsensitivities (measured as percentage stcadystate current response compared t o 100 I ~ MNMDA in ~ g ? ' - f r e emedia supplemented with 10 1cl.r glvcinc)'": 100 /(M NMDA (controll, 100Yh; 10 /lM I.-glotamate, 212 1 1 So!,; 100 11" ihotenate, 71 ! 211"L; 1 0 0 I ~ Mquisqualate, 43 ! IO":,; 100 1.-homcrcystcatc, KS t 1Y"O; 100 I ~ M NMIIA without glycine (controll, .ZS t 6%. Other :~gonistsare ineffective in cornparison ( - Soh) and include 500 /(hi k;~inate,50 IIAI AMPA (r-amino-.Z-hvdroxy-5methyl-4-isox;1zolcpropion;1tc,s i p c j EL(; CAT G L I I AMPAIKAIN, cBntqr071, 100 !chi IS, .ZR-ACPD ( I I-amino-cyclopcntyl-I,.Z-tlicarhoxylatc, an rnGluR ;~gonist)and 1 rnhl GARA. M i x c d r l ~ o n i s r nncross iG1ull r?7olcc'ul~rsuht)y>c.s 08-50-08: In nati,vc tissr~cs,the dicarhoxylicacids L-glutamateand L-aspartatcarc mixed aRonistsi of the NMIIA-, ktiinatc- and AMPA-sclcctivc receptors and thcir effects :ire parti;~lly inhihitcd hy all sclectivc antagonistst. 13cfinitc incrcases in hoth thc opcn timci and open-state prohahi lity 1 of NMDAopcrntcd channels h;wc hccn shown t o he intluccd hv pro101i+yd application of g l ~ ~ t a n i a to t c hippocampal slices in .sit~l."'. In spinal cord ncuroncs, I.-proline elicits inward current that can he pc~rtic11Iv antagonizctl hy ~-APs.'"'.
Evidcnc-(J for bit-nrl~ontitr ion (1,s (1 c o - f a c t o r i n R S ~ ~ ~ ~ S ~ - ( J C ~ J J C ~ ~ I ~ J C ~ glutclrncltc r7rurotoxicit,v 08-50-09:The ahility of the (exogenous)neurotoxic glutamate agonist RMAA (/IN-mcthylamino-I.-alanine) to opcn NMDA and AMI'A channcls in isolated mernhranc patches is strongly potcntiatcdt hy hi~arhonate.'~'.T h e nenrotoxic nr.-2,4-diami:~ntlneuroexcitatory effects of two stmctural a n a l o n ~ c s oBMAA, t nnhutyrate ; ~ n d!)I.-2,3-diaminopropionate,arc ;~lsopotentiated hy hicarhonatc. Nor(': IjMAA is ;I neurotoxic glut;~matc agonist implicated in ncuronal denenemtion fount1 in the Guam :imyotrophic lateral sclerosis-l'arkinsonismdementia complex (Guam disease) - FCC c l ~ ~ ~ r ~ ~ c t ~ rIistrd i x t i ( in . . srrl.'"?
entry 08
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Inhibition o f pre-synaptic glutamate ogonist relense by riluzole
-
08-50-10: Sodium channel hlockcrs, including the anticonvulsantt and
n e ~ r o ~ r o t e c t i v ecompound t riluzole, hlock rcsponscs to NMIIA (ICscl 18.2 I ( M ) following expression of rat whole brain or cortex mRNA into Xenopus oocytes3"'. Riluzole also hlocks rcsponscs to kainic acid (kainatc, , (ICqn = 0.04.3 MI. 2-APV lCso = 167 I~M), CNQX (ICso = 0.21 p ~ )NRQX (we Receptor anta~onists,08-5 1 ) yields an lCsIl of fi. 1 ,IM in this ~ ~ s t c m ~ ' ~ " . The inhibition by hoth riluzolc and 2-APV is rcvcrsihlc and docs not appear to he use-dependentt, unlike that of the channcl hlockcr MK-801 (dizocilpine). Riluzolc acts in a direct hut non-compctitivcT manner and does not interact with any of the known ligand-recognition sitcs on either the kainate or the NMDA receptor'". Characteristics of riluzolc and other antagonists of glutamatc release have havc hccn rcvicwcd"'.
Synaptic release o f glutamate in hippocampal slices occurs in an 'allor-none' manner 08-50-11: The substantial differences in reported sensitivities of the NMDA
-
and non-NMDA receptors to glutamatc agonist sumest that changes in transmitter concentration in the synaptic cleft can result in differential modulation of thcsc two components of the EPSC~.Howevcr, pharmacological manipulation of pre-synoptic receptors affecting glutamatc release in CAI pyramidal cells of guinea-pig hippocampal slices 1i.e. baclofen antagonism of GABAR receptors and non-sclcctivc agonism of adenosine receptors hy theophylline) result in parallel c h o n ~ e sof NMDA and nonNMlIA receptor-mediated components of EPSCs over a 16-fold range.'"". Induction of long-term potentiationt ( L T P ~ in J this preparation (hy lowfrequency synaptic stimulation in conjunction with depolarization to +30 mV, see Fig. 1) leads to differential rnhrlncemrnt of the non-NMDA receptor-mediated component of the E P S C ~ .Thus L T P ~appears to occur through either modifications of post-synaptic receptors or through prcsynaptic changes involving increased transmitter conccntration in the synaptic cleft""'.
1
Receptor antagonists (selective) Pharmacologically distinct sites for an tagonism of NMDAX responses 08-51-01: The multiple sites for antagonism on thc NMDA receptor complex
havc hccn rc~icwed~'""'~. Conventionally, thcsc havc hccn separated into pharmacologically distinct sites haset! on thc characteristics of hlock and the pattcrns of additivity or overlap in dose-response experiments. These sitcs include (il the transmitter-recognition site, (ii) the ion channel site, (iii) the glycine (co-algonist) site, (iv) thc zinc (modulatory) site, and [vJthe polyamine (modulatory) site. The present 'classification' of sitcs is largely hascd on pharmacological criteria, and further discrete sitcs havc hccn Many studies provide support for a n t a ~ o n i s m at the sitcs rcprcsented schematically in Fig. 6. In due course it may hc possible to resolve some of these discrete sites of antagonist action ti) dcfincd amino
entry 08
Figure h. l'ostlllrrtrrl crrrTs o f rrrltrr.yorlrst rrc.rron m t the NMDA reccptor ~ . o ~ i i p l ~Kcl(~fivr~ x. sizil.r: orlri positrons o f sitrs on protclin doni(~in.sR ~ C shown on (1 /typotl~i,!icr~/ r/ioEyrrlmrtirrticcross-srction througli ( I N M l ) A l < . Thrs sitc posilioris rrrrJ not rr/~.soIrrtr~ (lnri rrrrT {or i//l~ctrtrtionori1t7 (s(>rp trxtl. (Fro117 OX-5 1 - 0 1 1 acid loci ( c . ~ hv . i n a ~ n sof site-tlircctc(l mutagcncsist). T h e figilre is a compilation of sirnilar ones ptthlishcd in scvcrnl reviews '*". '". *'-.
."'
Gcnernl notus on rnodc.s o f mcrion for cornpcritivr versus noncornput i t ivc NMDAII nntrrgonists 08-51-02: Ry definition, cornpetitivet antagonists for receptor agonist sitcs clan potentially inhihit ntlrmal physiological ;acivities in one hrain region nt r Glutamate accumulation conccntmtions which do not affcct o t h ~ rcgions. to high levels within thc synaptic clefti in conditions such as strokei may nlso 'out-cc~mpctc' t h c cffccts of such conlpctitivci nntagonists"". Ry contrast, non-ct~mpctitivcinntngonists acting ; ~ t'rnodulatc~ry' sitcs have a potc*ntinl thvrapcutic advantage in that they arc ahlc t o inhihit cffccts of cxcc.;s ~ l i t t ; i ~ n a In t c ';~ffrctctl'arvas of thc hrnin, with rrI:ltiveIy little direct influcncc on nclrm;jl rcccptor function2". Examples of such 'rnorlnlatnry sites' incl~ttlc. t l i ~ ~ srvc g ~ ~ l : ~ t cI,y( l polyaminesi, redox rcagentsi, channel ions itntl p~'"''. A .;umm;iry ot cc~mpoun(l.i shown t o h l o ~ k n r l ~zinc , ;~nt;lgonizcN M l l A responses is listed in Tahlc 1 0 , c;itcgc~rizcdhy their sitc ot ;lctio~i.For J T I O ~ P ~ . o r ~ ~ ~ ~ r ~ rrat,ic~v~, , ! i ~ ~ r YW i , ~r i~~, f~~ ~. )'i-'i". ' ," ~
Table 10. Known NMDA receptor antagonists a n d their fpnturcs (From 08-51-02) Antagonist
Fcaturcs
~ o m ~ c t i t i v e n-2-Amino-5-phosphopcntanoic t acid (D-APS)was antagonists at the first NMDA rcccptnr-specific antagonist (pA2 ~ l u t a r n n t e - 5.2-5.9)(see Protein interactions. 0831). Similar [~indingsite compounds are dl-2-amino-5-phosphonovalcric n-AP5 acid {2-APVJ,11-2-amino-7-phosphonoheptanoic 2-APV acid [AP7 or i>-AP7)and 3-(2-carhnxypipcrazin-4CPP yl)-propyl-l -phosphonic acid (CPP).Othcr CGS-19755 cxamplcs arc CGS-19755 (cis-4-phosphonomcthylCGP-37849 2-pipcridine carhoxylic acid, pA2 6.0). CGP-37849
Refs 38"
LY-235959
{n~-~E~-2-amino-4-mcthyl-5-phosphono-3-pcntanoic
SDZEAA494 MDL100453 DAA
acid) and longer-chain glutarnatc analogues (c.g. 11n-aminoadipate, D M ) . Generally, thcsc compounds do not possess sippificant channclhlocking activity. Structure-activity relations of thcsc anrl othcr compounds has hccn rcvicwcd"". Fnr molccular 'loci' of agonist-hinding sitcs, see Domnin functions. 08-29
Competitive anta,yonists at multiple sitcs DDHB and suhstituted dcrivativcs
Suhstitutcd hcnzazcpincs arc class of glutamatc receptor antagonists that show compctitivct action, significant potency at mr~ltiplcsitcs, and a high tdcgrcc of lipophilicity. 2,s-Dihydro-2,s-dim3-hydroxy-lH-hcnzazcpinc[DDHB)and thrcc suhstitutcd derivatives, 4-hrorno-, 7-methyl-, and X-methyl-DDHR, inhihit thc activation of NMDA rcccptors at hnth thc NMDA rccomition sitc and thc glycine allostcric sitc
Noncompetitivct (usedependent. uncompetitive) openchnnnel a n t a ~ o n itss Dizocilpine (MK-80 1 ) Ketamine Tiletamine Phencyclidine SKF10047 Dextrorphan Dextrometharphan Desipramine
"W Dissociativc anacsthctics including dizocilpine (MK-801), (tj-5-mcthyl-10,11-dihydro-SH-dihcnzo"" cyclohcptcn-5,lO-iminc malcatc), phencyclidine (PCP, 'angcl dust'), ketamine, tiletamine anct SKF10047. High-affinity dcxtrorphan binding in rat hrain has been localized to non-compctitivc antagonist sites also recognized hy nanomolar 1-(I-(2concentrations of dizocilpinc and TCP (['HIthicnyl~cyclohcxyl]pipcridinc. Dextromethorphan and rernacemide arc wcll-tolcratcrl clinically. Dizocilpinc has hccn uscd to soluhilizc NMDA rcccptors from rat and porcinc hrain and in its tritiatcd form is a common radioligand for rcccptor hinding and distribution studies (scc I'mtein distrihrrtion, 08-15).Thc S-cnnntiomcr of kctarnine is mtwicc as potent as the R-cnantiomer in exhibiting a voltagc- and use-dcpcndent hlockade of NMDA rcccptor currents. Calculated relative
"I"
"'"
Table 10. Continclcpri Refs
Antagonist
Fcatilrcs
Memantine CNS1102 CNS1505 M ~ " Rcrnacemide/ FPL12495 2%'' (high /m) ~ n ? *
forw:lrd and backward rates suggest that cnantiomcr confonnntional diftcrcnccs influence t h e dissoc~~(ltir~n from t h e hinciing sitc more than the ;~ssociationwith it. T h e tricyclic ;~ntideprcssantdesipramine (DMI, 20-50 ~ l h r lis a potent sclcctivc :~ntagonistof responses t o N M D A in m o u s e hippocnmpnl ncuroncs. T h e potency of DM1 a s a n NML3A antagonist is hixhly v o l t n ~ c - d e p e n d e n twith t h e k',, increasing c p - t o l t l per .Zh mV tlcpolariz;ltion. At 60 I ~ V t,h e I<,,for IjMI hlock of responses t o N M D A is 10 /,rri ;ind in t h e presence of ~ g ? ' hlock, channels do not hind DM1 hut d o s h o w a dccrcasc in t h e opcn t i m e and hurst length distributions, consistent with binding of DM1 t o open chnnncls. For det;lils of hlock I>y ket;imine ant! memantine, rr,c. Hloc.kc~rs,OX-4.3. For molccul;~rIoci of porc-lining tlom;lins, scr. ~ ~ 0 1 1 ~ (~' O1 f ~l V1 ~ 1' ~ \ f ( l ~ l O 08-28, ll, (lI1d i ) o r r ~ ( ~ i t ~ Ilrnc.rior~l;.08-20. For t h e dual blocking- and potentiating ; ~ c t i o n sof ~ n ? ions, ' .sr.rpRlockrrs. 08-4.3, i1nc1 Clic1nnc.1r n r ~ ( l z ~ l r ~ ~ 08-44 ior~. E l c c t r o p h y s i o l o ~ land biochemical studies have demonstrated that pyrazole, an inhihitor of alcohol dchytlrogcn;lsc ;Inti ;I proposul therapeutic agent for treatment of alcoholic intoxic;ltion ( i l ;~ctiv:ltcsNMT3A receptors a t low (- 0.5 / I M ) conccntrations hut [ i i l blocks N M D A receptors non-con~pctitivclya t higher concentrations. <:o1711?(1r(1tit~* note: Pyr;izcilc docs not intcrnct signiticantlv with thC end-pl;itc nicotinic : ~ c c t y l cholinc receptor (.ACliRl
Co~?ipc't ititrcp nnlogonists, gl\~-incsitc Indole-2carhoxylic acid Methoxyindole carhoxylic acid
"IV
Indole-2-carhoxylic acid (I2CA) specifically and ."" conipctitivclv inhibits glycinc potentiation of NMIJA-gated current. In solutions containing low lcvcls of glycinc, I2CA completely blocks t h e rcsponsc t o NMDA, s u a c s t i n g that N M D A lone is not sufficient tor channel activation. Methoxyindolc carhoxylic acid is n low-nffinity c o r n p e t i t i ~;~nt;lgonist ~ [A',, 5 m ~ atl t h e glyc~nc-bindingsitc of t h e NMDAR and can he ilsctl t o eliminate spurious gating d u e t o glycinc contamination from solutions, which is a c o m m o n technicill prolllcm (scmc. Art irwt ion, OX,?.?/. For molecular 'loci' of t h e glycinc co-;~gonist si tc, scpr' Ilon~irinfunct ioris. 08-29
-
-
Table 10. Continued Antagonist
Features
Refs
Antagonists at Compounds include HA-966 (3-amino-1-hydroxy- ""72'* the glycine (D- pyrrolid-2-one),n-cycloserinc, MNQX (S,7-dinitro- ",' serine) site quinoxaline-2,.7-dione),7-dichlorokynurcatc (7-ClHa-966 Kyn), 5,7-dichlorokynurcatc and L-689,560 (+/-4MNQX trnns-2-carhoxy-5,7-dichloro-4-plicnyl~ 7-CI-Kyn honylamino-l,2,3,4-tctrahydroquinolinc). At hi~11 Lh89,560 concentrations, the non-NMDA rcccptor antagonist CNQX can reduce NMDA responses non-compcti(CNQX) L(1874 14 tivcly hy competing with and hlocking the Fclhamate enhancing action of glycine on thc NMDA rcccptor. AC.PC For further details on the glycinc co-agonist site, see Receptor agonists, 08-50. Felbamate is a tolcratcd, FDA-approved anti-convulsive ngcnt which in addition to its antagonism of 'strychinc-insensitive' glycinc receptors (i.e. NMDAR) may also hlock voltage-gated sodium channcls
I
I
Redox modtrlltory site Nitroglycerin Nitroprusside Glutathione
For mechanistic details of modulation at thc rcdox site, see Channel modulotion. 08-44. Compounds that can act as antagonists at this sitc includc nitrnglycerin, sodium nitroprusside, glutathione and pyrroloquinonc (PQQ).Notahly, agents affecting thc rcdox sitc arc wcll-tolcratcd in humans
H' ions
A fall in pH decreases channel activity noncompctitivcly (see Channel modulation. 08-44)
Competitive nntrtxonista, polvr~mincsite (putative) Arcaine Ifenprodil Elipmdil/
Arcaine, a putative competitive antagonist at thc polyaminc sitc on thc NMDAR inhibits polyaminc cnhanccmcnt of NMDA-induccd [,'HI-dizocilpinc (MK-HOI)hinding and also dcprcsscs hinding in thc absence of polyamines. In cultured hippocampal neurones, arcaine produces a concentration- and voltage-dcpendcnt hlock of NMDA-cvnkcd inward currcnts (Kt, 61 ~ I Mat -60 mVJ. Increasing thc dizocilpine conccntrntion partially overcomes the arcainc effect, indicating a compctitivc interaction hetween arcaine and dizocilpinc. Arcainc has also heen shown to hlock thc open NMDA rcccptorchanncl, an action that is independent of thc polyaminc sitc. For further dctails of polyaminc modulation, see Cllnrlnel modltlntion. 08-44, Ifenprndil can discrirninatc hctwccn different combinations of hetcromcric NMDAR: The affinity of recomhinant NRIAlNR2A receptors cxprcssed in oocytcs for ifcnprodil (ICSo = 14611~3 is -- 400-fold lower than that of NRI A/NR2R rcccptors (ICso = 0.,341~~1. Part of the mechanism c ~ action f of ifcnprodil at NRlA/NR2R rcccptors may involve non-compctitivc antagonism of thc effects of glycinc
SLX20715
m..m.
""
""
-
'"*
0.z.31
Table 10. Continuud Antagonist
Features
Compct itivc A pcptidc frt~inConus ,qco,qraphl~.~ (cone snail) vcnom, conantokin-G (CntxG),competitively peptldc wit11 high affinity and specificity NMDAR( ~ n t n ~ n n ~ q t qhlocks , polvomtnr mediated currents in hippocampal neuroncs and has heen reported as a 'rcliahle' prohe for detcrClfC Cnnantokin-G mination nf NMDAR distrihution (sec Protcin Conantokin-T distrihution. 08-1.5). Conantokin-G pcptide Argiotoxin inhibits only 70% of thc clcvation of intracellular free calcium produced by NMDA in cerehcllar granule cclls. The highly-related polypeptide conantokin-T also acts as n potent non-compcti tivc inhi hi tor of polyaminc responses of the NMDAR-channel. Chemical substitution of the highly conscrvcd ?-carboxy~lutamatcresidues as well as modification of the N- and C-termini of conantokin-C, al>olishcs these responses. Thc cicrivativc TyrO-cr~nantokin-C.has hccn found to cxhihit pcllyaminc-like actions at 7-fold greater potency than spertninc. Argiotoxin-636, a component of the Arginpr spider venom, has a higher affinity for the NMDAR than for kainatc receptors, hlocking the corresponding ion channels in a vo!ta~c-dcpcndcntmanner. Modifications of the polyaminc tail or the terminal argininc rcsiduc clf synthetic argiotoxin-636 strongly reduces the hlocking pntcncy. The hiology of Conrls (cone snniI1 vcnom pcptidcs which act on ion ch;~nncls including the NMlIAR has hcen reviewed."
lR2
-
""'
--
other Biclxanthmccncs of microbial origin (thc ES-242s) rlntrl,yoni.~l,s n t rcpruscnt novel NMDA receptor antagonists which mtlltrplr sitc5 interact with both the neurotransmitter recognition site and the ion channcl domain
.'""
,3.75
"For further details, scu rct~~*~~'"-."".
T h e therapeutic ,significnnceof NMDA receptor ilntcl,yonists
08-51-03: N M D A R antagonists have hcen examincd as candidate neuroprotective agents for pathological ctlnditions associated with acute snd chronic 'overstimuIatinn' of NMDAR and ~~~-NMDAR~~.'~~~""~'.'~'-'~"' (SL'L, (11so ~ E ~ C ' ~ E I I C under CS I'hcnntvpic cxprcssion. 08-14). NMDA channcl hl(lckcrs can provide n 'thcmpcutic window' hv preventing 'cxcitotoxic' calcium influx during globalt or focalt brain ischaemiaf elicited hy conditions such as strnket. Thc ro1c of cxcitatnry amino acid receptors in epilepsy, and the cffcctivcncss of NMDAR antagonists in various in rrirm and in vitro
entry 08
-
models of epileptic seizuret have been revicwedf2'. Effective therapeutic compounds may act to directly antagonize excitatory amino acid agonist hinding to receptors or may modulate uptake of endogenous agonist within the synaptic c ~ e f t f ' ~ ?The chemical structural requirements for the development of potent+ NMDA receptor antagonists havc been discu~sed""~.
General note on the relationship o f antagonist selectivity and their molecular 'targets' 08-51-04: Thc existence of multiple subunit isoformst (and splice variantst) within functional extracellular ligand-gated channel complexes (coupled with known variahilitics in expression patterns within the CNS) raises the possihility that selection of antagonists capahlc of modulating subsets of hrain functions is a rational goalf2".
Antagonist sensitivities for recom hinant NMDARs - examples 08-51-05: ~ o r n o m e r i cNMDA t receptors (NRI ) expression-cloned in Xenopu.~ oocytes from cDNA in plasmid vector pNhO (see Isolntion prohe, 08-12) display the following antagonist sensitivities (measured as percentage reductions of the steady-statc currcnt response compared to a control of ~ (control, without 100 , r ~NMDA in ~ g " - f r e e media'": 100 l t NMDA antagonists), 100% I0 [IM D-APV, 27 f 6%; 1 I ~ MCPP, 47 f 7%; 1 IIM CGS19755, 43 f 5%; 50 I'M 7-CI-Kyn, 2 i 1%; 1 {tM (+)MK-801, 7 f 4%; 100 / l M GAMS, 97 *2%; 100 nM Joro spider toxin, 83 1671,; 100 IIM CNQX, 70 ho/oln. Note: Differential sensitivity to the open-channel hlockcr dizocilpine (MK-801) hy certain NMDAR subunit comhinations has been rcportcd2"' (for detnil.~,see Nlockers, 08-4.3).
*
Effects of N M D A nntngonism in vivo - hehavioural studies 08-51-06: A systematic study of relative potencicst for glutamate receptor antagonists arlministcred in vivo reported MK-801 and CGP-37849 to havc relatively high potcncy (EDso < 1 mg/kg following thrcc different administration proc~dures).'"~.A range of NMDA channel antagonists (including several listed ahovc plus ifenpmdil, f-N-allylnormetazocine and dextrnmethorphan) prnducc impairment of learning and muscle relaxation when administcrcd i.c.v.t in rats.'.?". 1n addition to 'tcmporary psychiatric disorders', systemic administration of allostcrict and isostcrict NMDAR antagonists has also hccn associated with disorders of cardiorespiratory regulation"4", neuronal degeneration*7qfant! reductions in synaptic strengtht4'. NMDA receptor a n t a ~ o t ~ ineuroroxicily st dependent on neuronnl interconnections
u
08-51-07: Certain NMDA antagonists cause neurotoxic side-effects consisting of pathomorphnlogical changes in ncurones of the cingulate and retrosplcnial eerehral corti~cs""~.FolPowin~ low doses these changes appear to hc rcversihlc, hut higher dnscs can cause irrcvcrsihle neuronal necro~is.'~'. The neurotoxic action of MK-801 in adult rat cingulatc cortex is potentiated hy pre-treatment with thc cholincrgicT agc~nistpilocarpine, and can he aholishcd hy co-admini!tration of the cholinergic muscnrinic antagonist scopolamine and agonists' which act at the GARAA reccptor (c.g. diazepam and b a r b i t ~ r a t e s l This ~ ~ ~ .~rcvenriono f NMDA nntoeonist-induced nr1troroxic:itv
----0
-0 -00 -0
Cingulate neuron
k2 GABA
AW
muscerlnlc \receptor
I
Figure 7. NMDA receptor antagonist neurotoulcltv dependent on neuronal lnterconnectlons Glutam~ner~qrc (Glui axon collateruls o f cingulote neurones 11 I feed hack to NMDA receptors (21 expressed at GARAergzc IGARAI neurones (31 to s at MI muintazn toxic inhibitory control orer the release o f acct~-lchollnc(ACht from chollnerglc neuroneu ,I41 and ~ t actionr cxpretred on the clngulate neurone 1tce1f Phormocolo~calblockade o f the ,hr.h4DA receptor (2) muccannlc receptor\ /i,' crhol~\hes the inhll~ltorvcontrol o w r ACh rcler~seand suhiects the clngulutc neuronc to LI ctute o f perrlrtent cholrnergic h-yperstimulotion This hrperctzmulatron hor been proposed as the maln cause o f the pothomorphologicol effectrIn the czngulate ncurone tollo117ngNA4DA receptor-channel hlockclde lsee Receptor clntagon~etc.08-571 licstorutron o f GABAergic tone fe g bv adrnlnIctration o f barblturc~tecopenlng the GABA receptor-channel at /6]. etTenIn the ahrence o f GARA / w c ELG CI GARAA, entrr 10) or brr MI-se1cctlr.e ~lntugonlsmat [5jt hnr been chorvn to protect cingulate neuroncr against the neurotoxic d e - e f f e c t s o f S M D A ontu,qon~$ts(Rased on doto hom Olner- er rl. 11991, Sclencr 254 151.5-18) (From 08-51-07,
entry 08
-
hy M 1-selective anticholinergic and harbituratc drugs has heen explained hy considering the specific set of connections which modulate glutamcric cingulatc ncuronal activity (fora summary o f these inten~ctionx,scr Fig. 7)*12.
Receptor inverse agonists I'utc~tiveinverse axonisis at the polynmine sitc 08-52-01: Voltage-independent, dose-dependent, non-compctitivet antagonism of NMDA-mediated currents hy chloride transport hlockers has hccn shown in cultured spinal cord neuroncs""'. These agents include furosemide (a widely-used loop diuretic), and the relatcil compounds pirctanide, bumetanide, niflumic acid and flufenamic acid. The action of turoscmidc could arise from interaction with the z n L ' inhibitory sitc (as hlockatle of NMDA-induced responses hy furosemidc and ~ n ? 'arc additive). These agents may act as inverse agonists of the polyamine site ant1 their action may explain the 'protective' effect that has hccn shown for some of these drugs in neuronal degeneration3"' (see I'l~rnotvpic ~xpression,08-14). 08-52-02: 1,lO-Diaminodecane (DAIOI acts as an inversct agonistt at the palyamine recognition site of the NMDA receptor.'"'. The cffcct of DAlO is not mediated by action of DAlO at the binding sites for glutamate, glycine, ~ g or~ z n+2 ' hut is attenuated hy diethylenetriamine (DET). In hippocampal ncurones, NMDA-elicited currcnt is decreased hy LJAlO, an cffcct opposite to that of spermine. The effects of spcrminc arc also selectively hlockcd hy DET'"' (scc cllso Chr~nnclmodulntiori. 08-44).
*
),a4:c-,x
-
a
14: I
Database listingslprimary sequence discussion
08-53-01: The relcvant datahase is indicated hj, the lowcr cclsc p r ~ f i x(c.,~. XI':) w1iic.h should not he typed (see Introduction c*) l(lj~o1lto f cntries, cntrv 02). Dattrh~~se locus ntlmes clnd c~cccssionnrlinhcrs immcrlicltcly follorv the colon. Note that a comprrhciisivc listiri~ o f (111 tiv(~il(lhIe~ ~ ~ c e . s , s i o n nun117er.s is superfluous for location o f rcll~r~clntsrcluences in (;cnliaiik" resources, which arc norv avnilclhlc wit11 porverful in-huilt neighhouringt analysis rontincs (for dcscription. sec the Dtrfrlbclsr Iistin~s i~c'lflin thc Introdr~ction el layout o f entries. cntry 02). For cxmniple*, sc.eluer1c.c.s o f y t c(ln 11e nvrldi1,r c-ross-spcrics vorianta or reIntr(j Rent ~ m i ~ n~c~inl)rr,s 17,. one or two mun(is o f nei,qhl,o~~rin,yt c,nc~Ivsi.( ~ v h i c hr~re1)asrei i~c.cc*s.scd on prc-computed ctlignrnents perforrnrcl sins the HLAST~olgorithrn I)y the N C R I ~ ) This . featurr is most 1lscr111for retric~r~cll o f scqtrcncrp entri(*s depositc~tlin dotnhases 1cltcr thon thosc listrti l ~ r l o n Tlitls, ~. r(ppr~s~ntrrtivc 171c1171~7~~rs o f known scrloencc hornolog~~ groupii1,qs rrrr listrti to pcrrnjt initi(11 e1irc~c.t retrierra1.s lw occe1.s,~ionr i ~ ~ i i ~ l v( ~ r ~, t l i o r / r ~ ~ / c ~or r(~i~c(~ -(1ir~~c.f -(~c.c.cssion, horc,c,vc~,nc.icq/~llorrrii~,yt trncll~sis iioriirnc.li~t~~rr. Follo~ting to iclrntify--r~r.rvl\~ rcpportc.ti rlr~tlrc,lotc~isc~luc~ncc,.~. - is stror~,ql!,rr~commer~clc~el --
--
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--
-
-- -
-
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~
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~-
entry OX
Nomcnclaturc
Spccics, IINA
0rlgin:ll isolate
Accrsslon
Scqurnce/ tliscussion
gh: LOX?2X
Hollmann, Nruron (l99.3) 10: 94\1-54.
- - - - -- -
Rat NMDA receptor
NMDARl NRI
NMDARl (rat\ NMIIARI gcnc cxons I through 22 NMDARI-LL
R;lt. 'Funtl;~rncntnl .;uhrln~t' cxprcsf~on-clonccl from 3 r;lt forchr;~incl)NA lihwrv Ivoltrtitrn pnrlv.. OH- 12)
Altrrnntivc splice variant (rat1
YhO aa; comhigh: Xh5227 natorinl R N A splicing altering t h e s11rf:lcc ch:lrgc o n t h r NMllAK
Annntharam, F E U 5 1.t9tt
1 I 9921 30s: 27.?O.
NMDARl (rat1 NMIIARI-la suhunit
Y*39;):I; zinc gb: UIIJIX potcntintcs sxrvnist-inrli~crd currrnts ; ~ crrtain t splicr v:~ri:~nts
Sullivan, 1.M. andRoultrr, unpi~hlishcri (~rrlrlrl 1994).
NMDARl (rat) NM1)ARI-la .;uhunit
q,lO a;l; zinc ~ h : UOX2h I potmtlntcq a~onist-in(lucrd currents : ~ tccrtzlin .;pllcr v;~ri;ints
Hollmann, Nrvrrron ( 199.3) 10: 94\1-54,
NMDARl (r:~tI NMIIARI-lh sr~hun~t
'150 na; zinc potcnt1;ltru ;~gonist-indrlccd currents : ~ tccrtiun splicc variants
NMDARl (ratl NM1)ARI-lh suhunit
gh: Sh78 14 Kusink, Hrain R P S . Mol.
Rat hrain c D N A I~hr;lrv
Hrc1i111 1 ~ ~ . ( 1Y9.71 20: h470.
NMDARl (rat) NMI)ARI-~:I
90 1 aa; zlnc potcntiatc.; i1gonist-1nduci~c1 ctlrrcnts at certain splice vnrl:ints
Nomcnclnturc
Species, DNA sol~rcc
Original isolate
Acccssion Scqucnce/ discussion
NMDARI (rat) NMDARI-2h suhunit
Sprague-Dawley strain; cDNA lihrnry
923 aa
gh: U08264
NMDARI (rat) NMI)AIII-33 suhunit
Spraguc-Dawley strain; cDNA lihrary
923 aa; zinc potentiates qonist-induced currcnts at ccrtnin splicc variants
NMDARl (rat) NMIIARI-.3h suhunit
Sprague-Dawlcy strain; cDNA lihrary
944 aa; zinc potcntiatcs agonist-induccd currents at ccrtain splice variants
NMDARI (rat) NMDARl-4a suhunit
Spraguc-Dawlcy strain; cDNA lihrary
886 33; zinc potcntiatcs agnnist-induccd currents at ccrtain splice variants
Hollmann, Ncbrlron( 1993) 10: 943-54.
NMDARI (rat1 NMDAKI -4h suhunit
Sprague-Dawlcy strain; cDNA lihrary
907 aa; zinc potentiates agonist-induced cilrrrnts at ccrtain splice variants
Hollrnann, Nruron ( 199,3) 10: 943-54.
Human hrain cDNA lihrary
886 aa plus 17 aa signal pcptidc
Hollmann, Nruron (1993) 10: 94,3-54. Hollmann, N ~ ~ r l r o( n1 993) 10: 943-54.
gh: LO5666
PlanellsCases, Pmc Not! A r d Scj (JSA (199.3) 9 0 5057-61.
NMDARI (human)
Human 'key' suhunit
9.38 na (9Y0&nmino gh: acid homology t o D1,3515 rat N R I )
886 aa hNR1-1 (human) Homcl snprens (cDNA I~hrary: Stratagcnc 9362051 femalc infant hippocampus
gh : L1.3266
hNR1-2 (human1 Ilomo s o p ~ ~ n g 604 sa (cDNA lihrnry: Stratagcnc 9.36205) fcmalc infant hippocampus
gh: L1,3267
hNR1-3 (human) Homcl .snpri.nr 929 an (918 aa (cDNA I~hmry: mature chain] Stratagcnc 9.36205) femalc Infant hippocampus
gh: LI3268
Foldcs, Grnc (IYY.3)131: 293-8.
Foldcs, ( : m r (lYY.3)131: 293-8.
Nomcncl:~turc
Spccics, IJNA cot~rcc
Orlginnl isolate
Acccss~trn Scq~lcnccJ d~scuscion
hNR1-4 {hum:~nl tforiio \oprc.n\ 2 I p ~ r t (cllNA 11hr;lrv: \cqucncc Str:~t:~~c.nc c).if~20i)
gh: IJOX 106
NMDAR {humanl h u m ; ~ ntcr;ltoc:lrclnomn line NT?.
101 ;I;) pnrtinl .;cqucncc
gh: S57708 Younkln, /'roc. N ( I ~Ac.i1(1 / .Scr r rsn 1 I 90.31 90: 2174-8.
h N R l N [huni;~nl GRIN1 spllcc variant
196 ;la partiill scqtlcncc
gI1: UOX 107
Foldcs, (;rpnr. ( 1 0041 In prcs.;.
zeta 1 1- i l l GluR suhunit NMDARI NR1
Motrsc. NMI)A 'fundn~ncntnl'or 'kc\,' s u h u n ~ t : isolatcd through cros.;-l>ornolo~y scrccnlng
9.M nn, A4, 105 SW
ah: 1110028
Yamaznki, FFIj,? /,ctt [ 1 992) 300: .3945.
NMDARl splice variants NR1a ancl NRl h
NRI spllcc See I r o t gh: t , n .6.32 v;~rinntsNIZ 1:1 and ] ? / ~ o ~ p / ~ c ~ n ~ / ( l t iLO1 OX-.??, clnil NRlh / k ~ r r!rifc.rr.ric~e,\, cepen (;/~r~nric./ (.'epnv o r , y ~ r n ~ . - ~ ~~t ~i ~o no .( / ~ ~ / O,Y.4,1 ot~r~n,
Durantl, I'ro~. N ~ J IAc.(lt/ I Sc.1 IISA (19421 89: <)~3?9-6,Z.
-
Foltlcs, ( ;e,r~c* [ 11)L)41 In press.
OX-20)
receptors w ~ t h . ; u l ~ u n ~NIII; t lionlolog\,-l>:~.;c-tl I'CII scrccn NMIJA 'potcnt~.~t~ng suhun~ts'"
I\olatctl hv I'CK ~ ~ l / r hnr nt ~ n cl3NA l~hrarv
gh: MVI 56 l gll: M1)l 5h2 XI>: MU 156.3
Monvcr, ( 1 902) S(.~r,r~c.c* 256: 1217-21.
gh: 111.321 1 gh: 1)1.3212 gh: l>I.321.3
Ishil, I H ~ o l C.'/1(~1711 l99,Zl 268: 28,3643
gh:
1)1.7214 Epsilon 1 suhunit Mouse. Homology- 1464 an (1445 an, I NMI)AK?.AI hnscd PCR .;crccn mnturcl
gim: 405314
Mcguro, Ne~trtrr.(1992) 357: 70-4.
{
Epsilon 2 sc~hanit Mouse. H o m o l o p - 1482 ;la NMDARLIXI h:~sctl I'C1lZ .;crcc.n
gim: 4052
K~~tsuwndn, Nrrtrlrc. ( 1 992) 358: 36-4 1 .
Epsilon 3 sohonit M o ~ ~ stclc~riiologv. 12.19 nn 1 NMI>ARI('\ h;~scilI'CK scrccn
gim: 305.725
Katsuwad;~, Nc~tz~rtp 1 I 0911
-
Nomenclature
Spccics, IlNA
Original isolate
SOLI~CC
Epsilon 4 suhunit M(luse. Homology- 1%12.3 an
hnscd PCR scrccn Ligand-hintiing prclpcrtics not (C;Rl') of NMDA rcpnrtcrl; nn dirwt reccptor complcx cvidcncc of p l r ~four ~, channcl activity o r NMDAR cDNAs NMnA pharmacological spcciftcity
Glutamatebinding pmtein
Pre-synaptic
glutamatc rcccptor-channcl
-
-
-
Molcculnr mass 57 kDa [including signal peptitlcl. No homolorn to any other CluR or kainatc-hintling protein
Accession Sequence/ discussion not found Ikcdn, FEIZS Lcrt (1992) 313: 34-8. not fount1 Kumar, Nrrttrrc (1991) 354: 70-3.
not found Smirnovn, Scicnce ( 1993) 262: 4.30-3.
Notr: T h e above tahle lists genes encoding subunits NMDA-selective ionotropict glutamate receptors {iGluR\. In datahasc searches, these should not he confuscd with thc gcnc nomenclatures uscd to rlcscribc the C, protein-linkctl (mctahntmpictl g1ut;lmate rcccptors in the r n G I ~ ~ - r n ( : I scries t ~ ~ (see Appendix A - Ir~dcxo f C pmtcin11nkc.d rc.r-cptor.s, entrv 50, a n d rhc norrs l~clowtile nwrn t o l ~ l rin 1)armhnsc Irrtings undcpr ELC: CAT G L 1 I AMI'AIKAIN. 07-53).
Related sources & reviews 08-56-01: SEC OISO Ti~hJc4. Ma.Jorquoted sources for this
128~"6"'"M;
NMDA rcccptor physiola~yrc~icws"~'~"~'."~'~"'~"'~~ , functional distinctions of NMDA and non-NMnp, rcccptor~hannels241.255 259 262.263. .'(a. 3~p8-.37n. physiological and pathophysiological roles of cxcitatory amino acids during d c ~ c l o ~ r n c npharinacc~logy t~~; of thc glutamate receptor NMDARs and Hehb-type synaptic pfasticity"'; excitatory amino acid receptors in cpilcpsy"'; cloning of cxcitntury amino acid r c ~ c ~ t o r s ' ~commentary ~"~~; on original descriptions of cloncd NMDARS""; sites for antagonismt on the NMDA receptor complcx~'"; glutamate ncurotoxicity and ncuronal dcRcneration4~~62. ~2,10~.~.~.~-124,374, , pharmacological strategies (including
NMDAR antagonism) aimed at limiting CNS tissue damagc following traumat'z3n,'.'$ cornmcntarics on NMDARs in synaptic excitatinni"7s'"7~; physiological roles of second mcssengcrs in rewlation of NMDAR function5; reviews on rolcs of GluRs in CNS function and memoq~~.'-"~"*74*18"'""; phosphorylation of iGluRs in synaptic plasticityt65; protein kinasc C function in LTI' inductionrq" the glycine site of thc NMDAR.~'~"""'~; polyamine ; as 3 pharmacological target for modulation of the N M D A R " " . ~ ~ ~NMDAR ethanol2*"; in viirn and in vivo ncurochcmical chnrnctcrization of N M U A I ~ " ~ ' NMDAR ; ktimulus-transcription coupling' in neuroncs (it'. trans-synaptic contrt~lof Kcno c ~ ~ r c s s i n n ) ' ~comp~itational ~~'~; implications of NMDA rccuptor-~hanncls'~'.~'~; permeation pathways of neurotrnnsmittcrgstcd ion chnnnuls'"". See r11so rrvfcw.< l ~ s t c du n d r ~ rEi.C: CAT CLO AMI'AI L 'Ion C channel /look rr/crcnccs. erltry 00. - KAIN,e n t r y 07,clnd R I ? S ~ I I E~ -
cntry OX
Rook reierences 08-56-02: Hall, Z.W., Sargent, P. R., Schcllcr, R. H., Kuss, E. M., Kennedy, M. R., Vale, K. n., Ranker, G, Lemkc, C;., Anderson, D. J., Patterson, P. H., Mardcr, E. and Rrcakcficld, X. 0. [ 1992) An Intmduc-tion to Molcculmr Nc~lrolriolo~qy. Sinaucr Associates, Sunderland, Mnssachusctts. Kozik(iwski, A. P. and Itarrionucvo, G. (crlsl (199 I ) Nrnrol~iolo,yyo f the NMDA Rci:cptor: From Chcmisrry to thr Clinic. VCH Vcrlagsgcscllschaft, Germany. Krngsgaard-Larsen, P. and Hanscn, 1. 1. (cds) (1992) Exr.itcltorv A ~ n i n oAcid Rcccptors. Ellis Horwood. L o d ~ c ,D. (cd.) (19811) Excitlitory Amino Acids in Hcnlth r~nd Discrtsc. Riolo~icalCouncil Symposia on Drug Action. John Wilcy & Sons. Plaitakis, A. (1984)In The Olivop)ntoccrrhcIIll~rAtn)pl~ics(eds R.C. Duviosin and A. Plaitakis), pp. 2254.7. Kavcn Press, New York. Watkins, J.C. and Collingrirlgc, G. L. (19891 Thr NMDA Rcccptor. IRL Press.
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entry 08
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entry 08
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Cline, I Nc,rrrosc.i (I')Y01 10: I 197-216.
'"Cline, Nrnrrron (19891 3: 41.3-26. 4')
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entry 08
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cntry OX
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Mchain, Physiol R e v (1944)7 4 72\7-60,
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'"' Thomsnn, Trr.nd.7
'"
(Ohcnaus, Net~rtlsr~i Lcrt (1989)98: 172-H. Hnrvcy, Neurosci Lrtt (19921 139: 3'17-200. '" J ( ~ l ~ n s t oAnn11 n, Rev Physinl (1992)54: 489-505. lno Knmatsu, I Neurophysiol ( 1 9921 (17:401 -1 0. '" Kullrnann, Nerrron [ 1992) 9: 1 1 75-83. '" Grover, Nrrturr (1990)347: 477-9. ln3 Miyakawn, Nr>rlron (1991)9: 1 167-73. "4 Kellcr, EMRO /(I9921 1 1 : 891-6. I65 Asztcly, Errr Nilrrrr~~ui [ I 9921 4: (18 1-90. '" Aniksztcin, Nrrturc (1991) 349: 67-9. I67 Malgardi, hmturc ( 1992) 357: 134-9. Calahrcsi, Eur Ncurosci (1992)4: 929-35. IbY Walsh, Nr>rrro.fc:ir,nr:r [ 1 993) 57: 24 1-8. 17" Thclinpson, Nri!tlro (1992) 359: 6,384 I . 17' Pcr(~uansky, IJhysiol Loncl(~n( 19'1,3) 465: 22,744. Rckkcrs, C o l d S p r t n ~Hmrhor Svmp CJr~nnt/ 19901 55: 1.7 1-5. Mott, Sr:icnue ll9VI) 252: 171X-20. " ' Rmcsrtl, N e ~ ~ r t l(1993) n 11: 751-7. 17.5 Izumi, Nrurnsci Let! (1991) 122: 187-90. "'C o l l i n ~ r i d ~Ien, ! Acod niorned D n ~ qRcx (1991 ) 2: 41-9. 177 Radpour, Ncrrrosci Lc! t ( 1 992) t 38: 1 19-22. I 78 [ 1992) (17: 1009-1,7. Xic, Ner~rophj~.~ioI I7g Sak~trni,I Ncrrrr)c:hr'm ( 199.3)60: 1344-5,Z. "" Harris, Ncrrrosc*i I,ct t ( I (lX6) 70: 1,32-7. '" t h r i ~ N(1tt1ro , ( I 09 I 1 354: 76-%[I. Rcnkc, Proc Null A c d Sci USA (1993)90: 781 9-2-3. IH.7 Fnrsythc, I Ph,vsAl Lonrl ( 1 9X8] 396: 5 15-.3,1. lR4 Rckkt'rs, N ~ I ! I (1989) I ~ P 341: 2.10-3. ""~alcn kn, MoI Nrumhir,l [ 1 99 1 ) 5: 289-95. '" Izqiiicr~i(~, T r c ~ l dPhnrrnrir:ol ,~ Sci (1'191 ) 12: 12R-9. IH7 D'AIIKL'IO, NNt lire [ I YYO! 346: 467-70. lnR nckkcrs, Prou NarI Acmrl Sui USA ( I99 1 ) 88: 78.74-8. lAPTskano, Riochem Hioph~?.~ Kes Cornmsln (199,1)f 97: 922-6. IW Nakanishi, I1roc N r ~ t lAcad Sci IJSA (1992) 89: 8 5 5 2 4 . 'Of Anantharam, FEHS Lcr t [ 1992)305: 27-+Z0. I*' Sugihnra, Rbchcm Kiophys Rcs C o m n ~ u n( 1 992) 185: X26-32. Hr,llrnann, Nromn [ 199.3)10: 94.1-54. i5H
'"
"' '",
"'
Rai, Riochim Riophys Act0 (1993)1152: 197-200. Planellscases, Proc Natl Acad Sci U S A (1993)90: 5057-6 1. Rurnashev, Science ( 1992)257: 1415-1 9. IP7 Lee, FEHS Lett (1992)311: 81-4. 19' Sakurada, Riol Chern (1993)268: 41&15. 199 Lcster, Annu Rev Riophys Riornol S t n ~ c (1992)21: 267-92. Mnri, Nature (1992)358: 673-75. M t Durand, Proc Nat1 Acad Sci lJSA (1992)89: 9359-(~3. M2 Ikeda, FERS Lett (1992)313: 34-8. M3 Hahn, Proc Natl Acad Sci U S A (1988) 1988: 6556-60. Chctknvich, Proc Natl Acad Sci USA (1991)88: 6467-71. Silva, Science ( 1992) 257: 201-6. Zcilhofcr, Nct~ron(199.3)10: 879-87. M7 Cherncvskaya, Nature (1991)349: 41 8-20. 2"18 Simpson, 1 Neurochern (1993)61: 760-3. 2"9 Conquet, Nature (1994)372: 23743. "* Chcn, Ncriron (1991)7: 3 19-26. Artola, Nature (1987)330: 649-52. Olncy, Science (1991)254: 1515-18. Novclli, 1 Neurosci (1987)7: 40-7. 'I4 Gnrthwaite, Netlroscience (1988)26: 321-6. ""~arthwaite, Trends Neurosci ( I 991 ) 14: 60-7. "'"adison, Annu Rev Nemosci (1991)14: 379-97. 2'7 Malinow, Nature (1990)346: 177-80. 2'8 Pekkcrs, Nature (1990)346: 724-9. 2'9 Kullmann, Nature (1992)357: 2404. 2M D R V ~ CNature S, (1989)338: 500-,3. Halp~in,Ncumn (1990)5: 237-46. 222 Lichcrman, Nnturs (1994)369: 235-9. 22" Roscnmund, I Physiol Lond (1993)470: 705-29. 224 Okada, hkurosci Lett (1989) 100: 141-6. 22s Williams, Neurosci Lett ( 1989) 107: 301-6. 2Z6 Lynch, j Neurochem (1991) 56: 1 13-1 8. 227 Oliver, Brain Res (1989)505: 2.33-8. Huang, Neuroscience ( 1992) 49: 8 19-27. "' Kelso, 1 I'hysiol Lond (1992)449: 705-18. 2430 Hu, Nature (1987)328: 4269. 2.3' Malenka, Nnture (1986)321: 175-7. 2.72 O'DCII, Nature (1991)353: 558-60. 2'3 Rading, Science (1991 ) 253: 912-14. 234 Lcstcr, Nature (1990)346: ,565-7. ""~ustr, Neuron (1990)5: 247-53. 2'%G 1 Ieurosci (1994)14: 4561-70. 2.3 7 Clcmcnts, Neuron (1991)7: 605-13. 23R Jahr, Science (1992)255: 470-2. 239 Gihh, J Physiol Lond (1992)456: 143-79. ""dmonds, Proc R Soc Lond /I
IPS
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"' ""
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Rcichling, ] 1Ihysjo1 Lond (1993)469: 67-88, Schinder, FERS Lett (199.1)332: 44-8. Vyklicky, / I'hysinl Lond [199;1)470: 575-600. 2'" Chizhmakov, I'hvsir~l Lond ( 19021 448: 453-72. "' Lcgcndrc, Nrurosci (199,3) 13: 674-84. '*' Hcstrin, Nrrrron (1Y9219: 991-9. *' Lconnrd, Nrurtln ( 1 990) 4: 5,1-60. Roscnmunt!, Nrurr~n[ 1993) 10: 805-1 4. ."' Schncmcnhurgcr, Neuron (1993) 11: 163-4.3. 2"2 HOWC, / I'hy.~inlLand ( 1991) 432: 14.3-202. Cull-Candy, 1 I'hysicll Land (lYK9)415: 555-82. '"'jnhr, Nrrturc8 (1987)325: 522-5. '"" Cull-Candy, Nrl!urrJ (1987) 325: 525-8. "" Cull-Candy, l'hv.siol Lond ( 1YRX] 400: 189-222. 2.5 7 Aschcr, 1 I'hysiol Lond (I9881 399: 207-26. "' Smith, 1 N e u r o p h ~ ~ s i (19911 ol 66: 369-78. ""Aschcr, 1 Phy.~iolLand (I9RX)399: 247-66. 26" Kellcr, Physiol Lond ( 19'1 1 ] 435: 275-93. - o1l21 70: 28,1-8. Wnng, Crln f'hysir~lP h ( ~ r r n ( ~ ~( 19' '"'Mayrr, Nritllrr (1984)309: 261-,3. '"? Nowak, Nritllrc. (1'184) 307: 462-5. Legcndrc, lJh?.siol i.onr1 (1990)429: 429-49. '"" Wcsthrocik, N n t t ~ r i(1987) ~ 328: 640-3. 266 Gottcsman, Nerrrphy.~inl(1992168: 5964304. "' Yamakur;~,Neurorcport ( 1 99.3) 4: 6X7-90. "'Macnonald, 1 i1h!~riolLond ( I 99 1 ) 432: 48.1-508. '"'L(idgc, Trrnr1.q Pl~mrrnnuolSci (1990) 11: 8 1-6. Chen, 1 Nt7uro.~ui [ 1 992) 12: 4427-,16. Lin, FA.TEI3 1 ( 1Y9,1] 7 : 479-85. '"Nctzrr, E~ir]1~1~17rlrn10ct~l (19931 238: 209-16. 273 Wright, 1 IJhy.~jol Lond (199 1 1 439: 579-604. "' Malcnka, Nr~rrrrrj(1989) 340: 554-7. 2'5 Aizcnman, Nrriron (1989)2: 12574.1. 27f1 Tang, Mol I'hrrrrn~tcr>l [ 1993) 44: 47.1-8. ? ' Lei, N I ~ I I ~( 1O992) I I 8: l(l87-99. Lipton, Nnttrrc (lYt)3) 364: 626-32. 9 ' 2 C,111s . herg, Stroke (1976) 7: 125-3 1 . '"Reynolds, Rr 1 Phr~rrnnrol(19901101: 178-82. "' Lcvy, Npurosri Lett (1990) 110: 291 4. "'Suchcr, N c ~ ~ r o r ~ p [nl990] r t 1: 29-32. "' Tang, PItysjol Lond (1993)465: ,3Ot3-Z3. '"'Wicrnszkn, Ne~lrcln(1993) 10: 55.1-7. Williams, Lif? Sci (1991)48: 469-9R. Yoncda, N ~ I I T I ) RC.F . F C(~1 99 1 ) 10: 1-3,3. "' Scott, Trcnds Ncurosci (1993) 16: 153-60. '** Rock, MoI IJhnrmocol ( 1 992) 41: 83-8. Rock, Mrrl I T I t o r n ~ ~ c( o1 992) i 42: 157-64. I\cnvcnistc, 1 IVtysiol Lorlrlon (1993)464: lC11-6.1. Bird, 1 Riol Chrrn ( I Y9.1] 268: 21486-8.
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entry 08
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Morgan, Riochem Soc Trans (1990)18: 1 0 8 0 4 . Vyklicky, Physinl Lond ( 1 990) 428: 3 13-3 1. ' "Weight, Ann NY Acad Sci (1991)625: 97-1 07. 29.5 Lovinger, Science 11 989) 243: 172 1-4. Conzalez, Trends Phnrmucnl Sci (1990)11: 137-9. 297 Skecn, Mol Phormocol (1993)44: 44,3-50. 29n Rowlhy, Mol Pharmacol ( 1 99.3) 43: 8 13-1 9. 299 Vyklicky, Physiol Lond (1990)430: 497-517. ""O Traynelis, Nature 11 990) 345: 347-50. "Of Kwiecien, Eur Pharmacol (199.3)249: 325-9. .?02 Yonrda, 1 Ncurochem (1993)60: 63445. Franklin, Mol Pharmacol(lYY2) 41: 1,34-46. ""O Draguhn, Neurosci Lett (1991)1 3 2 187-90. " " ~ u m u i sNature , (19881 336: 68-70. Nicholls, Nature ( 19921 360: 106-7. Linden, I Neurosci ( 1 9921 12: 360 1. "OR Herrcm, Nature ( 1992) 360: 1 6 3 4 . .209 Rusin, J Neurochern 11993) GO: 9 5 2 4 0 . ."" Miller, Notrrre (1992)355: 722-5. "" Wong, Annu Rcv Phnrmacol Toxicol (1991)31: 401-25. "I2 Watkins, Trends Ncurnsci (1987)10: 265-72. 313 Maycr, Neurophysiol (1988)60: 6 4 5 4 3 , 314 Watkins, Trends Phhormacnl Sci ( 1 990) 11: 25-33. 315 Swartz, Mol Phurmacol ( 1992) 41: 1 130-41. "" McKernan, 1 Ncurochern 11989)52: 777-85. "'? Zcilhofor, Ellr J Phnrmncol (1992)213: 155-8. .?" Scrna~or,Neuron (1989)2: 1221-7. "Iv Pcrcira, Pharmacol Exp Ther ( 1 992) 261: 3.31-40. "zn Hucttner, Science ( 1989) 243: 161 1-1 3. Grimwood, Mol Phnrmncol( 1992) 41: 923-30. "22 Johnson, Nature (1987)325: 529-3 1. "" Klcckncr, Science (19881 241: 835-7. 324 Kcmp, Proc Not1 Rcnri Sci USA (1988)85: 6547-50. .325 Moroni, Eur Phrtrrnacol (1992)218: 145-51. 326 Kcmp, Trends Phnrnlncol Sci (199,1]14: 20-5. ".' Whitc, Epilepsid (1992)33: 564-72. McCahe, 1 Pharmacol Exp Ther ( 1 993) 264: 1248-52. " .' Tang, Proc Not1 Acnd Sci USA (1990)87: 6445-9. ?' Doncvan, Mol Phnrmacol ( 1 992) 41: 7 2 7 4 5 . ""'Williams, Mol Pharrnacol(199.7)44: 851-9. "32 Olivera, Science 11990)249: 2 5 7 4 3 . "'.' Haack, Nwrosci Lett (19Y,3)163: 6.3-(,. "54 Chandler, 1 Riol Chern ( 1993) 268: 1 7 1 73-8. """oki, Riol Chem (1992)267: 14884-92. Rogawski, Trend,$Pharmacol Sci (1993)14: 3 2 5 3 1 . "? ' Riggc, Riochem l'hormncol(1993) 45: 154741. 3" Risnga, Eur Phnrmncol ( I 993) 242: 213-20. 334 Murata, Eur I Phnrmacol ( 1993) 239: 9-1 5. .34" Ahmhams, Soc Nrr~rosciA h . ~ t r (19X8) 14: 937. 292
29.3
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entry 08
Olncy, Scicncc [19X9)244: 1.760--3. Allen, Scirncc (1990)244: IL360-,3. Mancv, FASER f(1990)4: 2789-96. 744 Engclscn, Actn N~irrolScrlnd (1982)74: 337-55. .'4"~nvcnistc, j Cprrh Rlood Florv Mrtah (198919: 629-39. Mancv, Mol I'hmrn~mcnl( I 9891 36: 106-1 2. ,147 Choi, 1 Ncrjrt~rci(19R7)7: 369-79. 3411 0 gurs, Exp Rrnin I l r . ~[I9881 73: 447-58. "4V Hc~lopaincn,Ner~rosc-i Lctt (1989) 98: 57-62. "'"Pauwels, Mu1 Pbrlrmtlcnl ( 19891 36: 525-3 1. "'I Pcrkins, Brrrin Kes (1982) 247: 184-7. IJllnrrnmctllE x p Ther ( l 983) 226: 55 1-7. .7,52 Pcrkins, .7,5" Maycr, Nrzturr (1989) 338: 425-7. ,154 Lester, Nc?uros~:i (1993) 13: lO8X-96. """ Zilhcrtcr, Nrurtlrl (1990) 5: 597-602. .7""~~valchuk, Ncuroscicncc (1993)54: 557-9. ,'"'Hcnzi, Mol Phnrrnlrcol [l992)41: 793-801. Wciss, Nc-trron ( 1989) 3: ,321-6. Dchonn, Etrr Phrlrrnacol (1993)235: 28%'). Pcrkcl, IJhvsiol Lond (1993)471: 481-500. Lcrma, M o l Phurrnuunl ( 1992) 41: 2 17-22. .''O Williams, Neuron (1990) 5: 199-208. ". Nictlll, Phy.crol R e v [ I 990) 70: 51.7-65. "' WWisden, Cltrr Opin Ncurol~iol(1993) 3: 291-8. Collingridgc, Ph~~.qio! Rev ( 1989) 40: 145-21 0. " " ( ' M ~ ~ cPros r , Ner1rr)hiol (l987]2R: 197-276. Monaghan, Annrr Ilev Phnrmnctll Toxic01 (1989)29: .365-402. Aschcr, 1 Phy.~iolLnnd (19X8) 399: 22745. Ozswa, Ipn Physinl (199.71 43: 141-59. "'Pctcrs, Scit~nct?(1987) 236: 589-9.1. Shinozaki, I'rog Ncumhiol(lY88) 30: 399-435. "','Hcnnchcny, Riocssnys [ 19921 14: 465-71. ", 3 Rarnard, Trcnd,s II'l~nrrnoctllSci ( I 992) 13: 12-1.7. "" Sicsiii, Ann NY R c r d Sci (19RR)522: 638-61. ,17" Rarncs, Science (1988) 239: 254-6. .T ' 6 Foster, N r ~tire t ( 19871 329: ,795-6. 177 Thornson, T r r n d . ~ Ncuro.~ui(1989) 12: 349-53. Rlatz, Trends Nrttmsci (191171 10: 463-7. Wood, Nr,l~rtlchen~ Res ( 1990) 15: 217-30. Murphy, Hr Phormacol I19881 95: 9.32-8.
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William J. Brammar
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Entry 09
Abstractlgeneral description 09-01-01: The nicotinic acetylcholine receptor (nAChR) is a ligand-gated, non-selective, cation channel that is activated hy hinding of the chemical neurotransmitter acetylcholine. There are two maior suhtypes of nAChR: the muscle type and the neuronal type. In vertchrate skeletal muscle, the nAChR is concentrated at the endplate of neuromuscular junctionst, where it hinds the neurotransmitter acetylcholine (ACh) and mediates excitatoryi transmission and initiation of the action potentialt. In neural tissues, the nAChRs are present on autonomic neurones and adrenal chromaffin cells in the peripheral nervous system and on many neurones in the central nervous system. Neuronal nAChRs are prohahly involved in the control of electrical excitation and release of neurotransmitters. 09-01-02: The nAChRs are targets for several animal and plant toxins, local
anaesthetics, sedativest and hallucinogenic drugs, all acting as antagonists and inhihiting channel function. Nicotine itself is a receptor agonist and prohahly has its addicting psychoactive effects through actions on nAChRs in the central nervous system. 09-01-03: In nearly all cases, both the neuronal and the muscle nAChRs
mediate 'fast' inward currents in response to activation by acetylcholine. The neuronal channels, unlike thc muscle isoforms, are modulated to pass increased currents hy external ca2+,and can play a role in activating ~ a ? + dependent pathways via ~a"-influx. 09-01-04: The nAChRs from Torpedo electric organ and skeletal muscle are composed of five suhunits, r2/l;.(S; in adult muscles of some species an c suhunit displaces the y suhunit. 09-01-05: The neuronal nAChRs show considerable diversity in their subunit composition and function. Two main types of suhunit have been identified, comprising eight distinct r and four P suhunits. The muscle a-suhunit is referred to as r l and the P-subunit as 111. The neuronal suhunits are designated 22-9 and P2-P5. The p-suhunits from avian systems tend to he called 'non-r' (nr). 09-01-06: The general structure of the Torpedo nAChR has been determined to 9 A resolution hy electron microscopy and image-reconstructioni techniques. The nAChR is composed of a ring of five membrane-spanning suhunits delineating a central cation-conducting poret. The channel opens transiently when acetylcholine is released from nerve terminals into the - synaptic cleft*, and depolarizes the post-synaptic memhrane.
Category (sortcode) 09-02-01: ELG CAT nAChR, i.e. extracellular ligand-gated cation channcls
cntry 09 -.
I
integral to the nicotinic acetylcholine reccptor. The s u ~ e s t e delectronic retrieval code (unique cmheddcd identifier or UEI) for 'tagqin~'of new articles of rclcpncc to the contents of this cntry is UEI: NACHR-NAT (for rcports on nativei channcl propcrtics] ant! UEI: NACHR-HET [for reports or reviews on channcl propcrtics npplicablu to hctcn)logouslyi cxprcssctl rccomhinantt For 11 tliscussion o f thc cjdvclntmscs suhunits encoded hy c l l ~ ~ osr gcncs't). t of UEIS nnd ~ t ~ i r i i ~ loinn their ~ s implem~ntotion,srv* tlw scctior? on Resource / rlnder lntmtiljction r~nrl Imyout. entry 02, clnd for fitrtl~crdftrlils, s r f Rt.sourcc. - Sec~rrhcritericl Q) C S N development, entry (7.5.
1 I
Channel designation Chnnnc.1 d e s i ~ n oions t presently in use
09-03-01: The ionotropici acctylcholinc-gated cation channels arc ~ c n c r a l l y
dcsignatctl as nicotinic acetylcholine receptors (nAChRs).The musclc form of the receptor cfoscly rcscmhlcs that found in the electric organ of the electric ray, Torprdn ci~lifornicrr,while the many isoforms found in the nervous system, collcctivcly known as 'ncuronal channcls', arc less closely rclatcd nnil arc clcctrophysiologically and pharmacologically distinguishahlc.
0
Current designation
09-04-01: Gcncrsllv of the form 1,,,
i.c.
Gene family
09-05-02: Scrlucnccs encoding isoforms of the suhunits of musclc and
nuiironnl nAChRs hnvc hccn detcrmincd for scvcral species, as shown in Tahlc 1 .
Xcnnpus muscle suhunits 09-05-02: T w n distinct musclc x suhunit C D N A S ~x ,l , and XI,,, dcrivcrl from
mRNAs encoded hy diffcrcnt genes, havc hecn cloned from Xennpus embryos. Roth n l , , ant1 a l h encode a protein with a prcdictcd signal peptide and a maturu protein of 4.37 amino acids'".
Subtype classifications
09-06-02: A changc in thc clcctrnphysiolo~ical propcrtics of thc nAChR channels in vcrtuhratc musclc is associated with the switch from thc gamma (;.Isuhunit in cmhryonic musclc t o the cpsilon ( 1 : ) suhunit in the ac1111t channclZ4(For detfljls. .FCC Developmental reayulotinn,09-1 1 . ) 09-06-02: The ncuronal channels can hc constructed from various comhinatinns drawn from eixht z variants and four diffcrcnt /I suhunits. Each known suhunit can affect rcccptor kinetic paramctcrs, receptor pharmncolrlgy and/or channcl c n n d u c t a n c ~ ~ ~(For - ~ f dctnil.~,net3 ELECTRO1'HYSIOLOC:Y and I'HAIIMACO[,OGY sections.)
Table 1. nAChR subunits specified b y cloned sequences from various species (From 09-05-01) Species
Torpedo
Subunit
a
P Y 5
Rat
a2 a3 a4 a5 a6 a7 a9
P2 P3 P4 na2 &
Transcript size (kb)
Encodinga Amino acids (aa)
Signal peptide (aa)
Mature peptide, M,
Refs
Chicken
a
a2 a3 a4 a5 a6 a7 08 P2 (ncr, n a 1)
P3 (n021 D4 (na31 Y 6
Human
a
a3 a5 a7 P2 P4
"Encoding data show the number of amino acid residues in the specified channel subunit, determined from the longest open reading framet and the predicted position of signal peptide cleavage, with signal peptide residues and the predicted molecular weight listed separately. In many cases the position of signal cleavage has not been determined or predicted, and the number of amino acids encoded is then determined directly from the open reading frame. Sequences encoding four different Drosophila subunits, one cockroach subunit, and four goldfish subunits, all of the neuronal type, have been determined and are detailed in ref.'.
entry 09
Trivial names 09-07-01: Endplate channels (nAChRs at the motor endplate); nicotinic
receptor.
Cell-type expression index 09-08-01: See mRNA distribution. 09-13, a n d Protein distribution. 09-15,
Channel density nAChR channel density at the vertebrate neuromuscular junction 09-09-01: ~ n d ~ l a t channels ei are 'by definition' expressed at the vertebrate neuromuscular junctioni in high density. Electron microscope autoradiography has heen used to show a nAChR density of 10000-20000 channels per pm2 at the neuromuscular junction2'.
nAChR channel density a t neuronal sites 09-09-02: The dcnsity of nAChR channels in the synaptic membrane of the chicken ciliary ganglion has been measured by quantitative electron microscopic autorad~ography and ['25~1-neuronal-bungarotoxin(n-Bgt), which blocks nAChR function in this tissue. The n-Rgt h ~ n d sto two sites, one of which is shared with a-Rgt. When the g-Rgt-hinding sites are blocked, [ ' " l ] - n - ~ ~hinds t selectively to synaptic sitcs at a density of approximately 600/pm2 ". 09-09-03: The total number of ACh-activatable nicotinic channels in bovine
adrenal chromaffin cells has been estimated at 260OZ9.
0 7 channels in neuroblastoma cell line 09-09-04: The human neuroblastoma cell line SH-SY5Yexpresses an z-Rgt-
sensitive neuronal nAChR with thc properties of the n7 homomeric channel. The channel density measurcd by [ ' 2 i ~ ] - r - ~binding gt is 2400 per cell2".
nAChR channel densities ohtained by heterologous expression 09-09-05: A high density of nAChR channels can be ohtained by transient expessiont of C R N A S ~injected into Xenopus oocytes. Estimates of 2 x 10' molecules of nAChR per cell (3.2 fmol per oocyte) were obtained by z-Rgt binding to oocytes 2 days after microiniection of cRNAs encoding all four mouse nAChR subunits"'. Expression of the human 87 cRNA in Xenopus nocytes produces 7.2 x loR r-Bgt-binding sites per oocyte, with 63% of thc expressed monomers assembled into pentamers at the cell
nAChR channels are associated with cytoskeletal components 09-09-06: Nicotinic receptors are known to associate with components of the cytoskeletont"'-"", and such interactions may control channel mobility, subccllular distribution and local channel density (for details, see Protein interactions. 09-31).
entry OY
Specinl note on determination o f channcl density hv immunoossr~y 09-09-07: There is an important cautionary note for the intcrprctation of data rclatine, to channcl dcnsity dctcrmincd hy ligand-hinding or quantitativc immunoassay. It is clcar that antiscra and ligand-hinding assays can dctcct hoth activc and inactive channcls at thc ccll surfncc, and that the quantitative relationship hutwccn the two forms can vary. For cx;~mplc, analysis of chickcn ciliary ganglion neurones grown in normal nnrl high K' mcdia shows that thc ACh scnsitivitv is rcduccd scveral-fold hy clcvatcd K', hut the numher of nAChRs dctcctcd hv antibody-hinding rcmains unchanged. Elcctrophysinlo~icalestimations of the numhcr of functional channels are 2000 pcr ccll undcr normal growth conditions, and 700 pcr ccll in high R' conditions. Thc numhcr of antihodv-hindin~,-lincng sitcs is 100000 per ccll undcr hoth conditions. The ciliary e,;in~lionncuroncs h:ivc two classes of nAChR on thcir surface: :I Iargc cxccss of 'non-activatahlc channels' and a small fraction of ACh-nctivatnhlc chnnncls, the size of thc lattur pool being sensitive to growth conditions.".
Cloning resource Orj,qinol isolat cs 09-10-01: Sequences cncoding suhunits of the nAChR wcrc first ohtaincrl
from ct3NA lihrarics prepared from poly(Alt RNA from the elcctric organ c ~ f thc electric ray, Torprdo cc~lifornicn"' or T. rnc~rrnorr~tc~~. Thc candidate clones were rcco~nizcdby their hyhridizationt to mixed synthetic oligonuclcotictc pr~)l)csid c s i ~ n c don the hasis of primary amino ncitl scclucncc from the Tor;lc~lr,r f , /I, ,i'and ;..''subunits, then verified hy IINA sccli~cncing.
Clonin,y .sccluc~nce.scncoding musclc and ncwronnl nAChR sr~Runif.s: 09-10-02: Clones cncoding the suhunits of musclc or ncuronal nAChR chnnncls were isolatcd from cDNA lihrarics prepared from the R N A of appropriate tissucs or from gcnomic DNA lihrarics hy thcir crosshyhritlizationi to prohcs derived from cloned Torpr.do sequences. The us: ot cloncd scqucncrs as prohcs t o screen lihrarics ; ~ trcdi~ccdstrinRcncyT ; ~ I l ( ~ wthc c d isolation of honiologoi~s~ scqurnccs.
CuIturctl crll lincs n s
(I
sourcc o f nAChK tnl
09-10-03: A nunihcr c ~ ccll f lincs in culture express the genes encoding forms o f the nAChR and can he used as a sourcc of mRNA from which to gcncratc cIlNAs corrcspon(1in~to chnnncl sr~hunits.The mouse myogenic cell line, Snl 8, ~ t n d c r ~ o c;Is elcvclopmcntal change in culturc and switches from cxprcssing the foctnl form of musclc nAChR, to the adult fcirm, 7?/it:ri.37.The human rhahdomyosarcoma cell line TE071 is a rich source of human musclc nAChR ~ R N A - " . The human neurohlastoma line IMR 32 is thc soiIrcc from which the human xS suhunit cL)NA was clonci12' and thc hum:~n small cell lung carcinoma cell line NCI-N-592also produccs high Icvcl.; c ~ f75 sithunit IIIKNA". The cl)NA for the human 77 subunit was grnrratud from rnRNA i.;c~l;~tcdfrom the SH-SY5Y cell line, dcrivctl frtlm ;I nci1rohl;1stom;1of a syrnp;lthctic ndrcncrgic g;inKlir)n2f". ~,/1;1,i,
-
Developmental regulation Developmental gene-switching in skeletal muscle 09-11-01: The p subunit of the embryonic or foetal muscle nAChR is rcplaccd hy thc h o m o ~ o ~ o u sr: t subunit in adult solcus muscle ~ A C ~ R * " .Direct comparison of the nAChR channels of foetal and adult hovine skclctal musclc by single-channel recordinRi with outsidc-outt patches shows that thc channels are distinguishable by their diffcrcnccs in single-channel currcnt, slope conductancct and their duration after activation by ACh (see Tflhle 2). Single channels obtained by expression of the cRNAs encoding bovinc u, p, y and ii suhunits in Xenopus oocytcs closely rescmhlc the channels from foctal musclc in all respects, whilc thosc derived hy cxprcssion of cRNAs encotling a, P, d and I: suhunits arc indistinguishahlo from the channels from adult hovine musclc" (sce Fig. I).
Table 2. Single-channel properties of two forms o f bovine nAChRs expressed in muscle c~ndXcnopus oocytes (From 09-11-01) nAChR channels from
Conductancc" 7 (PSI
Foetal musclc Adult musclc Oocytcs with AChRy Oocytcs with AChRf
40 (41 59 (2) 39 (7) 59 (6)
Avcragc duration" T
( - 100) (ms)
1 1.0 (4) 5.6 ( 6 ) 10.4 (7) S.3 (6)
H (mV) 108 9,Z 106 K3
rcprcscn 9 the slope of the linear I-V relationships determined with outsidc-out patchcs. "r ( - 100) represents the calculated duration of clcmentary currents at - 100 mV rncmhranc potential. H is thc shift in mcmhranc potential' that causcs an p-fold chnngc in the average duration of clemcntary currents. Data taken from r ~ f . " ~ . '?
i.
Changes in 7 and
E
mRNAs during musclc dc~velopment
09-11-02: Northern hlott analyses of RNA from hovinc diaphragm and Icg muscles,'R and from rat muscle show reciprocal changes in thc level of 7
and
r:
suhunit-specific rnRNAs during post-natal development.
09-11-03: The embryonic form of the channel permits more current flow
(smaller conductance hut a significantly longer open time], so miniature endplate currentst can promote spontaneous mitsclc contractions in developing m i ~ s c l c ~Spontaneous ~. mitsclc activity induccs differentiation of thc ncuromuscular junction.
Developmental re,qulation by clectricnl (~ctirfitv in primary cultures 09-11-04: In primary muscle cultures, spontaneous or stimulated elcctrical
activity of thc rnyotubes represses nAChR hiosynthesis, whcrcas treatment with pharmacolo~ical agents that inhibit elcctrical activity increases ) a 2.5nAChR Icvcls. The c a 2 ' channel blocker verapamil ( I 0 m ~causcs fold increase in surface AChR, rncasurcd hy 1'-"I]-r-Rxt hind in^, and a 7.9-
entry 09
-
fold incrcasc in .x suhunit mRNA in culturcd chicken myotuhcs4". The Na' channel hlockcr TTX (0.5 m ~generates ) 2.9-fold and 14.9-fold increases in tllcsc rcspectivc levels. These cffccts arc almost eliminated hy thc ca2+ ionophore A23187. 09-11-05: The cffccts of TTX in elevating nAChR and x suhunit mRNA arc cnhanccd hy 50 mM dantrolene, a hlockcr of ~ a ? 'efflux from thc sarcoplasmic reticulum4". c
a
40
20 ms
z
b
o
o
200
il
='20
400
h (mM)
V(mV)
-100 l
'
50
-50 L
8
t
l
t
L
c
a
-2
4
3 -4
.>
-
2
-
- -6
-100
-50
0
50
V(mV)
I'ropcrties o f ACh-activcrtcd singlc-chr~nnrlcurrrnts in Xcnopus r>or.ytclsinjtrctcld with cRNAs encoding 1,ovinr nACIiR ( 1 , ,{. -, rrnd ;4 srrl~units (ACIIH..) or with cKNAs cncodin4y t l ~ cr t . .I. /I and r suhonit.~ ( AChR, I. ((11 Sin~1t.-c.hannclc.urrclnts nctivntcd 1 7 ~0..5 l r h l ACh, rccordrd u . s i n ~orrtrirlc-out prltchc,s crt (1 nir17ihron~potential o( ( 1 0 171V. IJpj~cr frar'r, rccortling from (In ooc.vtcJ injected with AChR; RNAs: mcrrn current mnlplit rldc 2.4 pA. 1,owcr trr1c.r. r ~ c o r d i nIrom ~ (rn oocvrc injected wit11 AChR, KNAr: nlr(rn current clrnplituclc 3.6 pA. ( h ) Sin,yIc~-chmnnd I-V rr*lrttions o i thr AChR; (fillrd .syml~ol.s\ond thc AChl<, (open ryr7ihol.q). Thr slopc rontluc.trrnc.cs crrc .'ZY 17s rlnd 59 pS for tlir AChK. rlnd ACIIR, c.hrrnnr.1.~.rc.sp(*c-tir~c,h,. ( c ) Condlrctclricr (?I vrrsus Nil' irctivity ( ( I N , ] for AChR, (fillctl symholsl and AChR, (opcn symholsl. Half-saturating Na' activities arc 7.7 mM for AChR. and 46 I ~ for M AChR,. (d)Averngc duration ( t ) of clcmcntnry currents versus memhranc potential on scmilogarithmic coortiinatcs. Fillcci symhols, AChR, channel; opcn symhols, AChR, channel. Figure reproduced from Mishina r.1 (11.Notr~rc'(19X6) 321: 406-41 1, with kind permission. (Froni 00- 1 1-011 Figure 1.
entry 0'3
The region of the h suhunit gene promoter responsible for response to electrical activity 09-11-06: Using a reportert gene construction in which thc promotcrt c ~ the f m t 6 suhunit gene controls the expression of a lucifcrasc-coding sequcncc, a region of the promoter that confers regulation by electrical activity has hccn dcfincd4'. The electrical stimulation dcprcsscs cxprcssion of the lucifcrase reporter genc during transicnt transfccticln of primary rat myotuhes. ~eletion-mappingtcxpcrimcnts identify a minimal sctlucnce of 102 hp that confers regulation in response to electrical stimulation.
Involvement of PKC in the control of nAChR synthesis protein kinase C inhihitor staurosporine causcs a concentration-dcpcndcnt increase in surface AChR and a suhunit mRNA lcvcls. T h e maximal effect, a fivc-fold increase in surfacc rcccptor concentration and a 10-fold incrcasc in r mRNA level, is ohtaincd at staumsporine concentrations of l a 3 0 ng/ml, with a half-maximal response at approximately 1 ng/ml. (Staurosporine inhihits protcin kinasc C activity half-maximally at 1.2 ng/mI4'.) Prolonged treatment with thc phnrbnl ester TPA, which rcsults in down-regulation of PKC Icvcls, also incrcascs surface nAChR and r suhunit mRNA Icvcls. 09-11-07: Thc
Developmentnl regulation o f nAChR synthesis in muscle cell-lines 09-11-08: The le-~elsof the nAChR incrcasc dramatically in terminally differ-
cntiating muscle. In the skeletal muscle ccll linc C2 the lcvcls of thc nAChR as measured by a-Rgt hinrling incrcasc 10- to 100-fold during diffcrcntiation. Northern hlott dctcrminations show that the lcvcl of r and ci suhunit mRNAs incrcasc 10-fold and 15-fold, rcspcctively, during diffcrcntiation. Nuclear run-ont expcrimcnts show that thcsc incrcascs arc accounted for hy changes in the rntcs of transcription of the T and 5 gcncs"7.
The n suhunit mRNA is affected hv dectricnl activity 09-11-09: The mature rx mRNA and its precursor forms arc found tn vary in
parsllcl throughout all the trcatmonts that modulatc the clcctrical activity ot the cultured myotuhcst, supporting the idca that thc rcgulstion is occurring at the level of transcription4". Thc mechanism hy which PKC and ~ s "arc involvcd in the regulation of ACh hiosynthcsis hy clcctrical activity in developing musclc is not understood.
Developmentnl ~ene-switchingin cultured cell lines 09-11-10: Cells from the mouse myngenic cell line Sol 8 can hc culturcd with
a fccdcr laycr of cells from thc cmhryonic mcsenchymal ccll linu 10T1/2 and a scrurn-frce medium, to form contracting myotubest for 2 wccks. Undcr thcsc conditions, Sol H myotuhcs undcrgo a maturation process charactcrizcd by scqucntial exprcssion of two phenotypes: an carly 'cmhryonic' phcnotypc typified hy the exprcssion of thc nAChR ;. suhunit transcripts and lowconductancc ACh-activntcd channels, and a late 'adult' phcnntypc charactcrizcd by thc cxprcssion c ~ fnAChR t: silhunit transcripts, thc dccrcascd accumulation of ; suhunit transcripts and the nppcarancc of highconductancc ACh-activatcd channels. Thcsc ohscrvntions indicntc t h a t
I
rntry 09
-
cxprcssion of functional adult-type AChR is an intrinsic feature of the Sol H muscle cclls and docs not require the presence of the motor nerve4,'.
Influence ofsynflpticconnections neuronal nAChK
or^
thc drvc~loprnrntalr~~qulntion ol
09-11-11: T h e detection of specific nAChR niRNAs in thc tlcvcloping brain by
hybridizationt techniques has suwested that the cxprcssion of nAChR gcncs can hc increased, sometimes tmnsicntly, when ncuroncs make synaptic connections. 09-1 1-12: Neuroncs in the chickcn lateral spiriform nucleus do not express tlctcctahlc lcvcls of 2 2 rnRNA until cmhryonic tl;~y I I ( E l 1 J,whcn fihrcs
cnntaining cholinc acctvltransfcrasc first enter the 09-11-13: In the chickcn optic lobe, cxprcssion of the fl2 genc is incrcsscd -> 10-hlld hctwccn Eh and E12, after which it rapidly d c c l i n ~ s1n ~ ~situ
hyhritlizationi shows that /I2 expression occurs in a rostrocaudal gradient and is coincident with the invasion of the tectum hy retinal affcrcnts. Removal of the cyc cup ;it E2 results in 10-fold reduction ot exprcssion of /(7_ compared with controls. This suMests that p2 exprcssion in optic Iohc ncuroncs is tr;lnsiently stirnulatcd by arriving retinal affercntsR.
-
Developrnentcil r c ~ u l tnion o / (1 7 suhunit gene expression in chick optic ti?ctutn 09-11-14: Transcripts from the gene encoding the 27 isoform of chick nAChR transiently accumulate in the developing optic tectum hctwecn ES and E l h .
They arc present in 170th the deep and the superficial layers of El 2 tcctum". Thcrc is excellent correlation hctwccn the lcvcls of 77 mRNA anti r-Rgtbinding activity in the dcvcloping optic tcctum of the chick cmhrycl. Toxin-hind in^ activity increases sharply hctwccn E l 0 and hntching, dccrcnsing to a low plateau in the neonate and adult4". T h e decrcasc in toxin hintling may coincide with the maturation of cholinergict synapses4".
Hct,qcnert~tiono f optic ncrvcpstimul~ltesriAChR suhunit gcnc c>?rprc1.s.~ ion 09-11-15: In adult goldfish retina, cxprcssion of r.3 ant1 three /I gcncs is clcvatcd IS days after crushing of the optic nerve, whcn retinal ganglion
cell axons are reforming connections in the optic tcctum. Surgical sectioning of the optic ncrvc t o prevent the reforming of connections trom the retinal ganglia hlocks the increase in nAChR genc cxpressinn. It appears that retinal ganglion cclls arc sti~nul:~tcd to express thcir nAChR genes whcn their terminals reach thcir target4'.
mRNA distribution Ilistrihution o f muscle .subunit mRNAs 09-13-01: T h e mRNA for the nAChR r suhunit in chick latissimus dorsi is
I{)catcti in discrete regions that co-localize with neuromuscular iunctic~ns irlcntifictl I>y histochemical staining for a ~ c t ~ l c h o l i n c s t c r ~ s ~ .
entry 09
mRNA.7 encoding muscle suhunits in Xenopus 09-13-02: The genes encoding the two Xcnopus muscle a suhunits show different patterns of expression. The rl, mRNA is found in skeletal musc!e at all dcvelopmcntal stages and in oocytes: the ctlh mRNA is also exprcsscd throughout muscle development, hut is not dctectahle in oocytesz3.
Distrihut ion of neuron01 suhunit mR NAs 09-13-03: Neuronal nAChR genes show differences in thcir patterns of expression among hrain regions, suggesting that different genes are exprcsscd hy unique suhscts of neuroncs. The sites of cxpression of the genes encoding thc various suhunits of ncuronal nAChRs are shown in Tahlc 3.
Phenotypic expression Frrnction of nAChR in the electric organ o f mnrine rays 09-14-01: The richest naturally occurring sources of the nACh receptor are
the electric organs of the marine electric rays, such as Torpedo californica or T. mnrmorata, or the electric eel, Elcctrophorua. The tissue of the electric or an consists of parallel stacks of flat cells (clcctrocytes or electroplax ), each ccll heinn inncrvatcci on one face hy a cholinergict nerve. Thc stacks of cclls arc such that current cannot flow from one side of a cell to the other. Simultaneous stimulation of the ncrvcs causes depolarization of the innervated faces of each electroplax, producing a potential difference hetween the extracellular fluid of each ccll in the stack. The transcellular potentials across each ccll in the stack add to produce a large electrical discharge, which in the electric eel can hc hundreds of volts.
P
Functional cxpression o f nAChR ~t the neuromusculmr junction 69-14-02: At the ncuromuscular junctiont, the nAChRs in the plasma mcmhrane of the skeletal musclc cells arc activated hy the ACh rclcascd from the motor neurones. The resulting transient opcning of the nAChR cation channcls produces an influx of Na' ions, causing localized dcpolnrizationt of thc musclc ccll mcmhmnc. This dcpolarization opens voltagc-gated Na' channcls, resulting in further Na'-influx and cnhnnccd clcpolarization. The crlnsctlucnt opcning o f ncighhouring voltage-activatcd Na' channels leads to thc self-propagating dcpolarization of the plasma rnemhranc characteristic of thc action patentiali.
Functional expression o f nAChR in neurones 09-14-03 Numcrous ncuronal nAChRs havc hccn rccognizcd by clcctro-
physinlogical methnds, immunological cross-reaction and hiochumical piirificatic~n. The ncuronal channels differ from the Torpccio and mosclc nAChRs by thcir containing only two typcs of suhunit, r ant1 If, and mnst of them arc not sensitive to r-Rgt. Scclllenccs cncodinfi eight ncuronal 2 suhunits and four ncumnal p suhunits havc hccn idcntificd hy molccutnr cloning. In sito l ~ ~ h r i d i z a t i o noft hrain sections with prohcs for diffcrcnt sulwnit mRNAs shows that each r and /I suhunit gene is cxprcssctl in ;1 iliffcrcnt, rc~ionally specific manner. The functions of the diffcrcnt mr>lcculnr spccics of nAChK in the diffcrcnt arcas of thc brain arc ~loorly untlcrstood.
s of Tahle 3. Sitr's oi r>.uprrassionof thc ,ycnc.s c,r~c.ocljr?!: v ( ~ r i o ~ lstrl~units nruronrl! n A C h R s ( F r o n ~09- 1.7-0;3) Suhunit (12
03
(14 0.5
Cell or tissue tvpc
Rcfs 44.44
Literal spirifnrm nllclel~sof chick dicnccphnlon (El l to ncon;~tcl;Ncuroncs d o not cxprcss dctcctnhlc Icvcls of (12 mRNA until cml~ryonicclay I I (El I I, when fihrcs contilining cho1inc'ncctyltr~nsfcr;iscfirst enter thc nuclcus. T h e r;lt 112 prohc highlights ;i s11i~11numhcr of Purkinie cells in the ccrchcll;~rcortcx. It is not known whutlicr this represents ;i distinct su11-popul:itic~nof cclls, or whether ;ill I'urkinie cells c:in tr:insicntly cxprcss the 112- gcnc Chicken ci1i:iry ganglion (EIX: 900 copies mRNA per ".'".47 ncitronc); si~pcriorccrvical ganglion (E10);goldfish rctinnl ganglia I4 Adult chicken ccrchrum, ccrchcllum, optic lohc Chicken ciliary ganglion ( E l % :200-,300 copies mRNA per ncuroncl; human ne~lrnhlastomacell line, IMR .32; Iiunian small cell lung carcinoma cell line, NCI-
"*"
N-592. 11
7
IIX
09
,I2
In . \ i t r ~hyhridizntiont shows that the rat 0 7 gene is
highly cxprcsscd in olfactory regions, the hippocampus, the hypothalamus, the smygdala ;ind the cerehral cortex. Chickcn Ill 3 p;irnsympnthetlc ciliary ganglion I I X O O copies niRNA per nciironc); ;im;lcrinc ;inti ,qi~nglioncells in chick rctin;~cxprcss the 117 suhunir, detected hv immi~nohistochc~mistrv T h c I,% suhunit is the mnior suhtypc in chick retina, wticrc its distribution has hcen stuilictl hv i~nnlunohistochcniistrv (.so(, I'rotc~rr~ cli~tril~rl!ion. 00- 15) Hypnphyseal gland o f the r:it cnihryo at stage El(,: thc m R N h I S restricted t o the pars tuberalis ot thc :icicnoIivpophysis: : ~ l s ofound in the adult r:it pars tiihcmlis, ;it tlic vcntr:il surt;icc ot the median cniincncc. T h c rt'l subunit mRNA is ; ~ l s odctcctnhlc in the E l 6 rat nasal epithelium of the olf;ictory turhinntcs ;inti in t h e skclctal muscle of the tongue of the dcvcloping rat. In s ~ t l rhvhri~lizationof scrinl sections of t h e adult hrain failcd t o ilctcct r,') mRNA. T h e o c ) sul?llnit mRNA can he dctcctrd in r:it cochlea hv RT-PCRi, using 119specific primers' dcsignctl t o span an intron' of the 09 ncnc nntl thus distinguish cDNA from gcnomic LjNA. I n sitrr hvhritlizntion on sections of rnt cochlcn shows that the r t Y gene is cxprcsscd in hoth the inner and outer hair cells of all cochlcnr turns Chick ci1i:iry ganglion ( E l H: 200-.Z00 copies per ncuronc): In the chickcn optic Inhe, expression ot thc .I2 gcnc is incrcasctl xl0-foltl Iwtwccn E 6 ;ind E l l , after which it rapidly declines. I'hc .f1cxprcssion occurs in n rnstrocal~clalgradient and is coincident with invasion of
'."".62
"' "R
"*'
-
Table 3. Continued Suhunit
Ccll or tissuc typc
Rcfs
thc tectum by retinal afferents. Removal of the eye cup at E2 results in >I(]-foltl reduction of expression of i12 compared with controls. This suggcsts that ~j2 cxprcssion in optic lohe neurones is transiently stimulated hy arriving retinal affcrcnts ;j4
Chick ciliary ganglion (E18: 200-300 copies mRNA per neurnne); superior cervical ganglion (EIO);Purkinic cclls of rat cerehcllar cortex
"0.'".49
An autoimmune disease involving antibodies against nAChR 09-14-04: Myasthenia gravis, an autoimmunct discasc charactcrizcd hy muscular weakness and fatipahility, results from a hreakdown in immune tolerance of the nAChR. ~utoantihodiesito the nAChR are found, and immune complexes ( 1 g ~ tand complcmentt) are deposited at the postsynaptic mcmhrancs, causing interfcrencc with and suhsequcnt destruction of the nAChR (rcvicwcd by "1. A numhcr of infcctious agents, including H S V ~ 1" and several cram-ncgativct hactcria".', cncodc molcculcs that immunologically cross-react with thc nAChR r-chain scqucncc and might initiate thc hrcakdown of self-tolerancei. 09-14-05: Immunization of mice with nAChR purified from Torpedo electric
organ causes a disease similar to human myasthenia gravis, tcrmcd experimental autoimmune myasthenia gravis (EAMG), susccptihility to which corrulatcs with thc H-2 liaplotypei. Pepti~lus derived from the murinc musclc nAChR r subunit strongly stimulntu T hclpcri cclls from - immunized H-2d m i ~ c " ~ .
-
Protein distribution Distribution o f nACkRs in adult skeletal muscles 09-15-01: Thc nAChR channels at thc ncuromuscular junctionj, localized hy
immunogold clcctron microscopyt, are conccntratcd at thc crests of the postsynaptic folds and immediately surrounding mcmhranc foIding~',~.
Distrihution o f nAChRs in foetal muscle or denervated adult muscle 09-15-02: In foctal skclctal musclc, the nAChR protein is founri throughout
the muscle cell memhrane. If adult skeletal muscle is denervated, nAChRs arc synthcsizcd and appear throughout thc entire surtacc of the musclc. This increase in cell surface receptors is prcccdcd hy an accumulation of nAChR mRNAs in the muscle sf^.
Distribution of neuronal nAChRs 09-15-03: Many of the nAChRs in hrain appcar to he located on ncrvc
-
terminals, whcrc their role is presumed to he the modulation of transmitter rclcasc. Radioligand-hincling studies in the cat visl~alcortex dcmonstratc the prcscncc of pre-synaptic ~ A C ~ R S " ' Immunochemical . studies with
entry 09
-- - -
monoclonal antihodics against electric organ nAChRs show labelling of t h e lateral spiriform nucleus (SpL) nntl specific lavers of t h e chicken optic tectum, w h ~ c his t h e principal site of termination of SpL ncuroncs ;lnd contains axonT tcrn1inals with nAChR i ~ n m ~ t n o r c a c t i v i t y ~ .
nACllKs on tlic non-xyntlplic .sur/~luc~.s in lhc optic tccrum 09-15-04: Electron microscopic cx;~min;ationof irnmitnr~lnhcllcdnAChRs in
t h e optic tectum of t h e frog shows that t h e rcccptors arc prcscnt o n t h e non-svnaptic surfaccs of vesicle-hearing prc~filcs"". It is suggcstcd that ACh relcasctl from cholincrgict terminals in t h e nucleus isthmi of the optic tcctum hintls nAChRs on retinal affcrcntst and motlitics their release propertiesx.
Suhunits c.ornhinntion,s in ciliary grrn,yliir 09-15-05: At least five ncuronal nAC:hR gcncs,
7.3, 25, 27, /12 and 14, are exprcssctl in chick ciliary ganglia. ~ m m u n o p u r i f i c d ~nAChR from emhryonic chick ciliary ganglia has hccn shown hy Wcstcrn hlottingt with subunit-specific monoclonal nntihotlics t o contain ~ 3r5, ant1 P4 ~ u h u n i t s ' ~ . Antihodv specific for t h c 7.3 s l ~ h u n i tremoves XO'X, of t h r P4 suhunit ant1 7,Z":, of the 75 suhunit from a mcml,r:anc extract; s i ~ ~ ~ i l a ranti-114 ly, rcmovcs .ZXo:, of the .;uhunit ;~ntl 56"1, of ttic 7.5 suhunit. Sequential immunonffinityi purification ot t h e rcccptors using anti-23 followctl hy anti-114 nntihodics viclds receptors that contain sul,stanti;al a m o u n t s of t h e 75 gcnc p r o d ~ ~ cThese t. findings support t h e conclusion that n significant proportion of t h c receptors fro111 synaptic sitcs in chick ciliary ganglia cont;ain t h e co-;asscmhlcd 7.3, YS and /I4 suhitnits. T h e same n A C h receptors lack t h c 77 suhunit, Ilut this is prcscnt in t h c distingitishahlc, r-Rgt-hiniling nAChII from non-svn;aptic sitcs in chick ciliarv ganRli;~"'.
Ncrironcll ( 1 7 .suhzir?it.s 09-15-00: T h e scrccninv of a chick brain c D N A lihrary with synthetic
oligonuclcc~ticlc~p r o h c s hascd o n N-terminal pcptidc sequences of a n 1hungarotoxin-hinding protein sithunit Icd t o t h e ist~lationof a c D N A clonc encoding ;I novel Y . ; ~ ~ l > i ~tcrmcd nit, 77'".
l)rc\~alcncc~ o f ( 1 7 .sr~l~unit,s ill nAChI1 from rcri~hrlhrm 09-15-07: T h e receptor affinity purificti fro111 chick cerebellum hv hintling t o Y-hung;~rotc~xin contiains ;at least three suhunits c ~ :Ippnrcnt f mol. w t 51000, 5 7 0 0 0 ant1 67000. T h e i ~ s coi monocl(~nalantihotlics specific for t h c 1 7 suhunit ticmonstratcd that 75% of t h e I ~ I ~ I C C I I I C S prcscnt in t h e purified prcpnration ;Ire of t h e 27 suhtvpc nntl that this antihodv lahcls t h e 5 7 0 0 0 I,;~nd in ;I Wcstcrn hloti. Rcconstmction cxpcrimcnts in planar lipid I>il;~versshow t h ; ~ t thi.; 7-llockcd hy (~t)-tnhoc~~rarine"'.
Ilistrihution o f niluronal 08 .suhut?its 09-15-08: Low-stringcncv scrcrniny: of
-
;I chick hrain c D N A library with n cl)NA prohc for t h e 77 suhunit rcvcalcrl ;I second cDNA clonc encotling :I tlistingt~i.;hnhlc 2-rcl;~tedsuhttnit, now tcrlncrl r ~ ' " . f m m u n o p r c c i p i t ~ i o n t ; ~ n d i r n n i i ~ ~ i o h i . ; t o c t ~ ~ n ~ i s li;~vc t r v ' icicntificd n nAChK sithtvpc that
entry 09
contains 28 suhunits, hut not a7 suhunits, as the maior suhtype in chick retina. This suhtype has a lower affinity for 2-Rgt than does the subtype containing only '27 suhunits. The suhtype containing only a7 suhunits compriscs 14% of the a-Rgt-scnsitivc nAChRs in hatchling chick retina. The suhtypc containing r8 suhunits (hut n o 27 suhunits) accounted for 69%, and thc a7z8 suhtypc accountcd for 17%"2. 09-15-09: Amacrine, bipolar, and ganglion cells display r8 suhunit immuno-
reactivity, and a complex pattern of labelling is evident in both the inner and outer plexiform layers. In contrast, only amacrinc and ganglion cells cxhihit 27 suhunit imniunorcactivity, and the pattcrn of r 7 dctcction in thc inner plcxiform layer differs from that of a8 suhunit lahclling. Thcsc dispritics suggest that tho z-Rgt-sensitive nAChR suhunits arc differentially cxprcsscd by different populations of rctinal neurones. In addition, the distribution of sc-Rgt-scnsitivc nAChR suhunit immunorcactivity differs from that of 2-Bgt-insensitive nAChR suhunits".
Suhcellular locations Subcell~rlarlocation o f nAChR in ciliary ,panglion neurones 09-16-01: Two classes of nAChR havc hccn itlcntificrl on chick ciliary ganglion neurones, where they occupy diffcrcnt suhccllular locations. One class is concentrated in post-synaptic membrane and is rcsponsihlc for mediating synaptic transmission through the ganglion. The other, which hinds r-hungarotoxin, is locatcd predominantly in non-synaptic membrane",'.
Transcript size 09-17-01: The sizes of the mRNAs cncndinp. suhunits of the nAChR arc as
f(1llows: z suhunit
/lsuhunit
;. suhunit ii suhunit r 5 suhunit
r 7 suhunit
(T. cnli{ornicn) (mousc) (chickcn) (T. coliiorniorr) (T. ciiliiornica) (T. califomica) (mouscl (human) (chickcn) (human cell line)
2.8 kh' 1.8 kt?' 2.8 khf4 2.0 kh" 2.1 kh" 6.0 kh" 3.3 kh"' 2.7 kh and 2.1 kh2' 7 kh (major)and 3 kh (minor]" 5.9 kh, 2.6 kh and 1 .,? khZry
Chromosomal location Location o f gcnes en cod in,^ n ~ ~ ~ ssuhunits cle in the mouse 09-18-01: The chromosomal locations of the gcncs encoding four muscle
suhunits of the nAChR in thc mousc havc hccn dctcrminctl hy R F L P ~
entry 0 9
n
-
analysis of DNA from crosses hctwccn Mu\. n~l~sc-nlis tiornr*stic.~~s (DRA/2) and MIIS.sprrP!lrs(SPEI. T h e r gene maps to chromosome 17, the If-gcnc to c h m m c ~ s o ~ n11 c and t h r ;* ant1 rF genes arc closely linked on mouse chromosome I '".
Encoding 09-19-01: Sec Cknc f(in?iJy,09-05, for ( I list o f the pmtrlns encoded hv
the ,yenes of thc nAC17R f n n ~ i kin . vnr~oussprcicr Cl(lssificntiono f suhunits
( I S (t
or
.j
09-19-02: Assignment of a neuron;ll subunit to the ' 7 ' class is haset! on the conservation of the adiaccnt Cys residues at the positions h o r n c ~ l o ~ o utos
Cysl92 and Cysl9.3 of the Torpr,tlo Y suhunitl. Subunits lacking the two adiaccnt Cys rcsitlucs arc generally designated P, hilt investigators working with scrlucnccs from chick and goldfish prefer the term 'non-r'
Mrlsclr subunits rc.cluircd for func.tion 09-19-03: Fully functional nAChli ch:lnncls arc cxprcssctl hy the co-injection of t l ~ ctour cRNAs cncclding the 7, / I , ;* ant1 (5 sl~htlnitsof the musclc channel into Xrnopris oocytcs(". When combinations of RNAs cncc~dingonly a s u t ~ s e t
of t h r suhi~nitsarc inicctctl, the Y suhunit is essential for activity, t o ~ c t h c r with either thc ;*or 15 subunit: the /i suhunits are dispcns;lhlc"6~"'. 09-19-04: Six different comhinntions of thrrc or more suhunit RNAs produce
significant n ~ ~ m l ~ eofr sfunction;il channcls. T h e order of combinations yielding the greatest ;imount of current is rk. -. y/hi rrSt: r15;)r. rtS > T;,. T h e extent t o which a channel type with three different subunits is cxprcssctl is highly dependent upon the ratios of RNAs coding for thc diffcrrnt suhunits ;ind is critic:iIIy tlcprndcnt upon the order of inicction of thc K N A S ~ ~ .
-.
Nr~umnol.sul?units rcquircd for chclnnel f~rnction 09-19-05: Thc RNAs encoding only two nruml suhunits, r and non-lx (alsr~ called /!I, arc sufficient t o encodc functional channels In Xcnopus oc~c~tcs'. Eight distinct Y ant1 four diffcrrnt / I suhunits havc hccn itlcntifictl in ncuronal t i s s ~ ~ cNrural s. /{ suhunits can substitute for muscle /I suhunits in
forming functional channcls in Xcnopuq oocytcsl". 09-19-06: At least five ncuronal nAChR genes, 7.3, 75, r?, /l2 and P4, arc
cxprcsscrl in chick ciliary ganglia. ImmunopurificdT nAChR from crn1,ryonic chick ciliary gangli:~has I,ccn shown hy Western blottingt with subunit-specific monoclonal antihotlics to contain 7.3, 25 ant1 P4 s u l ~ i ~ n i t s " ~ /.SPL, I'roti,1n (l~,s!ril?t~! 1011, 09- 15).
Thc~( 1 7, ( 1 X (lnd 0 9 subunits form homo-oli~yort~cric ch(lnnc:ls 09-19-07: Thc chick 77 and 78 ant1 the mt 70 nAChR suhunits asscmhlc into
functional homo-oligomcrid channels, responding t o acetylcholine, whcn thc corresponding cRNAs arc singly inicctcd into Xcnopus oocytes17~"7v2'R.
cntry O'I
-
Most rv-R,qt-bindingproteins in hrain contain the ,r 7 suI?unit 09-19-08:The mature r 7 protein (479residues) has modcratc homology with all other 1 and non-1 nAChR suhunits and prohahly assumes the same transmemhrane topography. A hactcrial fusion protein containing residues 124-2.39 of 17 hinds lahelled z - ~ g t ' ~In. sit11 hybridizationt maps of 17 mRNA closcly rescmhle the pattern of ['2'~1-r-~gt hinding in rat hrain, sug~cstingthat most rRgt-binding proteins in thc tissue contain 1 7 suhunits. The rR suhunits occur less commonly, representing only 15% of the I-Rn-hintling complexes and tcnding to he associated with the morc abundant 17 s~~hun~ts''.
nAChH from insect CNS 09-19-09:A nAChR purified from thc cockroach CNS hy a-Rgt-binding has an ovcmll size of ahout 300 kDa, hut produces a single hand of 65 kDa on denaturing gels"v. Reconstitution into lipid hiIaycrs prnduccs channels that are gatcahlc hy nicotinic agonists and blocked by thc antagonist, (+)-tubocurarine7". Sequences encoding a cockroach suhunit, zL1, havc bccn cloncd ant1 expressed in Xenopus oocytes, where they specify functional channels gated hy nicotine and hlockcd hy a-Rgt and n - ~ g t . "
Subunit composition o f functional neuront~lnAChR channels 09-19-10: The singlc-channel conductancrt and currcnt arnplitudct of ncuronal nAChR can he manipulated hy c h a n ~ i n gthe chargccl rcsidilcs immctliatcly downstrcsm of the M2 region. Changc of E266 to K in the ~4 suhunit rcduccs thc single-channel conductancc nf z4/[11 channels, while change of the analogous residue (K260) in pl to E incrcsscs the singlcchannel conductancc. When a comhination of cDNAs encoding n4E266 and r4K266 is co-injected with cDNA encoding plE260 into Xcnoplls oocytc nuclei, channels with three different amplitudes arc detectcct in insidc-out patchcs7'. This finding is the prediction if the functional nAChR contains two z suhunits.
Evidence for the pentnmeric nature o f neuronr~lnAChR 09-19-11: Similar experiments co-iniccting cDNAs specifying two distinguishnhlc P suhunits plus onc 1 suhunit ([I!, lIlE260 plus 14) result in four distinguishahlc current amplitudes, as predicted if thcrc were three /i suhunits per functional channel. Thus the functionnl ncuronal nAChR is a pcntarncr, of composition z2/i,372.
-
n
09-19-12: When the nAChRs synthesized following injection of chicken r 4 and /{2 mRNAs into Xennpus oocytes arc la!>cllcd with [,3S~]-methic~ninc, 1.46 times morc lahel is found in /I suhunits than in r suhunits, after correction for their methioninc content7-'. This ratio is very close to the value of 1 .S expected for a stoichiomt.tryt of .x2/i,1.
Gene organization Introns and exons in the
tu
subunit genes
-
-
09-20-01:The chickeni4 and human'" 1genes havc nine coding exonst. In the human r gcne, the lengths of the eight intronst arc 4 . 9 kh, 1 1 1 hp, 1.7 kh, 3 . 1 kh, 0.4 kh, 3.4 kh, 1.2 kb and ,324 hp in the 5' tn 3' directinn'".
-
-
-
-
Intron-rxon structure o f the 02, r r 3 09-20-02 T h e chickcn 22,
y.3,
nrlrI
r r 4 suhunit genes
34 and nan-x gcncs and the rat r2 and 33 gcncs
all have six p r o t e ~ n - c n c n d i ncxonsr, ~ the fifth of whtch 1s large and cnccldcs prntcin sequences h o m o l ( i ~ o u sto th(lse s p c c ~ f ~ chy d cxnns 5 through 8 of the r gcnc'J. T h e pnsltmns of the exnn-intmn houndarics in 12-75 anti nx 1 -nr
Intron-exon structure o f the o 7 sr~hrlnrtxeric. 09-20-03: T h e c h ~ c kr7 gcnc c o n t a ~ n s 10 exonst, the frrst four of w h ~ h cxnctly match thc c t ~ r r u s p n n d i ncxons ~ In o t l ~ c rnAChR subunit T h e other slx cxans do not corrcspontl to any of thc cxons In the other rnu.;clu or ncuronnl nAChR jicncs.
Intron-cxon strr~cfurcof t h e r k 9 suhurlit gene 09-20-04: T h e mouqe gcne encoding the xY suhunit of the nAChR has fivc cxnns s n d an intmn-cxon structure that ciiffcrs fram that of all nthcr known nAChR genes. In contrast wlth othcr nAChR gcncs In which thc tntron-cxon hoi~ntlnricsof thc f ~ r s tfour cxons arc cc~nservcd (SCC II~>OVL'], cxons Ill and 1V in the xY gcnv nrc ft~.;ctl"~. [ N o t e . T h c mtron-cxon structurc c ~ fthc r 9 gcnc wnr rlctcriil~nctlhy coniparlnK r;lt cI3NA scqucncc wath n>otrscbgcnomic scquuncu2IH , so that the information presently available strictly appllcs only to thc tnotlrc rY gunc.]
Intron-exon structure of the h ond 3 suhunit Kenems 09-20-05: T h e chick gcncs encoding thc ii ant! ;. s u l > ~ ~ n both i t s contain 12 exonsl. Thc hornol(~gouscxons of the t w t ~gcncs arc vcry similnr in sizc and thc splice sites1 arc ~ x a c t l yconserved. The corrcsprlntlinp, intronsi (if the two gcncs differ sharply in length anti ~ c ~ u c n c c T' ~h c. two gcncs arc vcry closuly linkcd in the chickcn genomc, with only 740 hp I>ctwccn the last cod(ln c ~ fthe ii gene and the tmnslation-initiation codon of the ;. gcne. T h e intergenic region cclntnins n single cnnonical polyadenylationt site, 77 hp downstrcatn ot thc 6 gcnc tmnslntinn-tcrininnt~oncodon''. TIlc human ~5 gcnc ;11so contains 12 cxons, :in
The ( 1 stlhunit ,qene promntcr confcr.s tissue specificity 09-20-06: A rcgion c ~ upstrcarnt f scqucncc of thc chickcn
,Isubunit gcnc lying I~ctwccn 1 10 nnJ 45, was shnwn to confer tissuc- and stagc-specific Rcne tmnsfcction into chickcn primary expression on a rcportcr gcnci f{~ll(~wing myotuhcs cjr thc mouse C2.7 rnyogcnic cell line75. This region ctf DNA interacts with several nuclear proteins from rnusclc cells nntl diftcrentiatcd myotuhcs, including an ~ ~ 1 * - 1 i factor ke and a G stretch-hinding protein, which hind to overlapping sites irnmctliatcly upstrcam of thc T A T A ~ box. Several protcins intcractinl: with n s c r ~ ~ ~ c nsimilar cc to thc SV40 crwc cnh;inccrt appear during in vitrtl diffcrcnti;~tion c ~ t myohlasts into tnyotuhcs, nntl thc concentration of sonic of these increases nftcr dencrvation t)f Icg rnusclc in ncwl,orn chicksT5. -
cntry 09
-
The h subunit gcne promoter 09-20-07: The romotcr rcgion of the chicken nAChR (5 suhunit genc lacks typical TATA and CCAAT~hoxes, and transcription starts at six major and seven minor sites hctwecn -1 10 and -.30 with respect to the translation-initiation sitc. T w o sites, at positions -77 and -66, givc risc to ahout 50% of all transcripts. A transcription enhancert was located hy dcletion mappinRi to the rcgion from -207 to - 146. The enhancer is active in fihrohlasts and diffcrentiatcd musclc cells, hut not in myohlasts7'.
f
The transcription-start site for the h genc 09-20-08: Thc transcription-start site for thc mousc (5 gcne has heen mappcd 55 hp upstrcam of thc tmnslation-initiation codon. A scqucncc TAAACCA at positions -33 to -27 rclativc to the transcription-start sitc is presumed to servc as thc TATA hoxi, and a CATTG sequence, complementary to the C A A T C ~ hox, occupics -66 t o -62. CG-rich scquences having homology with known AP-2t-binding sites are centred at positions -55, -180 and -210~~. A sequence in the h-gene promoter necessary for muscle-specfic expression 09-20-09: A 54 hp rcgion of 5'-flanking DNA from the murinu gcnc encoding the ii suhunit, occupying - 148 to -95, is ncccssary and sufficient for musclespecific gene-expression. Deletion of this sequcncc results in a 50-fold rcduction in cxprcssinn in myotuhcs, whilc fusion of the scqucncc upstream of thc c-fos hasal promotcr confers myotuhc-spccific gcnc expressinn on an othcrwisc wcak, non-tissuc-specific promoter. Thc musclc-specific transcription factor MyoDl d c ~ snot hind to the 54 hp rcgion, hut other nuclear proteins from myotuhcs arc ahlc to hind this clument7'.
Consensus sequences for muscle-specific regulatory motifs in the promoters of genes encoding nACI?R suhunits 09-20-10: Thc consensus sequcnccs for nAChR gcne enhancers contain scvcral motifs that havc previously hccn implicated in tissue-spccific gcnc cxprcssion. Thcrc arc two copics of the CANNTG motif prcscnt in t h e MyoDl target scqucncc characteristic of many musclc-specific regulatory regions: thcsc flank n 'M-CAT' motif and an overlapping TGCCTGG scrluencc, both of which havc hecn proposed as muscle-specific regulatory motifs7'* 74. 1
A sequence common to several nAChR suhunit sene promoters 09-20-11: A sequcncc common to the chickcn z gcne and the mousc PI ;-and (1 gcncs has hccn termed the suhunit homologous upstrcam clement (SHUEJ box. The 13 hp SHUE hnx scqucn<e, all four copics of-which contain CCCTGG/C, is locatcd at -15.5 in the mousc ii gene and -75 in the chickcn sr gene7'. The function of thc SHUE hox has not hccn dctcrmincd.
I
Close Iinknge o f the h and 7 suhnnit gencs 09-20-12: Thc penes cncotlinc the ;iand :-subunits arc vcrv closclv linked in
the chickcn, mouse and human gcnomcs. T h e ;, gene lies 740 hp downstrcam of the ;igene in chicken" and 5000 hp downstream in mouse7'. This, t o ~ e t h c r with the high degree of sequence homcllogy and identical exnn-intmn structure, supports the idea that the two Rencs havc undergone a rclativcly recent tandem duplicatinnlR. T h e human ;, and 6 gcncs can he isolated on a single genomic DNA fragment of 15.7 kh, with B upstreamt of the y
Clr~sterin~ of the no,?, 03 r~nd05 genes in chicken 09-20-13: The threc gcncs encoding the nr3, 7.7 and r 5 suhunits arc clustered in thc chicken genome. Gene nr;i lies 5' of r.3 and is tmnscrihcd in the same .rlircction, whcrcas r 5 is located on the %?'sidec ~ x.3 f and is tr;~nscrihedfrom thc c~ppositcDNA strand. T h e 112.3 and 23 genes arc separated hy ahout 5 kh, and 2.3 anti r 5 hy less than I kh'". There is also an 13 gene cluster, containing gcncs encoding P4, 2.3 and 15 snhunits, in the mt genome7.
Homologous isoforms Homology amongst n~usclrsuhnnits 09-21-01: The musclc r, /I, *; and (isuhunits show scqllcncc similarities with
cach other and with suhunits from othcr channcl types, including the mouse serotonin-gated ion channcl and several glvcinc and GARA receptors. T h e {r' suhunits show approximately 42-45"!, scqucncc idcntity, the ii suhunits havc ahout 405, idcntity and the ;, suhunits .l7-40"/n idcntity with the r suhunits.
Homolo~yhetwccn
(111
nAChR subunits
09-21-02: There is strong conservation of amino acid scqucncc amnngst all
known suhunits c ~ fthe nAChR. This conservation is not evenly distrihutcd throughout the polypeptide chains, hcing strongest in the region that includes the transmemhranc domains M I , M2 and M,Z (see Domain ~.c~nscrvrttio~l, 09-2XI.T h e percentages of amino acid idcntity hetwecn the suhunits of the muscle and ncuronnl nAChRs are shown in Tahlc 4.
Xcnopus mrrscle n suhunits 09-21-03: T h e two Xrnol~upmusclc r isoforms are 89'3'0 similar t o cach othcr,
and they show similar levels of similarity (XS-XY%) to the mammalian or T(>rprdcT suhunit2,'.
Protein molecular weight (purified) The nAChR purified from Torpedo electric orgnn 09-22-01: The nAChR purificd from Torpedo electric organ is a 250 kDa pentamer, formed hv four different types of subunits ( r ,1,;., dl with x2P;d stoi-
chic~mctr~".The individual suhunits, cach c ~ fwhich is glycosylatcd~,have apparent molecular masses of 40 (71, 50 (111, 60 (;.I and 6.5 (h) k ~ a ' ' +
Thr: chicken hrnin nAChli 09-22-02: An nAChR i r n r n ~ n o ~ u r i f i e dfrom t chickcn hrain is cnnstitutcd of
subunits of 49 k n a and 59 kDa mcasurcd hy elcctrnphorctic mohility on
Table 4. Amino acid identity (%) between nAChR subunits (From 09-21-02) n
h
36"
n2
03
n4
n5
n6
a7
32'
08
.I
,?2
,'33
,
j4
f
41"
Footnotes quoted are the percentage amino acid identities between different subunits of rat, chicken ('1 or Torpedo ("1 nAChR subunits.
entry 0)'
-
denaturing gelsR2. Antihodics raised against this material arc ahlc to i m m u n t ~ p r c c i ~ i t arcccptctrs t~ containing 49 kiln and 75 kDa components. In cach case, thc l a r ~ c rs ~ t h u n i t sarc affinity lahcllcdt hy ('HI-MRTA and havc N-terminalt sequcnccs homol(~goils to thosc of r suhunitsH'. Subsequent studies havc rcvealud that thc N-terminal scqucncu c ~ fthe 75 kDa hand corrcspnnds to that of thc r 4 suhunitx" and the N-terminal scqucncc of the 4Y kDa hand to that of thc 112 sul,unitH5. T h e 59 kD8 hand prohahly includes hoth 12 anrl 3.7 su huni tsHh. 09-22-03: T h c 2 7 suhunit irnn~unr~pitrifiedtfrom chickcn h r ~ i nhas an apparcnt M , nf 57 k n a r,n rlr-nnturing gr.lsH7.
l'urificd nAChK from c h i c k e n c i l i ( ~ r vg n n ~ l i n 09-22-04: Purification of nAChRs from extracts of chickcn ciliary ganglia yicl(ls a fr;lction showing thrcc componcnts on denaturing gels, at 40, 52 and 60 knaRH.Thcsc havc hccn identified, using subunit-specific antihodics, as the 2.5, if4 and 2.7 suhunits, rcspcctivclv. It has not hccn cstahlishcd that thcsc components asscmhlc into a singlc nAChR.
The nAChR pzrrified from rnt l ~ r r ~ i n 09-22-05: An nAChR immunopurificd from rat hrain with anti-chicken nAChl< mAh 270 contained sul>units of apparent M, 52 and 80 kDaHV.T h c N-terminal scqucncc c ~ the f XO kIla suhunit corresponds to that of ~ 7 and 4 ~ that of the 52 kDa suhunit to the N-terminal scqucncc of the P2 suhunitH5. Antihodics to thcsc two components rcmnvc ; ,90% of the high-affinity ['HI-nicotine- or I . ' ~ 1 - c ~ t i s i n c - h i n d isites n ~ from detergent-soluhilizcd rat hrain cxtracts, sumcsting that nAChRs cnmprised of 74 and P2 suhunits arc the maioritv spccics in mt hrainY1.
Protein molecular weight (calc.) 09-23-01:Srv, r3(1frr I I I TrlJ~lr'I r1ndr.r (;c8nc f(1rn11v.00-05
Southerns Sout h e r n b l o t s o f c h i c k e n g e n o r n i c D N A 09-25-01: Rlots of chicken gcnomic DNA digested with EcoRI, RrlrnHI and ffinti111 showcd singlc hantls hybridizing t o 5- and ;,-specific probes. T h e two prnhes rcvcal the samc-sizcrl EcoR1 hand, hut distinct hands on an EcoR1-Hind111 double-digest, consistent with the two gencs hcing very 09-20) closcl y linkcd on n singlc Er.oRI fragmcn t (sce Cknc r~r~qclniz(~tion. and with thcir hcing untquc in tlir chicken gcnomclR. Southern rlnrn!y51,s of Xcnopus ,pcnornrc D N A 09-25-02: Southern blot4 of Xcnoptr\ gcnomic DNA prohcd with 7 I , and x 1 h prohcs l n d ~ c a t cthe prcscncc c ~ two f dlftcrcnt Rcncs, cach prcscnt in a ~ ~ n g l c copv pcr gcnnmcz'.
entry 09
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Domain arrangement Transmembrane domains 09-27-01: Each nAChR suhunit contains a large, hydrophilict, extracellular N-terminal domain, four hydrophohict transmcmhrane domains and a short, extraccllular C-terminal region. The scqucnccs of the four strongly hydrophobic scgmcnts (MI-M4) and that of a rcgion that suggests an amphipathict helix (MA) arc strongly conscrvcd amongst different suhunits and across species. Covalent lahclling of all four scgmcnts with photoreactivet phospholipids supports thc contention that the MI-M4 regions arc mcrnhr;~nc-spnnnin~~*.~~'.
Secondary structure of transmembrane domains 09-27-02:There is no direct evidence that MI-M4 arc z-helical and they are at least candidates for fi-sheet-formers according t o secondary structure prediction algorithmsA".
The MI domain is occessihle to open-channel blockers 09-27-03: The proposed open-channel structure of the nAChR s u ~ e s t that s the putative r-helix MI is exposed at the interstices (clefts] hctwccn the M2 helices. This interpretation is supported hy lahclling of MI helices with open-channel hlockcrs such as quinacrine (see relsv".v7).
Domain conservation Conserved Cys residues in all subunits 09-28-01:All the suhunit genes encode proteins with two cysteincs separated hy 1.3 residues that align with C128 ant1 C142 of the muscle z suhunit. Inv~iriantCys I92 and 193 in tr snhunits 09-28-02: All r suhunits from muscle anrl electric organ have adiacent cystcines at positions corresponding to musclc 1 192 and 193. Neuronal suhunits with this feature arc designated as 2 suhunits; those without it arc designated non-T lavian species) or fl (mammalian species). (Note that the desi,r.nation '11' docs not mean that such a subunit most closelv resembles the musclc p subunit. The neuronf~l/Isnhunits reprc.scnt memhers of o hetero*qeneorrs Rrorrp united hy their common Irlck of the two adincent cvsl r i n ~ s ! ) Similarities between mr~scleand ncuronal suhunits 09-28-03: Thcre is approximately 60% identity hctween the musclc and neural .x suhunits over the first 320 residues and in the MA and M4 regions near the C-tcrmini. The ncuml sequences contain an insertion of 60-160 amino acid residues in thc cytoplasmic region hctwccn M3 and MA. The similarity between muscle and ncural non-z sequences is slightly less - (about 44% in the first 350 r c s i d u c ~ ) ~ " .
entry 09
Overall sequence conservntjon 09-28-04:There is striking conservation of sequence amongst all the suhunits of nAChR channels. Amongst the first 320 amino acids, one-third arc conscrvcd. The strclngcst conscrvation is hetween rcsiducs 224 and 320, which includes the M1, M2 and M3 regions, where there is ahout 50% identity. Thcrc is also ah?ut 25% similarity in the first 223 rcsiducs that constitute the N-tcrminali cxtraccllular domain. The conscrvation is much less for the large cytoplasmic domain hetween M3 and ~ 4 ~ " .
An invariant Pro-Cys in M I 09-28-05:Thcrc is an invariant Pro-Cys (221 and 222) in the centre of the MI helices of the 2- and non-r-suhunits of the nAChR. The Pro will introduce a bend of ahout 20 into an z-hclixi, disrupting local hydrogen-hondingt and leaving amidc and carhonyl groups free to interact with water or a permeating iongH. It has heen s u ~ c s t c dthat the Pro may he a focus for conformational changes involving cis-trans isomerizationT of the peptide bondH".
Similarity bet ween nAChR and ryanodine receptor 09-28-06:The region encompassing segments M2 and M.3 of the nAChR show some sequence similarity with the M2-M
Domain functions (predicted) Acet~~lcholine-hindjng sites on the n snhunits 09-29-01: The acetylcholine-binding sites arc primarily located on the r suhunits, though since the two sites are not equivalent they may also involve residues on other suhunits"". 09-29-02: Amino acid residues involved in forming the hinding site have heen identified by covalcnt affinity l a h c ~ l i n ~ t ' ~and ~ ~ "hy~ site-specific mutagenesist"" to he Tyrl90, Cys192, Cys193 and Tyr198 on the Torpedo z suhunit. Thcsc residues, which arc conservccl amongst all known r suhunits, lie within 10 of a carhoxylate group that is involved in hinding the quaternary ammonium group in acetylcholine.
Aromutic residues implico ted in ACh binding 09-29-03: In vitro mutagcncsist experiments have implicated aromatict rcsiducs in the vicinity of the two Cys rcsiducs (192 and 193) as being part of the ACh-hinding siteJn2, or of heing involved in the coupling of agonist
binding to channel a~tivation'"~.
-
09-29-04: Two other peptide loops, one including Trp86 and Tyr9.3 and the other Trpl49 and TyrlS1, are also acccssihlc to hound affinity lignnds'"4*J"". Mutations suhstituting Phc rcsiducs for the amino acids homologous to TyrY.3, Trpl49 or TyrlPO in the neuronal homo-oligomeric x7 receptor decreased the apparent affinity for acetylcholine 10-100-fold, as well as affecting the binding of r - ~ c t ' " ~ .
-
Acid residues on the 6 and E subunits are important for ACh binding 09-29-05: ACh and all other potent agonists and competitive antagonists contain a positively charged quaternary ammoniumi group. Thcrc arc negatively charged residues in the 6 subunit sufficiently close to zCys192/ Cys193 to contribute to the binding of ACh. These were first located within the Torpedo 15 subunit between residues 164 and 224 by usc of a cross-linkingt agent that reacts with sulphydryls at onc end and with carhoxyls a t the other1"'. Each of the 12 acidic residucs in this region of the mouse 6 subunit was changcd to the corresponding amide. The mutation 6D180N led to a 100-fold decrease, and the change cE18YQ to a 10-fold dccrcasc, in thc apparent affinity of the receptor for AC~'"'.
ACh-binding sites are at the interfaces between subunits 09-29-06: Taken together, thc data suggest that the two non-identical AChbinding sites are formed at the interfaces between the z and ;and the z and (5 subunits. Residues identical t o iiAspl80 and iSGIu189 are prcscnt in all 6, 7 and r: subunits, and this conservation is consistent with the location of these residues at the ACh-binding sitcs. In contrast, in all muscle-type P subunits, His or Asn corresponds to cTAsplR0 and Gln aligns with dClu189. This may account for thc inability of P subunits to form an ACh-binding sitc with z " " ~ . ~ ~ .
rrTyrl90, rrTyr198 and rrAsp200 are involved in the coupling o f ACh binding to channel opening 09-29-07: Wild-type Torpedo nAChRs expressed in Xenopus oocytcs are half-
maximally activated ( K l l z Jby 20 JIM acetylcholine with a Hill coefficient of 1.9. Substitution of rY190 and zY198 with Phe residucs (zY190F, zY198F) or aD200 with Asn (zD200N) altered the K I l 2 to 408, 117 and 75 ,IM, respectively, with no effect on the Hill coefficientt.
Mutant receptors with altered response to partial agonists 09-29-08: The above mutant receptors show altered responses to the partial agonists phenyltrimethylammonium (PTMA) and tetramethylammonium (TMA). While wild-type receptors are half-maximally activated by 73 J(M PTMA and 2 mM TMA, zY190F, zY198F, and rD200N receptors are not activated by FTMA and TMA by concentrations of up to 500 JIM or 5 mM, respectively. However, PTMA and TMA bind to the mutant receptors with the same affinity as to the wild-type, acting as competitive antagonists. The rY190F, (xY19RF and zD200N mutations thus have their major effect on the coupling of ligand binding to opening of the channel"".
cr-Bgt-bindingsite: (rCys128 and rrCys142 are essential for rr-Rgt binding 09-29-09:In addition to the two Cys residucs (192 and 193)conscrved in the 2 subunit sequences, two other cysteincs (C128 and C142 in z subunits) are found at homologous positions in all AChR suhunits, as well as in GARA and glycine receptors. Mutant forms of thc T. californica nAChR with substitutions of Ser for Cys128 or Cys142 in either the r or /Isubunits are able to associate with other normal subunits, although the efficiency of
entry OY
association of thc mutant r s i ~ h i ~ n iwith t s the (5 subunit is rcduccdzr'. T h e mutations in the a suhunit abolish dctcctahle r-Rgt hinding in whole oocytcs, whereas the mutations in the fl suhunit result in dccrcnsetl total h i n d i n ~of a-Rgt and n o dctcctahlc surface hinding. 09-29-10: All the subunits mut;ltcd ;lt residues 128 or 142, when co-exprcsscd with the other normal suhunit.; in Xcnopt~soocytcs, produce small acctylcholinc-activated currents. (This ohscrvation is at variance with carlicr studies"' where mutation of C128 or C142 aholishcd the rcsponsc of thc rcccptor to ACh.) T h e functional ACh-gated channel formcd with mutant T suhunits, hut not rnut:lnt /I suhunits, is not blocked by r-Rgt. Thus, 3 rlisulphidc bondi hctwccn Cys128 and Cys142 of the nAChR a o r P suhunits is not essential for acetylcholine hinding, hut this rlisulphidc hnnd on the r subunit is important for formation of the a-Rgt-binding site"'.
Determinr~tinnof thr Ioctltion o f the o-Rgt-hind in^ site 09-29-1 1: T h e location of the a-Rgt-hinding site in the Torpcmdo 1 suhunit has hccn determined hy assaying the hinding of the Iahcllcrl toxin to overlapping synthetic pcptidcs cach of 20 :~niinoacids. The pcptide 2181-200 is by far the most efficient in hinding t h r toxin, with 255-74 also showing significant
binding"^'.
Thc influence of thc oxidation strite of oCys192/Cvs193 on rv-R,pt hindin#y 09-29-12: T h e importance of the potential disulphidc link hctwccn the ndiacrnt Cys rcsiducs a192 and 19.3 for a-Rgt hinding can hc asscsscd fn)m tlic effects of chemical oxidation, reduction and cnrhoxymcthylation~of the ~ 1 8 1 - 2 0 0 pcptidc on the efficiency of hinding. Neither oxiristion nor reduction of the Cvs rcsirlucs ;~ffccts ~ R g thinding, hut binding is tlmstically rcrluccd hy carhoxymcthylation of the reduced -SH groupsi'*. Clearly the Cys192-Cys193 disulphide hridge is not important for the r Rgt-binding reaction, hut hinriing I.; affcctcil hy modification of thcsc two Cys rcsirlucs. Thcsc findings suggest that the hinrling sites for acctylcholinc ant1 r-Hgt ;Ire vcrv close to cach other anrl must ovcrl:~p.
Affinity-lohellin!: of rcsiduc~sin t hc porc 09-29-13: Affinity-1;thcllinl: tcchniqucst togcthcr with thnsc of site-directed Inutsgcncqist and heternlogous expression have helped define the structure o f the ~ A C ~ R " ' . T h e transmcmhranc domain M2 has hccn shown to lahcl with compounds known to hind within the pore"".'". Noncompetitive inhibitors hind with high specificity to a dcfincd region of M2 of several subunits, particularly Scr, Thr ant1 Leu rcsiducs within thc consensus MTLSINVLL ScrIiIcncc at the hcginning of M2'".
Evidence for the o-hclicnl nnturc of M2 09-29-14: The cholincrgict nnn-cornpctitivc~antagonist+ 3-(triflunramethy1)3-(m-('25~1-iodnph~nyl)dia~irine ( 1 ' 2 5 ~ 1 - ~ hinds ~ ~ ) to thc nAChR with similar affinity in thc prcscncc ;ind ahscncc of 50 /!M carl~amylcholint.,and acts as a pl~otoaff~nityi 1;tbcl. In the ahscncc of agonist, I ' ' G ~specifically l - ~ ~ ~ lahcls
cntry 09
-
homologous aliphatic+ residues (PL257, iiL265, PV26l and iSV269) in the M2 rcgion. In the presence of agonist, lahelling of these residues is reduced approximately YO%, and the distrihution of lahelled residucs is h y d c n e d to include a homologous set of serinc residucs at thc N-terminus1 of M2. In the /I suhunit, residucs PS250, /iS254, pL257 and PV261 arc all lahellcd in thc prcscncc of cnrhamylcholine. This pattern of lahelling supports an 2helical model for M2, with the labelled facc fnrming thc lumeni of thc ion channcl.
Agonist causes rearrangement o f M2 09-29-15: The redistribution of lahel in the resting and desensitizedt states provides direct evidence for agonist-dependent rearrangement of the M2 heliccs. Thc efficicnt lahelling of the resting statc channel in a rcgion capahlc of structural changc also suggests that the aliphatict residues lahcllcd by [ " 5 1 ] - ~form ~ ~ a permeability harrier to the passage of ions that is removed on gatingt the channelffp.
Ne~ntivelycharged rings flankin,q M 2 enhnnce permeability to cations 09-29-16: Mutations of amino acids including those polart groups within'" and ncgativcly chargcd rings hrackcting"' seginent M2 affect ion permeation at thc single-channel Icvcl. A change of Scr248 (within M2) to Ala decreased the outward single-channel currents and the rcsidcnccT timc of the open-channel blocker QX-222"". The negative chargcs at zAsp2Z8, zGlu241 ant1 rGlu262 wcrc also implicated as determinants of thc channel's pcrmcahility"l. Thcse negatively chargctl residucs flanking M2, which arc largely conserved among the various subunits, arc hclicvcd to confer a net negative charge to the channcl entrance and enhance the pcrmeahility to cation^'^^-^^'.
Residue trThr244 is involved in ion selectivity 09-29-17: Mutations of zThr244, within M2, affect the channel's ability to tliscriminatc amongst monovalent cation^'^^'^". 09-29-18: The T244D mutation in the M2 scgmcnt of the neuronal 27 nAChR suhunit changes the selectivity of the homo-oligomeric channcl cxprcsscd in Xrnol?usoocytcs. Thc r7D244 channel cxhihits lnrgcr currcnts than the wildtype r 7 channcl and is activntcd at lowcr ACh concentrations. The relative ionic pcrmcahility of wild-typc AChRz7 to K' is PK/PNa= 1.2, and to ~ a " , P ' H ~ / I ' N-~1.4. The z7D244 channcl is less selective in discriminating hctwccn K+ and Na', I'K/l'NA= 0.95, hut cxhihits a markcd incrcasc in pcrmeahility to ~ a " , I)'R~/I'N.,= 3.7. In addition, only thc mutant rcccptors arc pcrmeahlc to ~ g '27. "
rvLe11247lies in the lr~meno f the channel
J
09-29-19: In the homo-oligomcric nAChR composcd of the chick brain 27 suhunit, mutations of thc highly conserved Leu247 residue in the M2 segment suppress inhihition hy the channel hlocker QX-222, indicating that this residue, like others from M2, faccs the lumcnt of the channelf2". 09-29-20: Thc same L247T mutations also dccrcasc thc rate of desensiti-
entry 09
zationt of the response, increase the apparent affinity for acetylcholine and aholish current rcctificationi. Moreover, unlike wild-type xi', which forms channels with a single conductancc level (46 pS), the Thr247 mutant has an additional conducting state (XO pS) active at low acetylcholine concentmtions. In addition, ;~ntagonists of the wilil-type receptor, dihydro-11erythroidine, hexamethnnirlm and (t)-tuhocurarine, act ;IS agonists with the L247T mut;lnt and activate the novel conducting state1'". It is sumcstcd that the L147T mutation makes one of the high-affinity dcscnsitizecl st;Ites of thc. nAChR conductive1'".
oTl?r264 is located in thc ~?arrnr4~ rcgion o f the porr 09-29-21: Suhstitution of the Thr264 in the transmcmhrane scgmcnt M2 of the x suhunit of the rat nAChR affects channel conductancet. Mutation of the residues at homologous positions in the /I, and c i suhunits shows the conductancc t o he inversely relater1 to the volume of the amino acid residue, sumcsting that rrsirlues at this position form part of the channel narrow region. Exchanges of residues hctwccn suhunits docs not change the conductancc, suggesting a ring-like structure formed hy homologous amino acids in thc suhunits of the pcnt;~meric channcl. Channels in which the narrow rcgion is formcd hy four scrincs and one vnlinc have the same conductancc if the valinc is located in the 7 , /I, or ;. suhunits, hut it is smaller if the v;~lincis loc;ltcil in the (5 suhunit. These results sitmest a structural asymmetry of thc AChR channcl in its narrow region formed hy the hvdn)xvl;~tcdTaminc~;lcitls of 7 , ;, and (5 suhunits, whcrc the 6 suhunit scrine is ;I main tletcrrnin;lnt of the ch;~nnelconductance"". In addition, for a given size of sitlc-chain, the conducti~nccis consistently higher with a polart r;lthcr than ;I hyctrophohici side-chain'.", suggesting a 'catalytic' role for the polar ring.; in thc tr;lnslocation of cations through the channel pore1.". $;,
Mutmtiorls c.rlrlsin,c:chc~n,c:c~s fro???cc~tionic. l o clnionic sc1cctivity 09-29-22: T h e M2 rcgion of nAChR has strong homology to the an;~logous rcgion of the anion-sclcctivc glvcine ant1 CARA,, receptors. Substitution of amino ;lcirls within or near the M 2 rcgion of the 17 nAChR suhunit hy different rcsitli~cs from the GIVR r l suhunit tlrastic;~lly changed the properties ot the chilnncl, converting its selectivity from cationic to anic>nicl.'.'. Thrcc amino acid differences, changes of Clu237 to Ala, Val251 to Thr nnrl the addition of n Pro after residue 2.36, arc sufficient to produce channcls that were 500-folti more sensitive to ACh, are activated by the competitive antagonist dihydro-8-erythroidine (DHPE), n o longer show inward rectificationi of whole cell currents and arc onion-selective. The M I t o M2 spc~cingc~ffccts ion .selectir~ity 09-29-23: Deletion of the extra Pro residue from this mutant r 7 channcl gives functional channels that show the enhanced sensitivity t o ACh, d o not desensitize r;~pidly, arc cation sclcctivc hut do not pass ca'' currents. Inversion of inn selectivity is also achieved hy the addition of cithcr an Ala or a I'rc~ rvsirluc, fc~llowingposition 2.76, to ;I cation-selective 77 mutant. These d;~tapoint to the irnportancc. of the length of the segment spacing M I and M:! in determination of the ion sclectivity'"~'.
entry 09
The tr7 T251 channel shows additional conducting states 09-29-24: The single substitutions, Clu237 to Ala or Val251 to Thr, do not
invert the ion selectivity, but the Thr251 mutation docs change the apparent affinity for ACh, response to DHPE and rcctificationt properties of the channel. Single-channcl recordings from outsidc-out+ patches containing the Thr251 a7 nAChR show multiple conducting states, including one of 54 pS, similar to the wild-type A state, and one of 86.3 pS, corrcspnnding to a desensitized D' state of the nAChR sccn with the Thr247 r n ~ t a n t " ~ .
The CIu237Ala change in ru7 nffects cap'-selectivity 09-29-25: The alteration of Glu217 to Ala in the 27 nAChR results in a
receptor that responds to ACh normally and shows rapid dcscnsitization~. Although this mutant channel is permeahle to cations, it has lost the ahility to conduct Cab '
s
~
.
Conclr~sionsfrom observations with mutnnt channels 09-29-2fk Structural interpretations of the data ohtained from studies with
nAChR channels altered in the pore region indicate that the wide entrance vestibules of thc nAChR pore contain net negative charges which can attract cations and are particularly important in attracting divalent cation^'^.'. Upon channel opening, thc pcrmeant cations rapidly pass through the uncharged, tapering rcgion of the poret, lined hy the M2 sequences from each of the suhunits. The geometry of the M2 segments, influenced hy the M1-M2 spacer region, is crucial in tlctcrmining ion sclcctivity of the channcl. The narrowest rcgion of the open pore, in the rcgion of Thr244.1, is very short (estimated to contain as fcw as six water molcculcs) and is likely to he the only rcgion of the channcl whcrc strong interactions between permeant ions occur^'"^.
Closed structure o f the nAChR channel 09-29-27: The closed structure of the channel is more difficult to invcstigatc,
for ohvious reasons, hut experiments with M2 peptidcs havc suggested that an association to hlock the porct, with thc appropriate stahility, could he achieved hy interactions hctwccn the M2 r-helices of thc suhunits'.'". Thc clcctron microscopic images of the Torpedo AChR suggest that amino acid residues come closest to the axis of the pore just helow thc middle of the hilayer when the channcl is closed. This coincides with the position within M2 of the highly conserved Leu residues that are known to be in the narrow rcgion and to face the lument of the channcl (scc nbovc). It has hccn suggested that the bulky, hydrophobict Lcu side-chains of the five r-helices associate t o create a harricr of limitcd stahility that forms the gate1.'".
il
09-29-28: Thc MA segment, located hetween Ma?and M4, is part of the
cytoplasmic domain ant1 docs not form part of the poret. Deletion of scqucnces encoding part of MA in the r suhunit eliminates channcl function in the Xcnoptrs oocytc system. Whcn MA and the 20 preceding amino acids arc rcmovcd. 3% of the native activitv is ohtaincd'~".
entry OY
St ruct l ~ r m lchong~:s on riesensiljzrltinn 09-29-29: AEtcratir>nsin protein stmcturc prod~rccrlhy binding r)f cholincrgic :lgonists to purificd nACliR rcconstitutcrl into lipid vcsiclcs can he cIctcctcd hy Fourier-tr:lnsform infrarctl spcctroscopy~ and diffcacntial scanning caloriinctryt. Spectral c h n n ~ c sindicate that the exposure of the nAChR to thc agonist carharnvlcholinc, undcr contlitinns which drive the AChR into the desensitizcd state, protluccs nltcmtions in the protcin scct~ndary stmcturc. Uuantitativc estim:ltio~l of thcsc agonist-inrll~ccd alterations rovc;ils nt? significant changts in the percentage of x-hcliit, hut a decrease in /!-shcctk structure, concomitant with an incrcasc in Icss-ordered stnlctitres. A ~ o n i s t hintling :ilst, rcsults in a ct~nccntration-dcpcncIcnt incrc;lsc in thc pmtcin's tlicrni:~l st:ihility, ns inilicatctl hy thc tcmpcratl~re dcpcndcncc c ~ fthe infr;~rctlspcctrurn a ~ l dby calorimetric analysis, further sumcsting that nAC:hR desensitizatiani intlllcctl hy the cholincrgic :~gnnist involves si~nificantrcnrrangcincnts in thc protcin structurc'"'.
Predicted protein topography Lnhellin,p ,studies 09-30-01: Given fnur ~ncmhmnc-spanningpcpt i J c s ( M 1-M4) in cad1 suhunit (SCI' I l o r n i l j z ~~ l r r m ~ ~ ~ 09-27), ~ ~ r l ~t hc ~ ~Nn tand . C-tcrminal regions must hc on
thc snrnc sitic of thc iilci~lhranc. Antibody anrl chemical lahullinR"H~'~7Y specify the N-tcrmin:alt, ~ - t c r n I i n a l tand M2-M,? linker regions as cxtraccllulnr, with t h c srnall MI-M7. ant! largcr M3-M4 linkers intraccllular, This determination s u ~ c s t sthe MA region t11 ho intraccllu!ar, at or near the membrane-cytoplasm intcrfacc.
Elcctron micrnsr:oy?l'cnnrr1ysi.s of channel strr~cturrat 17 A resolrltion 09-30-02:E!cctrt~nmicroscopy and 3-dimensional image-rcct~nstructionthas providcil the strLtctitrc of tllc pnst-synaptic Tr~rpcdomnrrnormtrr nAChR channel to n resolution of 17 A". I'ost-synaptic incmhrancs forrncd into tuIn!l:ir vcsiclcs sitspcndccl in thin films of icc pcrrnittcd the receptors (organizctl into helical :irrays] to hc seen froln all nnglcs, revealing the r c l ; ~ t i o of ~ l the lipid I7ilaycr nntl thu peripheral protcin (Fix. 2). A pcntomeric, /7r1rrel-stnvenrmngement oJ subunits 09-30-03: T h e nAChR is s pseudosymmctric pcntamcr, with the suhunits in a harrcl-stave arrangcrncnt (.we Fi,y. 2). T h c channcl ct~mplexis ahout 120 A long, prt~icctingahout A0 A into the cxtraccllular space and ahaut 20 A into the intraccllular solution. T h e rliamctcr at the cxtraccllular cntl nf the channcl is ahout 813 A, narrowing to ahnut 50 A in tho mcmhranu-spanning reginn. T h e conduction pathway can he soon to consist of a narrow central porcl across the hilnycr, t c r ~ n l n n t i nin~ entrances 213-25 A wide (fnr rrvicw Si,F I ~ J J . ~ ~ ) J .
of the mntlsr muscle nAChK 00-30-04: T h e structure of thc channcl ohtainod by c x p r c s s i n ~cRNAs
Strtzcture
cncndinc, the four mrlusc niusclc subunits in Xenoprrs oclcytcs can he
cntry 0 9
I
L
0
a
;? h -
iI -
-
Figure 2. Axial section thro~rghthe cylindrically ~veragedstmcturc of the Torpedo marmorata nAChR. show in,^ details o f the channel in relation to the lipid hilayer and the peripheral 43 kDa protein. ot the hottom o f the figure. (Reproduced with permi.v,virn from Toyoshima (1988) Naturc 336: 247-50.) (From 0930-02) visualized hy atomic force microscopyt. Thc pcntamcric structure with a central porc is ohscwcd on the extraccllular facc of thc memhranc. Thc angle between thc two Isuhunits was 128 and thc unit cell ahout 10 nm diameter'".
Analysis of the Torpedo channel at 9 A resolution
09-30-05: Analysis at higher resolution (9 A] has hccn ohtained hy recording
images at diffcrcnt lcvcls of dcfocus and averaging data using helical diffraction1 mcthorfsidn. This method allows some identification of secondary structure, particularly thc r-helicalt rods within cach suhunit. In the synaptic part of cach suhunit there arc thrcc rods, oricntcd pcrpcndicular to the plane of thc hilaycr. T w o of thc rods line the cntrancc to the channel, with thc third on thc outside. In the region of thc rcccptor that spans the hilaycr, cach suhunit has only onc visihlc rod: sincc this forms thc lining of thc porc it is assumed to he thc M2 transmemhranc helix. This rod kinks and tilts near its midpoint, whcrc it is closest t o thc axis of the outwards on cithcr side. It is flanked on thc lipid-facing sidcs hy a continuous rim of dcnsity that is intcrprctcti to hc P-shcctt.
Position of the M2 domain in the stnrcturc 09-30-06: Alignment hctwt.cn thc thrcc-dimensional dcnsitics and the scqucnce of M2 placcs the charged rcsiducs at thu cnds of M2 symmetrically hcstridc thc hilaycr, and a highly consuwcd Lei1 rcsidl~c(Leu251 of the z suhunit) at the position of the kink. A model is s u ~ c s t c din which the side-chains of thc Lcu rcsiducs at thc kink proicct into the porct to form a hydrophobic ring, closing thc channcl hy ma kin^ a harrier that hydratcd ic~nscannot cross'40 (Fir. 3).
entry Oc)
-
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I
' \ ~ /
Figure 3. Crlt-c~wt~y modrl to show the' I~msic. fcv~turrso f thc nAChRc.11r1nnr.1. Thr, c.hmnnc~1is 17nilt from ( I p~ntamcric association of siniilar y the .sr~l,~rnit.s, mrrrrngc'd oro~rndrr crntrcll porrt thcrt forms the p ~ t h w c ~for t,crtions. Mo,st oT thc protc~inmtlrs o f thc chmnnrl is on the .svnaptic side o f the. hilnyer. ( ~ n dthis re,yion inc-Irrdes thr hind in^ sites for r~cctylcholin~ r~rltlrt-/?rrngcrrotoxiri,Tlic pore i.s n(~rrorv( I C ~ O S S t h lipid ~ hilmvcr mntl is c.1oscd hv tl~rpjrlxtrrposition o f hjrdrophol?ic nrnino rrcid sidc-chrrins in thc rr3,rion of the gatr. Thr widcr entrancr* clnd rxit from the pore contain c.hcrr,yct?.yrorrps to .sc.rc7c*norlt c111ion.s.Thr sizr o f thr porr at diffrrrnt 1rrrrI.s is tlrtr~rrnirlrdhy the shapr, c~ndorirpntntion o f the M2 hclix ond the natlrrr ol tlir, cln~ino rrc-id sidc-c.hoim projrctinx honi it. (Rr[~rodrrcrd rvitli f~r~rrnission froni l l r ~ w i r( ~1 99.31 Cell 72 (Srlpld.):.3 1 - 4 1 . ) (Froni 09-,?0-06) C/i(~r?iic(~I n?o(lific.r~t ion o f rc~idlrc.sin M2 09-30-07: A combination of in vitro mutagcncsist to gcncrntc cystcinc rcsitlucs in M2 of the mouse muscle 7 suhunit, followed hy covalent chemical modification of the cystcines with small, charged, sulphydrylspecific reagents srlEgcsts that residues Ser248 to Thr254 of the M2 domain constitute a /~-stmncl'~'.T h e side-chains of Cys residues substituting for Scr248, Lcu250, Scr252 and Tlir254 arc all exposed to the reagent in the closed nAChR channel, and that of Leu251 is exposed on channel opening. These results arc inconsistent with an 7-helical structure, and strongly sumcst a /I-strandt conformation for this part of thc M2 transmcrnhrane domain.
A synthetic pcpptidc model for the pore region o f the nAChR chnnnel 09-30-08: A synthetic channel protein, TSM2d, that cmulatcs the prcsumcd pore-forming structure of the nAChR has heen gcncratcd hy asscmhling five helix-forming pcptidc modulcs at thc lysinc ,:-amino groupst of the I Iresidue template {K*AKbKK*I'GK*EK'GJ, whcrc indicates t h r ;ittachmcnt sites. Hclic;il modules repmsent the sequence of the M2 scgmcnt of the Torpcclo c.mlifornic.n n AChR ii s u h u n ~ t .Purified TSM2ri mlgratcs in SIT-PAGE with an apparrnt M, of approximately 14000,
-
consistent with a protein of 126 resiclucs. When reconstituted in planar lipid hilaycrs, the T5M2d polypeptide complex forms cation-selective channels with a single-channel conductance in symmetric 0.5 M KC1 of 40 pS, a value close to the 45 pS characteristic of authentic purified Torpedo nAChR, recorded under similar conditions. These results support the contention that a hundlc of five amphipathict s-helices is a plai~sihlc structural motif for the inner bundle that forms the poret of the pcntamcric nAChR channel1".
Influence o f lipids on the structure o f the nAChR 09-30-09: The gross secondary structure of the purified Torpedo calilornicfl
nAChR has been examined hy Fourier-transform infrared resonance spectroscopyf in reconstituted dioleoylphosphatidylcholinc membranes in H?O and D?O. The secondary structure of nAChR in H 2 0 was calculated to contain ahout 19% 2-helix, 42% /i-structurcl, 24Yn turns and 15% unordered. The secondary structure contcnt in D 2 0 was estimated to he 14% z-helix, 37% p-structure, 29% turns and 20% unordered. In the presence of phosphatidic acid the p-stn~cturecontcnt in D?O incrcasecl significantly from ,Z7% to 42%. This suggests that an ionic interaction hctwccn negatively charged lipid hcad-groups and positively charged peptidc side-chains may stabilize a 1-structure conformation. The inclusion of cholesterol in the reconstituted mcmhrancs significantly increased the 2helix1 contcnt from 14% to 17%. These results support the hypothesis that cholesterol may induce a transmemhranc region to undergo an unorderedto-helix transition which is necessary to maintain the integrity of the ion - channel1'".
-
Protein interactions Synchronorls initiation o f subunit interactions in assembly 09-31-01: The assembly of Torpedo nAChR suhunits is temperature-sensitive
in vivo , a phenomenon that can he used to allow synchronous initiation of the assembly of subunits synthesized at the non-permissive
Order o f sub~mitinteractions in receptor assemhly 09-31-02: In the mouse fihrohlast ccll line, All-1 I, that expresses the Torpedo
nAChR subunits, the earliest identifiable complexes are sr/b trimers, which hegin to he detcctahlc within minutes of the temperature-shift. The 5 subunit is added next, 2Py1i tetramers heginning to appear at about 1 h, followed hy the addition of the second s subunit from ahout 12 h onwards to form the szP$ pentamer14". Studies with ccll lines expressing restricted pairs of suhunits show that the assembly pathway is kinetically determined, with s1icomplexes being formed much more slowly than 211, P;, or ry compIcxe~"'~.
Suhnnit folding drrring the association process 09-31-03: In addition to suhunit assembly, there arc disccrnihlc subunit
folding events occurring during the association process. Thus the formation
entry 09
-
of the a-Rgt-hinding site occurs on the r suhunit hours after it has asscmh!cd
into the trirncr. The formation of an cpitopcj rccognizcd hy a specific monoclonal antibody, MAhl4, is even further delayed, occurring just hcforc the c i suhunits arc ad(ictl t o the trimcrsl".
Thc extrncc~llulr~r N-tcrrninrll (1or11~in contnins thc infornlcltion for stlhrlnit ossocintion 09-31-04: Co-expression of truncated, N-tcrminalt fragments consisting solely of the cxtracellular N-terminal domain of the 2 , (5 or ;. suhunits of the mouse rni~sclcnAChR with the four wild-type suhunits in transtcctcd COS cells hlocks surf;lcc cxprcssion of the AChR. T h e ftlrrnntion of 26 hctcrotiimcrs, an early step in the nsscmhly pathway of the AChR, is inhihitcti. Immunoprccipitntion nntl sucrose gr;~dient scdimcntatic~n cxpcrimcnts show tlint the N-terminal fragment ot the r suhunit forms a specific complex with the intact (5 subunit. T h u s the cxtracc!!ular Ntcrminal domains of the r, (5, and ;- suhunits contain the information necessary for specific association of the nAChR ~ u h u n i t s ' ~ " .
A 4,7 kl>n m i ~ n ~ l ~ r tprotcin l n r ~ is r~ssr~ntinl /or nAChK m~grcgatinnjn post-synr~ptic.n ? ( ~ n ~ l ? r ~ n ~ , s 09-31-05: A 43 kDa peripheral membrane protein is present in ctluimolar amtiilnts with AChR in AChK-rich mcmhr;~ncs,in close association with the /I suhunit of the AChR. This 4,Z kDa protcin is essential for the characteristic aggregation of the AChR in the post-synaptic+ membrane. Cocxprcssion of cI)NAs for each ot the four suhunits of hc~thfoetal (r, P, 7 , 5) anti arlult [ r , (I, I:, ,il AChR with cxprcssion constructs encoding the 43 kDa protcin in fihrohlast cell lines Icnds to spontaneous clustering of h C h R patches, compared with the uniform distrihution ohtnincd in the ahscncc of the 4.3 kDa protein. T h e 4.3 k n a protcin co-localiscs with thc AChRs, and is nhlc to aggregate into ch;lrnctcristic clusters when cxprcsscd alnnc. T h e 4.7 k I h protein has hccn propocctl t o form the key link hctwccn the hChK ;~ntlthe tosk skeleton"^.
58 clnd 87 kncr prriphornl mcmhrclnc proteins 09-31 -06: Other post-synaptic peripheral memhnne proteins, with mnlccular mnsscs (lf 58 and 87 k l h , hnvc hccn i4cntificd in Tnrpcdo clcctric organ ant1 mnmmnlian ncuromuscular iunctions'. T h e 58 kDa protein is conccntratcd at thc synapsr hut has n hrosrlcr distrihution than the nAChR, including tissues such as kidney from which the nAChR is a h ~ c n t ' ~ ' .T h e 87 kDa protein is more restricted to the synaptic region than the 58 kDa protein, hut 1s also prcscnt in the sarcolcmmai extrasynaptically. It has a rcstrictctl tissue distribution, hcing cxpresscd in clcctric organ, muscle and brain, all tissucs t h i ~ texpress nAChR.
Dvstrophin o,csocitrtt.s with thc~58 k Da and 87 kDa proteins 09-31-07: Co-immunoprccipitntion with anti-87 kDa protcin nntihodics shows that rlystrnphin (n memhmnc-associatcrl cytoskclct;~lprotcin that is the prodi~ctof the I>uchcnnc muscular tivstrophv [DML)] I(lcusl and the
entry 00
1
58 kDa protein associate with the 87 kDa protcin in Torpedo post-synaptic m e m h r a n e ~ ' ~ ~ (The . ' ~ ~ 87 . kDa protein, the 58 kDa protcin and dystrophin have also hcen identified in vcrtchrate skeletal muscle sarcolcmma, whcrc dystrophin is proposed to link the extracellular matrix with the ~ ~ t o s k c l e t o n ~The ~ " . )87 kDa protein is a substrate for kinascs, and it is suggcstcd that its association with other synaptic protcins may hc rcgulatcd hy phosphorylation1"".
Protein phosphorylation Multiplc kinoses can act on nAChR 09-32-01: The T. cnlifornicfl nAChR is phosphorylatcd hy at least five diffcrcnt protcin kinases; PKA that preferentially phosphorylatcs the 7 and (5 suhunits'"; cyclic AMP-dependent pmtein kinase that phosphorylatcs mainly y and 15 suhunits'"; PKC that modifies (5 suhunits; a ca2*/ calmodulin PKII that phosphoryl;~tes I{ and 6 suhunits; and a tyrosinespecific PK that phosphorylates 0, (5 and *; suhunits'"" The nAChRs in skclctal musclr: arc also phosphorylatctl hy similar protein k i r ~ a s e s ~ ~ ~ ~ * ~ ' ~ ~ 09-32-02: Many of the phosphorylation sites have heen identified: thcy arc locatctl on the major intmccllular loop of each subunit, hctwccn thc third and fourth transmemhranc hcliccs. Thcsc phosphorylation sites arc conserved in the sequences of most nAChR suhunits from a wide range of spccics'"('.
Consensus sites for phosphorylntion on the rr7 subunit 09-32-03: Conscnsus sites cxist for the phosphorylation of rat rx7 nAChR suhunits at S36S hy cAMPdependent protein kinase, of Thr415 and Scr427 hy casein kinasc I1 ant1 of Tyr442 hy tyrosine kinase9.
Desensitization and phosphorylation 09-32-04 Nicotinic receptor-channels can he dcscnsitizcdt morc rapidly following CAMP-dcpcndcnt phosphorylation'"2~15'"1"X (see Rcccptorl tronsdtrcer interactions, 09-49). CAMP-dependent processes may incrcasc insertion of prc-cxistin~nAChRs into the plasma memhranc in the ahscncc of ncw protcin ~ ~ n t h c s i s ' ~ ~ ~ .
In vitro phosphorylation by P K A 09-32-05: Thc single-channel propcrtics of purified AChRs from T. cnlifornicci reconstituted in lipid hilayers are altered hy in vitro phosphorylation with PKA. Notahly, thc spontaneous open-channel probability of phosphorylatcd AChRs (1.0 mol of phosphate incc~rporatcdpcr mol of AChRJ is incrcascd 40-fold over that c ~ f unphosphorylatcd receptors. Channel activation hy PKA is correlated with AChR phosphorylation and is aholishcd hy ~ R g (200 t n ~ )Like . thc unphosphorylatcd AChR, thc phosphorylated channel has two distinct opcn states, short- and long-livcd. Thc , rclativc frequency of thc long openings and thc magnitude of hoth timc constants incrcasc 4-5-fold nftcr phosphorylation, as thcy do with ngonist- mcdiatctf activation1".
!'KC and n,p~rcgationoT nAChR 09-32-06: Agrin, a protcin isolated from the electric organ of T. crrlifornica, induces the tormation of specializationst on chick myotuhcs in culture, at which several components of the post-synaptic apparatus, including nAChRs, arc concentrateti. T h e proccss of accumulation of the nAChRs into agrin-induced specializationsi involves lateral migration of receptors already on the surface of the myotuhc, a process that docs not require protcin synthesis'"". The formation of agrin-induced nAChR aggregates is hlocked hy the phorhol ester TPA, an activator of PKC, which c;ln also disperse prc-cxist~ng;~~grc'gates'"".
Agrin .rtirnulc~tcnA ChR phosphorj~lrrtjon 09-32-07: T h e adtlition of agrin to o v c m i ~ h tcultures of chick myotuhe cultures, in the prcscncc of I ' ' P J - H ~ P ocauses ~, a three-fold increase in phosphorylation of the nAChR /isubunit, and a 20-,10% incrcasc in the phosphorylation of the ;. and subunits. An inhihitor of protcin scrinc kinases, H-7, hlocks agrin-induced phosphorylation of the ;. and (5 subunits, hut fails to inhillit inci~~ccd phosphorylat~onof thc /I suhunit or nAChR aggregation'"'.
Tyrosine phosphorylat ion prcwdcs ~,q,qrcga tion 09-32-08: Immunohlotting experiments with affinity-purified antiphosphotyrosinc antihodics demonstrate that tyrosine phosphorylation of the P suhunit is intluccd hy agrin-treatment of myotuhes. Three treatments that 1 hlock ngrin-iniluccd receptor aggregation, low pH (pH 6.5), TPA (50 n ~ and aililition of polyanions (0.05 mg/ml dextran sulphatc), also hlock the agrinintluccd incrcasc in tyrosinc phosphorylation ot ~ A C ~ R S ' " ' ,Tyrosinc phosphorylation of nAChRs prcccdcs the aggrcgation of nAChRs hy a few hours. T h e agrin-induced nAChR aggregates stain with anti-phosphotyrosinc antihotlies and correspond in location with the sites of lahclling hy rhodaminc-conjugated r - ~ ~ t " ' .
Rasic ljhroblnst ~ r n w t hfcrctor inducts nAChR ng~rt:,yntion 09-32-09: Rcacls coated with hasic fibroblast growth factor (hFGF) induce the nAChKs of cultured X C I I ~ J ~rnyocytes ~IS to aggregate at the site of the myocytc-hcarl contact. Thc bFGF receptor is a protcin tyrosinc kinasc, and tyrosinc kinasc inhihitors stop the intluction of clustering hy the hFGFcoatcd heads'"'.
Activation Activntion hy ncetylcholine 09-33-01: T h e Torj7cd0 nAChR expressed in Xrnopt~s oncytos is halfmaximally activated by 20 mM acetylcholineJ"".
Act ivntion o f nAChR b y cxtmcellulnr ATI' 09-33-02: Extracellular ATP (10 !)MI increases the spontaneous opcning fretli~cncy of nAC:hR of rat skeletal muscle cells in the cell-attached'
-
configuration from 0.3 to 4.7 s-', without changing mean opening times (0.6 ms). When delivered through a separate drug pipette after first forming a gigasealt, ATP increases ACh-activated single-channel open prohahility in a dose-dependent fashion. (100 ~ J Mand 500 I ~ MATP increascs ACh channel activity induced hy 0.1 /JM ACh hy 46% and 63%, rcspectively.) Thcsc concentrations of ATP are helieved to he in the range found in thc synaptic cleft immcdiatcly after rclease from the cholincrgic motor ncrvc terminal^'^^. 09-33-03: The non-hydrolysable ATP analogue, ATP-y-S, also enhances ACh-
activated channel opcning, but ADP, AMP and adenosine, up to 1 mM, are without cffect. Rccausc thc facilitating effect is ohsewed when the ATP is administered through a separate pipette, hut cannot he seen with outsideout4 patches, it is likely that the coupling of the ATP to the AChR occurs through an intracellular
Activotinn o f neuronal nAChR by external co2+ 09-33-04: Neuronal nAChRs (hut not muscle nAChRsl are strongly modulated hy physiological concentrations of external ca2'. For neuronal nAChR species z2P2, zL3P2,23/{4 and z4P4 cxpressetl in Xenopus oocytes, and for native nAChRs in hovinc chromaffin cells, ACh-induced currcnts incrcasc as the Ic~"],, incrcascs in the rangc 0.1-,70 mM. Thc cffect is c a Z ' specific, is not duc to ~ a ? carrying + the additional current and occurs despite a reduction in the single-channel current a m p l i t ~ d e " ~ .
-
Current-voltage relation Muscle nAChR channels show n Iinenr I-V relationship 09-35-01: Thc nativc nAChR channcls of adult bovine skeletal musclc show a
linear I-V relationship, with a conductance of 59 pS and an average opcn time of 5.6 ms3R.The channels of foetal hovine muscle have a conductance of 40 pS and an average open time of 11.0 ms. These values are accurately reflected hy those ohtaincd from thc cxprcssion of cRNAs encoding thc r , P, ii and I: subunits and the r , /I,;. and (5 suhunits respcctivcly inicctcd into Xcnopus oocytc~"~.
Neuronal nAChR channels show inward rectificntion 09-35-02: The currcnts through nAChR channels of centralt and pcriphcralt
neuroncs contrast with those passed hy the muscle nAChR in displaying pronounced inward rectificationf . In rat sympathetict neurones, rectification of whole-cell currents results primarily from a reduced prohahility of channel opening at depolarizing potentials'". In rat PC12 cells, channels closc more rapidly at positive potentials to cause the rectificationfM. 09-35-03: The whole-cell nACh
-
currcnts in cultured post-natal rat hippocampal neurones arc of two distinct types. One class exhihits rapid and profound desensitizationt and is sensitive to inhibition hy x-Rgt. Thc sccond class activates slowly and cxhihits n o descnsitizatic>n during prolonged agonist applications. This slow current is insensitive to 3r-Rgt. Both the fast and slow responses exhihit inwardly rectifyinRi currentvoltage relationships and pass little currcnt at positive mcmhranc potentials"".
Hornomeric
rt
7 r~ntl0 8 o l ~ ~ n n eshow l s inwvlrd rectificntion
09-35-04: T h c ACh-induccii currents through chick homomcric r 7 and r 8
channels cxprcsscd in Xr,nopz~s oocvtcs show very rectificationi at potcntials ;thovc 20 1 1 1 ~ ' ~ * ~ " .
strong inward
-
Currcnt-voltngrp r i ~ l n i i o n , s lo~ fi ~hornorncric ~ 0 9 c,hnnncls 09-35-05: T h c I-V curvc ohtaincd from Xrnopr~soocytcs injected with rat 29 cRNA is non-lincnr, with n maximal, ACh-induccd inward currcnt at
-50 mV. Currents arc strongly rcduccd s t potcntinls ncgstivc to -50 mV sntl a t more positive holding potentials up t o 25 mV. Strong rectification f then occurs up to :I holdinx potcnti:~lof 20 m ~ " " .
j
Inactivation Nouronr~ln AChli chr~nnclsshow. rr~ryjr7~ inr~ctirrotion kinetics 09-37-01: Thc r-13gt-sensitive nACh currents in rat hippocampal ncuroncs dccay rapitIly (fuw milliscconi!sl in the continuing prcscncc of aRonistl"Y, ;lnd the chick 77 and 78 homoniilltimcrsi cxprcsscd in Xrnopus oocytcs hch:~vc ~ i r n i l a r l v " ~ ~ Thir ' ~ . I~ch;~viour contr:lsts with th:~t of thc r-Rgtinscnsitivc nicotinic rcsponscs o f r;it hippocampal net~rones"" and the ccrchcllum c ~ fs t l ~ ~r:atsft", lt whcrc nicotine-~nrlucrclcxcit;ltions do not show rapid ilcscnsitization.
The I I A C ~ chrlr~ncl R uurrcnts o f cmhryonic musclc rrrpidly (lascllsit izr~ 09-37-02: I'ulscs elf ncctylcholine [AChl npplirtl to oiltsidc-outt patches of crnhryonic-like nioilsc i n i ~ s c l r mcmhrnnc elicit channel currcnts which dcclinc rapidly (rLl- 10-60 ills\ due to dcscnsitizationi. Ahout half of the channels accovcr from 11cscnsitiz:ltion in .JOO 111s'~'.
Kinetic model Clossicoi kinc.1 ic rnodrl 09-38-01: T h e cl;~ssicalmodel for ;ictivation of the ncuromi~sculnrnAChR
c;in 1~ rrprcsrntrd hy: A
t
K
k.,
AR
; ~ '
li
1
+
A
k .? li
?
/I A?Ry-A2R" 7
In this modcl, the closcJ rcccptor (Rl unticrgocs sequential hinding of two tnolcculcs of ;lgonist ( A \ , with association constnnts l i q l and k , ? and clissoci3ti(ln rntc const;lnts I; I ; ~ n dli ? , followed hy ;I conformational chanxr t c ~thc o p m state (Ii'),govcrncd hv opening ratc constant 11 and closing ratc constant T'~'.
Model nlk)win,q /or oprninx o f .sin,yl~7li,y[~tcdrcccptor 09-38-02: Thc :ahovc rnniicl i s ;~lmost ccrt;ainlv too simplistic: thcrc is cvillcncc th:~t short-tli~r:atic~no p c n i n ~ s can rise from singly ligntctl rcccptors, for cx:~mplc. This woi~l(lrequire t1i;it thc nh(lvu scheme hc
entry 09
n
extended to include the activation of AR to an AR* state, as wcll as the dircct conversion of AR* to A2R9 by hinding of a second molecule of agonist. For a discussion of kinctic models for nAChR activation see ref.".?.
Selectivity Non-selective cation currents 0940-01: The nAChRs are generally cation specific, hut relatively non-selective
amongst cations. Every monovalent or divalent cation that can fit through a porct of 0.65 nm diameter is permeant in endplatet channels. Permcant ions include not only the alkali mctalt and alkaline cartht cations hut also organic cations such as triaminoguanidinium, cholinc and hi~titlinc"~.
External ca2+modulates neuronal nACh Rs 09-40-02: Note that physiological concentrations of extracellular c a 2 ' can
hind to neuronal nAChRs (hut not the muscular form) and directly modulatc channel activity, independently of permeation or second messenger-hased p r o c e s s e s i 6 ~ s c cActiviltion. 09-.7,7).
ca2+conductance measurements 09-40-03: The elementary slope conductancct of the nAChR channcl from rat " m ~and ) 42 pS medial habenula neurones is 11 pS in purc external ~ a (100
in standard solution. Thc ca" influx through nAChRs results in the risc of t o the micromolar range. This increase is maximal hclow -50 mV, when c a 2 ' influx thmugh voltage-activated c a 2 ' channels is minimal. The ~ a "influx via the nAChRs activates a ~a"-dependent CI conductance and causes a dtcrcasc in thc C.ARAAresponse that outlasts the risc in Ic~"],"".
IC~'.']~
09-40-04: Thc a-Rgt-scnsitivc nAChRs of chick ciliary neurones, activatcd hy
1 1 1 nicotinc, ~ pass inward ~ a "currents, detectahlc with ~a"-sensitive fluorcsccnt dyes hut not by whole-cell recording. The effect is sensitive to 20 I(M (+)-tuhocurarine and to nM z - ~ ~ t ' ~When '. the ncuroncs arc trcatcd with 10 / L M nicotinc, the .I-Rgt-insensitive nAChR channels arc activatcd and lead to increased I C ~ ~ ' ] , ~ . ' .
~ 4selectjvity '
of the
ru 7
channel
09-40-05: The homo-olignrnerict r7 ncuronal nAChR is exceptionally pcrmeahle tn ~ a " ' .Determinations hased on the reversal potential shifts in 1 mM and 10 mM c a 2 + (+29 mV) sumest a permeability ratio (PC.l/lJN.l)of ahout 20 for the rat r 7 channelY. This contrasts with a pcrmeahility ratio (Pc:n/P~n) of 0.2 fnr the muscle ~ A C ~ R " ' , 0.7 for rat parasympathetic
cardiac neuronesR ancl ahout 1.5 for thc nAChRs prcscnt in rhromaffin or in PC12 cellst7'.
cells"'
cn2+influx through cr7 channels nctivntes n Cl channel 09-40-06: Whcn thc homo-oligomcric r7 channcl of the rat is expressed in
-
Xcnopus oocytes, a significant proportion of the currcnt thmugh the channcls is carricd by ~ a " :this c a 2 ' influx activates a ~a"-scnsitivc CI channcl. High levels nf r 7 transcripts a r t dctcctcd hy in situ hyhridizationt in the olfnctory areas, thc hippocampus, the hypothalamus, the amygdala,
; ~ n rtlh e cerebral cortex. These rc.;ults implv t h ; ~ tY?-ccrnt;~~nirig rccrptcrrs I ~ I ; I V pl;~y;I rcrlr 111 : ~ c t i v ; ~ t i nc;~lcium-tlcpc~i~lcnt g mcch;~nisrnsIn sl.rccific r i r i ~ r o n ; ~ l of thc ;~ilultr;lt limllic svztcm". pop~rl;~tions
l < ~ . ','s' c ~ l c ( ~ t i vofi t ~t11c ~ r(/t ( 1 7 c./i(~r~ric~l 09-40-07:Thc nicotine-;~ctiv;~tccl r;lt 77 ncuron;~lnAChR, cxl.rressctl in X(2nq~ur: rjocytcs, shows ;In ~ n w ; ~ r drrctifvinnt lv current in cxtcrn;~lIl;lriu~n:in calcilum tlic, response is 1;lrgc.r ; ~ n d1i;ls ;I l i n r ; ~ rILV rcl;~tion.Tlir pcr~nc;~l>ility r ; ~ t i I'l,,/ ~) I'y., oi tlic r;~t77 receptor h;~.;1.rcc.nc.;tim;~tcd; ~ ;~liout t 1 7'7W.
'I%(* 0 8 011tI o c ) 1~01no1~1(~ric~ (~hi11111~1,s (lrc / T ( , ~ ~ I Tt (o ~(:(I," (I~I~~~ 09-40-OR:TIicch~ckIS s t ~ l ~ ~ wliich ~ n i t . h;1zA2"r, ;~mincr;lcitlitlcntity w ~ t ht h e 7.7 sul>uti~t ; ~ n t l ; ~i(lcntic:~l n M2 d o m ; ~ i n;,~ l s oform.; Iiomomcr~cch;~nnclspcrmc;~l.rli~ t t r <:;I" when ~lrotlucctlIn, cslirc.;sion of I S cRNA in ,Yrrlop~~< cr~cvtcs"~.T h e more tlist;~ntlvr c l ; ~ t r d7') zr~l>unittrom r:lt, ~vhiclili:~s. 7 X 0 : , identity to t l ~ cr7 ; ~ n t lYS suhunits t \ i P ( ,Ilor~ioloyo~rx rxolort?~\.(10-2I1, ;11.;o fortiis ;I homonicric crocytcs2". In channel that 1s pcmic;~hlcto (:;I" wlic*n csprcszctl in .Yrjr~o;~~rs hoth tlicsc c ; ~ \ c s~, n f l u of s c:;I" tringcrs ;I (:;I "-scns~tivcC:l currrnt. Mur(lr?l r , / ~ r l n ~ ~ ttTitll i , l s ir~.rc,cl.cotl~ c ~ l [ ~ . t i/or \ f i r1ir7cllr1i~t t~~ c.rrtio11s 00-40-00: T h e rcpl;lccmcnt of Thr?.-$4 Ilv Asp in thc p i ~ t ; ~ t i vch;~nncl-f(rrnling c ML scgmcnl ot the c l i ~ e kncurcrn;~l 77 nA(:Iill sulitlnit produces ;I m;~rkcd clx~ngcin the .;clcrt~vitv ot the Iic)mo-oligoriicric ion cli;~n~icl protllrcrtl I>v exlircss~onIn .Y(*r~oprr\ crocvtcs. T h e rcl;~tivci o n ~ c~ c r ~ i i e ; ~ l ~of~ wild-tyl)~ litv A to K \ 1 I I.? I 1 I , I , I , 1.4. In ccrntr~lst, c tliscririiin;~ting Iwt\vccn K ' :1ni1 Nn', A(:Iil
Single-channel data Singlip i.l?rlrrnrls short, irirvclrtl rc~.tific.i~tion 09-41-01: In l.rotli ncuroncs ; ~ n dmuyclc, ion pcrmc;~ticrn t l i r e ~ ~ ~ gsingle li r i.; dcpcnilcnt (111 c h ; ~ n n c l s .;how.; i~iwarrl ritrtificstionl, h c I i ; ~ v ~ o utli;~t intcrn:~l M ~ - ' ' In . tlic ;~l,scncc. ot M ~ ' ' or (13'' on either sidc r r f t h e ~ n c m l > r ; ~ nsci n, ~ l c - c h a n n e lI-V plot.; ;Ire linc;lr, thou,qh whole-cell currents continuc t o how i ~ i ~ v r~~rcdt i t i c ; ~ t i o ~ i ' ~ " .
Sin~lr.-c.l~rlr~nr.I c.c~r1rl~ic.tr/11c~t'.\ o f rlr~~lrontrl ~./I~IIIIIL'~S 09-41-02: T h e unit:~rvconduct;~nccof nAChl1 ch;~nnclsin rat sympathetic niwronps v:~ricsfro111 2(, to J S [is, \vith ;I ~ i i c ; ~ of~.W.S i [is,in I IIIM <:;I", I:cmov;il ot d ~ v ; ~ l c cn ;t ~ t ~ o trow n\ t h e c s t c r ~ i ; soli~tion ~l incrc;~scst h e unit;~ry ch;mncl conduct;~ncc. Altcriri!: tlir m ; ~ i n pcniic:lnt icrn in div;~lcnt-frcc solutirrns g ~ v c zthe following contluct;~nce>ctlucncc: K ' ilj,Z PSI . (1s' (61 PSI N;)' 15 I pS1 . Ll'(2.1 1iSt. licpl;~ccmcntof N;I' I.ry C s ' In t h c cxtcrnal solution ctrnsiilcr;~hlvrctlucc.; the current cvokcd hv ACIi in \vliolc-cell rccorilings and t h e cli;~nncl-openingfrcqucnc\r in outside-out 1 l~;~tclicsfx').
entry 09
-
Single-channel condrtctances o f different subunit comhinations 09-41-03: Single-channel conductanccs of avian and rat suhunit comhinations produced in Xenopus oocytcs arc as follows: a4nz1, 20 pS; 92/12, 33.6 pS and 15.5 pS; 15.4 pS and 5.1 pS; 14/{2, 13.3 pS. The 94/12 comhination also has a secondary conductance state hut this has not heen ~haractcrized'~'.
Multiple conductances in a sin& cell 09-41-04: There is strong elcctmphysiological evidence for functional heterogeneity of neuronal nAChRs, hascd on sevcral open-channel conductanccs hcing observed under identical conditions in a single cell. Thrcc or morc populations of conductanccs are ohscrvahlc in rat PC12 cells'", bovine chromaffin cellsrn" and chicken sympathetic neuronesrN4.
Rrlrsting behaviout of nAChR 09-41-05: Roth neuronal and musclc nAChR channels display bursting behaviour. Rurst durations vary amongst different channcl types: several nAChR channels on autonomic neurones show burst lengths with time constants in the range of 5-10 r n ~ ' ' ~ . ' ~ " .
-
Blockers Non-competitive blockers 09-43-01: A heterogcnenus group of pharmacological agents, collcctivcly known as non-cornpetitivet blockers, can inhihit the ionic pcrmcahility of musclc and ncurt~nal AChR without affccting the agonist site. Such hlockcrs include aminated local anaestheticst ( e . ~ .procaine, lidocainc, dihucaine, proadifen], sedativest, such as chlorpromazinc, and various toxins. Studics with .4~-!ahcllcrlnon-competitive hlockcrs, including histrionicotoxin (a frng toxin], phencyclidine (a halucinogcn) and chlotprnmazine, define high-nffinity binding sitcs, sensitive to histrionicotoxin 0.1 h 11~1, and low-iiffinity sitcs insensitive to histrionicotoxin (K,, > 100 / I M ) . The high-affinity sites arc present in one copy per receptor pcnt:ltner, with thc low-affinity sitcs hcing 10-,3[)-fold morc
Non-competitive bIockers intetrlct with the open chnnncl
-
09-43-02: Lahellinp; of the nAChR hy photoaffinityt derivatives of nnncompetitive hlockers is enhanced by carhamylcholinc and inhihitcd hy histrionicotaxin. The rate of covalent association of chlorpromazine with the high-affinity sitc incrcascs 100-1000-fold (k,,,= 10' M . ' S - ' ) when acctylcholinc is added in the conccnt rat ion range cffectivc for activation of the channcl in vitro. Competitive antagonists hlock this effect. Tlic rate of ( 'HI-chlorpromazine incorpori~tion tlcclincs on prolonged exposure of the channcl to acctylcholinc, with a time course ant1 concentration dependence similar to those of the rapid desensitization1 of the ion-flux responses of the native mcmhrancs of T. crtli~ornicu.These observations sumcst that the non-competitive blockers frccly diffuse to their high-affinity hinding sitcs within thc porct of the open channel'".
entry 09
-- ----
-
--- -
..
A -
Spccific a m i n o acids confcrcted h y non-cot71p~titivch1ockc~r.s 09-43-03: T h e amino acids plio~olnhellcdthy ['HI-~hlor~romazine have hcen
irientified hy peptidc-mapping' and sequencing experiments. T h e rcsidircs ~ S c r 2 4 8 ,/ISer254, PLeu257, ;.Thr2Sc3, ;tScr257, ;,Leu260 and (iSer262 arc all lahellcd hy [ 4 ~ ] - c h l o T r o m a z i n cand protcctccl hv phencyclidine. T h e lahcllcd scrines on all suhunits occupy homologous p o ~ i t i o n swithin the putative M2 helix that lines the pore of the ~ A C ~ ~ R ' ~ " ( I . ' I4,)~. .Experiments with alternative non-compctitivc hlockcrs reach very similar conclusionsffn~ "".
l'rocyrstcronr as
(1
hlockrr o f niwronal nACl7R
09-43-04: T h e in;iinr hr;iin nAChR is ;isscmhled from two suhunits tcrmcd 74 CLEFT 7
P
A I L
CMOPLASM
Figure 4. Modc.1 ol the hi,qll-(rffinitysitc for c ~ h l o r p m n ~ c ~ within z i n ~ ~ the nA<:hH chmnnrl. Tllc M2 tiommir?.s clrr .sllorvn as trrinsmrrn1,ranc hcliccs. t~r~cr.si-.syn~rnr~tri(:(~ll\~ t~rron,q~,tl trrountl thc c.cntrr11 (/xis o f the rnolccnlc~. thr M2 hclicvps o f r h ~ , ond subunits crrc s h o ~ v nin cktcli]. Thc o c.mrl)ons o f the omino (~ciiisN ~ shown, L wit11 thc rcsidurs designated l1y thr ctrrnrl(lrt1 single-lrttcr code. Thr crntral sphr*rr rcprcscnts the spncc orc.upicd by rr chlorpromnzinr n?olrc.ulc in (111 po~sihlcorirntrltions nt its ct al. (1990) Proc Ilinclin!: sitc. (I
-
and n r l [Note nrl is equivalent to 112, see table 1, p235. Whcn produced in Xenopr~soocytcs, these s u h u n ~ t sreconstitute a functional rcccptor that is rnhih~tedhy progesterone concentrations similar to thosc found in serum. The steroid intcracts with an uxtraccllular sitc on the channel prntcin, and the inhlhit~ondocs not rcqulrc agonlst, 1s voltagc ~ndepcndcntand docs nnt affect rcccptor dcscnsitlzatlont . T h e lnhih~tlcin hy pmgcstcronc 1s not competitive, though ~t may involvc interaction with thc ACh-hindlng s ~ t c , and is indcpcndent of thc ionic pcrmeahil~tyof the r e c ~ ~ t o r ' ~ .
Toxins as blockers of nAChR 09-43-05: The ganglionic antagonist neoqurugatoxin ( 2 n ~ causes ) almost
cnmplcte hlnckadc nf ACh responses In Xenopus oocytcs cxprcss~ngr2/!2, d p 2 or 14/12 combinations, hut is ineffectivc against thc mvsclc rcccptor formcd by microinpction of XI,p l , ;. and 5 subunit RNAS"'. 09-43-06: Lophotoxin (10 I L M ) , an inhibitor of neurotransmission at ncuro-
muscular junctionst and autonomic+ ganglia, complctcly hlocks the muscle al/il;.$ and the neumnal z41j2 nAChRs, hut only partially hlocks thc z2P2 and r3P2 specie^'^'. 09-43-07: Thc a-conotoxinp C I A and M1 block the muscle z 1 PI 76 rcccptor at 100 nM, but arc ~ncffcctiveagalnst ~ 2 f l 2 ,3312 OF r4P2 cornhinations at concentrations up to 10 (IM"'.
n-Rgt-sensitive and rr-Rgt-resistant channels in hippocornpal neurones 09-43-08: Two types of whole-ccll nACh currents can hc distinguished in
culturcd post-natal rat hippocampal neurones: onc showing rapid dcscnsi tiza tiont and sensitivity to x-hungarotcix~n(iC511 of 1-2 n ~ ~thc ) ; sccontl activating slowly, showing no dcscnsitization and insensitive to the toxin. Roth thesc currents are blocked by 0.1-1 .0 m M (+)-tubocurarine and 0.1-1 .O mM rne~arn~larnine~~''.
A plant toxin inhibits ACh-induced currents in hippocnmpal neurones Picomalar methyllycaconitine, a toxin from the sccds of l)t.lpI~~nirrm hrownii, inhihits acctylcholinc- and anatoxin-induccd wholeccll currcnts in culturcd foetal rat hippocampal neurones. This antagonism was specific, concentratinn-dependcnt,rcversihle and voltngc-independent. The toxin also inhlhits I ' " ~ ] - s - ~ g tbinding to adult rat hippocampal membranes, protects against thc 2-Rgt-induced hlnckadc nf nicotin~c currcnts, and shifts the conccntration-rcsponsc curvc of acctylchol~ncto thc right, suggesting, a cnmpctitivc mode of action. Low concentrations of methyllycacnnitinc (1-1000 f ~ decrease ] the frcqucncy of anatoxin-lnduccd singlc-channel openings, with no dctectahlc dccrcase in thc mean channel open time""'. 09-43-01:
I
Alcohols ns blockers of nACh R 09-43-1 0: T h e n-alcohals hlnck thc activity of thc nAChR from cultured rat
entry 09
myotuhes. Intcrmcrliatc-chain nlcohr>ls (pcntanol to octanol) causc currents to fluctuate hctwccn thc fully open : ~ n dcloscd statc Icvcl, the number of gaps within a hurstt increasing with alcohol conccntration. Nonanol and dccanol reduce thc mcnn duration of Iwrsts of openings hut do not causc an increase in the numl-rcr of short closcd intervals w ~ t h i na hurst. Rcyond decanol thcrc is a dcclinc in thc ability of the n-alcohols to affect nAChR function, ant1 a satiiratcd snlutinn nf dodccanol has n o significant cffcct. The value.; rlctcrm~ncrlfrom thc cffcct on the total charge cartled per 0.14 ITIM; heptanol, 0.097 4- 0.02 mM; hurst arc as fr~llows:hrxanol, 0.53 octanol, (104 mM and nnnanol, 0.16 ! O.O,ii m M . The K,! valucs, dctcrrn~ncdfrom thc ratio.; of the hlock~ngand ~ ~ n h l o c k l nrate g constantst, dccrense with ~ncrcnsingchain Icngth from X W M for pcntanol to 0.15 mM for octanol'".'.
+
Antrest het ics tzs hlockcrx nT nA Ch R 09-43-11: The volatile anaesthetics halothane and isoflurane hlock the activated nAChR channels of culturetl I
Other hloukers o f I I A C ~ channels R 09-43-12: The alkn!clid strychnine, a classical hlockcr of glycinc-gated C1 channels, hlocks the rat 27 nAChR expressed in Xcnopus oocytcs with an lCill of 0..35 ,IM'. The homomeric YY channel from rat, exprcsscd in Xcnoprrs oocytcs, is strikingly sensitive to hlockagc hy strychnine, with an ICq) of 0.02 l , ~ 2 1 H ( S P C II~ccl>tor ~iritt~.yonist.~, 00-5 1).
09-43-13: Hexamethnnfum (2.5 / I M ) and decamethonium ( I 0 jlhll hlock the function of thc avi:ln 741-171 nAChR in a voltage-clcpenrlcnt manner, supysting thnt they cntcr the channcl porc'qs.
n
Channel modulation 09-44-01: SECProtcin phosphorylation, 09-32.
I?otentiation o f nicotinic response by external
cn2+
09-44-02: Thc nicotinic response of the nAChRs of neurones from the rat
medial hahenular nucleus (MHh) is strongly potentiated hy extracellular <:a". T ~ ampliti~rlc c of whole-cell currents evoked by acetylcholine is . potentiationt, which is increased up to ,i.ti-fc~ldhy 4 mM external ~ a ' +The rapidly ruverscti on removal of ~ a " , is due to a change in conductance, and is observed at both negative and positive potentials. The effect of extcmal c a 3 ' is similar when internal c a 2 ' is buffered hy 10 mM RAPTA or 0.5 mM EGTA, surncsting that the ~ a "acts at an cxtcmal site and not at an internal site aftcr cntry through the nAChR channel.
entry 09
-
ca2+increases the frequency o f channel openinq 09-44-03: Single-channel recordings with outsidc-outf patches show that the
trcqucncy of opening of ACh-activated channels is increased hy a factor of 3.1 after addition of 4 mM ~ s " , thc magnitude of the potentiationt increasing linearly with cxtemal [ c a 2 + ]up to 4 mM. The potcntiation is also achieved with sr2+(4.5-fold at 4 m ~and ) Ra2' (.3.2-fold at 4 m ~ )hut , not with 4 m M ~ g " . Thcsc data arc interpreted as indicating allostcrict modulnticln of nAChR hy cxtcmal ~ a "to favour a transition to a rlistinct, AChactivatnhlc statetY".
Negotive effect o f divalent cations on sin,qle-channelcondlrctrrnce 09-44-04: Notc that thc divalcnt cations c a 2 * ,~ r " , Ra" and MK" at 4 r n M all
dccrcasu thc single-channcl conductance of nAChR at negative (hut not positivc) potentials hy ahout SO%, a finding that has heen interpreted as a screening effect of the divalcnt cations at the entrance to the channel. This inhihitory ctfcct contrasts with the potentiationt that is sccn with Ca2+, ~ a ? and ' Sr?', hut not with M ~ " , occurring at both ncgativc and positive - potcntials'ph (see above).
-
EquiIihrium dissociation constant Dissocintion constant for rr-hungarotoxin 09-45-01: The dose-response curve for thc hlocking of thc nAChR of chick
ciliary ganglion neuroncs hy z-Rgt yiclds an ICso of 1-2 nM. This is in good agrccmcnt wlth the Kd of 1.4 nM dctcrmined from ~ c a t c h a r d tanalysis of I"'1j-r-Rgt hinding to the target on neurnncs""~~11~le51.
Tahle 5. Affiniti~s o/choIinergicfl.cents forncuronn1nAChR.v(From 09-45-01)
K, V ~ ~ L I C( Sn ~ ] Ligantl tr-Rgt Nicotine (-t)-Tuhocurnrinc rr-Cohratoxin Methyllycnconitinc
rr-Rgt-scnsitivc
tr-Rgt-inscnsitivc
0.7 18
.7 100
600 1.7 2.8
8300 I ls3 1,18
Ki values wcrc dctcrmincd h c uilihrium hinding experiments in which the lipantla competed against i"'~l-~-Rgt in the case of the n-l3gt-sensitive channels or against [ ' ? - ' l ] - n - ~ in g t the casc of thc (1-ljgt-inscnsitivc channels. Data taken frr~mrcf.'"l
rk-Rgt interaction with the t r 7 channel 09-45-02: The lCSo for the hlocking of thc chick 27 nAChR cxprcsscd in
Xcnopus oocytcs hy r-Rgt is 0.7,Z n ~ " , in close agrccmcnt with thc EC5(] (0..ZS-0.61 n ~of) the complex hctwccn the toxin anci its hinding proteins in CNS mcmhrancs4", suggesting that most of the high affinity 2-Rgt-binding proteins in chick hrain may contain the r 7 suhunit.
entry 0 9 .
-
.-
--
-
-
11i.s.soc.irition c+onstrintso f vririous r7AC171< S / I C C . ~ C Sfor r i ~ c t ~ ~ l ~ h o 1 i n ( ~ 09-45-03: T h e apparent dissociation constants o f acetylcholine for t h e nAChR vary with the species of receptor. T h e K,, v;~lucsfor ~411x1, 24n2,Z, r . Z n ~ l , r.3nr.3 :lnd 27 channels alrc 0.77, 5.8, 5.6, 158 and 115 , ) M , rcspectivcly1'", while that for t h e homopcntamcric Y X receptor is 1.9 lr~'17.
Binding o f rigoni.sts nnd r~ntn,qoniststo homomcric chrinncls 09-45-04: T h e binding characteristics c ~ af range c ~ agonists f ant1 antagonists to h ( ~ m o m c r i c77 (chicken and human], rX lchickcnl and 7'1 (rat) ch;lnncls cxprcsscrl in Xrrnollr~soocvtcs :Ire sllmma~rizcdin T;lhlc 6.
Table 6. IIir?rlir?,yol ~l,yoriivt~ on(/
to I ~ o r ~ ? o r ~~IACIIK ~~~ri('
I I I I ~ I I , ~ ~ ~ I . Y ~ Y
c~litrr~n(.l,s (Fror??0')-45-OJJ t ~ o m o n i c r i cchannel 0 7 (chick\**'
117 (human121y 118 (chick1217 ocl (ratlZfx
ECi,, o r lC50 Agonist Acetylcholine L-Nicotine Cyti.;inc I) M 1'1' TMA Antagonist I)-Rgt Atropine Curare Strychnine
-
110 7.8
1H .30h 800 0.00,Z 7.1 0. I4 0.52
79.1 40.1 71.4 25.5 I01 0.0021 115 0.7 7..i
(,r,\ll
I .O
10
1 .O 1 .O 6.5 10
.?(YJ
0.001 4 0.4 0.6 0.8
I
ca 50"
I ,.3 0..i 0.02
Nicotinc (0.1 lrr.4 to 1 tilh!\ ;lnd cytisinc d o not act as ; ~ ~ o n i sfor t s t h e 119 ch;lnncl. Nicotine rccli~ccsthe currents inrl~lccdhv ACh, with an 1 C ~ oof 30 , ~ b l ~ ' ~ . 1, I>MI'P (I,l-tl~mcthyl-4-phcnylpipcr;~zoniitnil is ;I wca~k,p:~rti:~l a~gonistwith chicken ( 1 7;lnd rat 119ch;~nncls,Ilut ;I full i~gonistfor hi~tnaln117c l l ; ~ n n c l s . ~ ' ~ ~ ~ ~ ' T h e IC:,,,, for 11-ltgto n O V channcls h ; ~ snot hccn ilctcrtnincd, hut the currents inducer1 hv 100 1 1 1 ACh ~ a~rccompletely clitnina~tcdI7y 100 nhl rr-~tgt"~.
Hill coefficient I f ill c.r~ftt'lic.ic~nt for o c r t ~ ~ l i ~ h o l i n c . 09-46-01: T h e nAChR ch:lnnels arc predominantly activated hy the hintling of t w o molecule.; of ;~cctylcholinc,with ;I Hill coefficient1 of 1.7-2.0'". Several types of c h ; ~ n n c lopenin,< can hc tlisccrncil clcctropllysiologica~l~ and it is likely th;it ;I cl:lss of vcrv hricf opcnings arises when only :I single molcculc ( S I Y Kir?r,tic ri?orh,1,09-(78). of :lcct ylcholinc i.; hoi~nrl"~'~
Hill coc~fficirr~t /or r , -Hgt hindins 09-46-02: T h e chick 27 channcl expressed in Xcnoptts oclcytcs is sensitive t o 7 1 C 0 . . 3 nni317; 0.7.3 nu"), with ;I Hill coefficient tluotcd as 1 ..1*" or at least 6'-.
Ligands Snnke venom neurotoxins 09-47-01: Two snakc venom neurotoxins, a-bungamtoxin (a-RgtJ and
neuranal hungarotoxin (n-Bgt; also known as h--hungarotoxin, hungarotoxin 3.1 and toxin F) are frequently used to characterize ncuronal nAChR suhtypes. The r-Rgt hlocks most muscle nicotinic rcccptors and somc, hut not all, cloncd or hiochcmically purificti ncuronal rcccptors from the chick, rat, and insect nervous systems.
Amino acid sequence motif common to (1-Rgt-sensitivesuhunits 09-47-02: Thc sequence motif Cys-Cys-X-X-Pro-Tyr
is common to all known r-subunits in ncuronal receptors hlocked hy r-Rgt. In r-Rgtinsensitive ncuronal rcccptors, the Pro is replaced hy Ilc: other amino acid suhstitutions may also he important. Neuronal hungarotoxin hlocks somc, hut not all, r-Rgt-scnsitivc and -insensitive rcccptor suhtypcs cxprcsscd in oocytes, and in hoth vertebrate and invcrtchratc nervous systems. Non-z suhunits hclp dctcrmine the ahility of ncuronal hungamtoxin to hlock functional ncuronal receptors2"'.
The rr7 channel is very sensitive to tr-Rgt 09-47-03 The r 7 channcl is distinguished amongst ncuronal channels hy its
sensitivity to r-Rgt. The chick 27 channel cxprcsscd in Xcnopus oocytcs has a K, of 0.73 nM for z-~gt", whilc thc rat 27 isoform is complctcly hlockcd hy nanomolar amounts of toxinp.
Sensitivities o f different suhunit comhinations 09-47-04: None of the rat or avian rcccptors formcd in Xenopu.~oocytes hy expression of RNAs encoding r2, r3, 24, /?2 and P4 suhunits is functionally hlockcd hy nanomolar r-Rgt (see Tnhlc 7). The homomcric chick a7 and rH
channels producctl in Xrnopus oocytcs arc hlockcd hy r-Rgt with ECqn valucs of 0.000.3 /IM and 0.0014 ,IM,rcspcctivcly2".
Tahle 7. Toxin scnsitivitirs o f nrnronml nAChli suhunit cornhinotions rxprrssrd in Xcnopus oocytcs (From 09-47-04) nAChR composition
tr-Rgt (0.1 / / M I
n-Rgt (0.1 / I M )
NSTX (2 n ~ )
LTX- 1 (10 PM)
n 2 d2 rt3,92 rr4;92 (~4ntb 1'' rr3 94" (k7"
0 0 0
0
+++ +++
++ ++ +++
rrR"
+ + +(O.Ol/ I M J +
(0.3 / I M ) 0
St+
0 (0.5 /,M\ 0
t t- .k
+++
"Avian subunit comhinations. 0, n o hlockadc at inciicated concentration; +, ++, partial hlockadc at indicated concentration; + + +, nearly complctc hlockadc at indicatcd concentration. NSTX, neosurugatoxin; LTX-1, lophotoxin.
entry 09
-
09-47-05: Neuronal Rgt strongly hlocks the rat r.Z/12 comhination nt 0.01
~IM,
and also antagonises the r4/12 combination, though with IO-I0O-fold less scnsitivi ty. (t
-Rgt -scnsit ivc centrril nicotinic rcsponscs
09-47-00: There arc a few cases in which r-Rgt hlocks central nicotinic
rcsponscs. In thc,rat hippocampus, for example, nicotinic responses arc hlockcd hy 20 nM n-Rgt o r . 3 0 0 nlv r - ~ g t ~ " ' Such . nAChRs, strongly hlockcd hy relatively low concentrations of both 7-Rgt and n-Rgt, have hccn characterized in chicken cochlear hair cellsz"' ant1 in insects'"'.
Kn1~17i1-flavotoxin ~ n t r ~ ~ o n io3 z c suhtypcs s o f nAChR 09-47-07: Kappa-flavotoxin (K-FTX), a snake neurotoxin that is a sclcctivc
antagonist ot certain ncuronnl nAChRs, is rclatcd to hut distinct from nRgt. T h e K-FTX hinds with high affinity to r,? suhtypcs of ncumnal AChRs. T h e region of the ncuronal AChR x? suhunit forming the hinding site for K FTX has heen identified using ovcrlapping synthetic peptides covering the 2.3 sequence. T h e 'prototopc' for K-FTX-hindingwas identified within amino acid rcsiducs 51-70 of the Y ~suhi~nit'"~. ?
Othcr toxins 09-47-08: Neosurugatoxin spccificnlly hlocks neuronal nicotinic receptors in
mouse ncuromuscular junctions1 (when partially hlockcd with tuhocurarine or with low cnJ'/high ~ g ? tyrodc ' solutionl. Ncosurugatoxin (
An immunoactirre polypcpti(1c hlocks nicotinic responses 09-47-10: Thymopoietin, n thymus-derived polypeptide involved in thc
niotlulation of immune function, inhibits the hinding of I " " l l - ~ - ~ g tto mcml~rancstliroughoi~tthe brain. Thymopoictin potently blocks nicotinic receptor-mediated rcsponscs in m i ~ s c l c cells, interacts with ("'I]-r-~gtbinding sites on rat PC12 cells and hlocks nicotine-induced inhibition of neurite outgrowth2"'. It has hccn s u ~ c s t c dthat cxtrasynaptic 2-Rgt-hinding protcins scrvc to monitor cxtrnsynaptic ACh levels and hence regulate ncurite outgrowth hy raising frcc intraccllular [ ~ a ? ' ] " " .
Chcmicml lignnds: cyclic compotrnds that activate 09-47-11: T h e cyclic compound 1,l-dimethyl-4-acetylpiperazinium iodide
and its trifluoromcthvl nnnloguc (F3-PIP) intcrnct with nAChRs from both 'I7orpcdoc ~ e c t r o ~ l a q uand c ~ RC3H-1 cells at lower conccntratinns than thc acyclic dcrivntivcs, N,N,N,N '-tctmmethyl-N'-acctylcthylendmine iodide ant1 its fluorinatcil analogue IF,?-TEn). In mcnsurements of the initial interaction with the nAChR, the PIP compounds hnvc an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incuhntions indicate that the I'IP cc~mpoundsarc ahlc
-
to induce a time-dependent shift in receptor affinity consistent with dcscnsitizationt, whcrcas thc TED compounds arc unahlc to induce such a shift. The activation of single-channel currents by the cyclic compounds occurs at concentrations in the micromolar rangc, approximately two orders of magnitude lower than for thc acyclic compounds, but the TED compounds cxhihit a larger dcgrcc of channcl blockade than thc PIP compo~inds~~".
Chemical li'ynnds: tricyclic antidepressants inhihit nenronal nAChRs 09-47-12: Two structur:llly rcl;~tc~l tricyclic antidepressants inhihit ncuronal nAChR currents in human neurohlastorna (SY-SYSY) cells. Roth desipramine and imipmmine rcversihly inhihit inward currents evoked by application of the nAChR agonist dimcthylphcnylpiperazinium iodide [,30,300 I I M ) with lCso values of 0.17 IIM ant! 1.0 IIM rcspectivcly (holding potential -70 mV). The dcgrcc of current inhibition is unaffected by agonist concentration. The effects of desipramine arc voltage-indcpcndcnt ovcr the rangc 4 0 to 1 0 0 mV, and inhibition caused by imipraminc only increases very slightly with mcmhranc hypcrpolarization~ ovcr the same range2"'.
1
Receptorltransducer interactions
09-49-01: At the neuromuscular junctiont, motoncurones release calcitnnin gene-related peptide (CGRP)~in addition to acetylcholine. Post-synaptic rcccptors for CGRP couplcd to G, and adcnylyl cyclasc can clcvatc the level of CAMP rcquircd for desensitization via phosphorylation (see I'rotcin ~ ~ h o s p h o r ~ ~ l o 09-,?2). tion,
-
Receptor agonists (selective)
The different molecr~larspecies o f nAChR show distinct n,qonist potencies 09-50-01: Thc rat r7 nAChR exprcsscd in Xcnopus oocytcs displays the following order of agonist sensitivity: nicotine > cytisine > DMPP > A C ~ ' (sce Er7uilihriurn dissociation constant, 09-45). The rcccptors from ncuroncs of the rat interpeduncular nucleus show thc ortier of potency cvtisinc > ACh > nicotinc; thosc on ncuroncs from the medial hahenula have nicotine > cvtisinc > AC~".'.
The 0 9 homomeric chnnncl has mixed nicotinic-muscrlrinic pharmacolo~qy
-
09-50-02:The homomcric nAChR channcl ohtaincd hy cxprcssion of cRNAs cncoding the rat 2 9 suhunit in Xennptis oocytcs shows an unusual pharmacological profile, with characteristics of hoth a nicotinic and a muscarinic acctylcholinc receptor (see Kccci~tor nntogonists (sclcclivc). 09-51). The channels arc activated hy ACh (ECstl 10 / I M ) , hut not by nicotinc or the nicotinic agonist, cytisinc. The muscarinic agonists, hcthanccol and pilocarpine arc also ineffective. Surprisingly, hoth the nicotinic agonist 1,l-
entry 09
--
1
dimcthyl-4-phenylpiperazinium (DMPP) and the muscarinic agonist oxntremorine M ( 0 x 0 - M ) act n s partial agonists, eliciting maximum responses of ahout 5'";)of that ohscrvcd with ~ ~ 1 1 "The ~ . unusual pharmnc o l o ~of the 70 channcl is strikingly similar to that of the cholincrgict receptor prcscnt in vcrtcl>aatr cochlear hair c c l l ~ ~ ' ~ .
-
Receptor antagonists (selective) Forskolin r~nta~onizes hy interaction with the . I suhunit o f nAChR 09-51-01:Forskalin at micromo!ar concentrations acts as a non-competitive inhilritor of the 'l'orprbrio clcctroplax [ K , 6.5 ~ I M )ant1 mouse muscle (K, 22 j 1 ~ )nACIiIis rxprcsscd in Xi-nop1r.s oocvtcs. The antagonist reduces the n l ~ m h c rc ~ f chnnncI opcnings prr unit tirnc hy interaction with the ;. sulrunit of thc ~ A C ~ R " ' . Inhihitors o f phosphntc-trrinsfer rractions as nnto~onistso f nAChR 09-51-02: T h e single-channel and macroscopic ACh-intluccd currents produccci hy cxprcsssion of the muscle channcl in Xcnopus oocytcs are niotlifictl I>y IRMX (3-isahuty1-1-mcthylxanthine),a phosphotlicstcrase inhi hi tor, and hv H-7 (l-(5-isaquinolinylsulphonyl)-2-methylpiperazinc], ;I non-specific inhil~itorof protein kinasc activity. Roth IRMX (IC:5,) 475 / / h l at . Z 0 rnVl and H-7 [1C5,1I60 phi) tlircctly inhihit ACh-induccd currents indc,pcpnric.t~tlv of thcir mction on phr),sphorylc~tir,~~. H-7 prcfcrcntially inhihits the open nAChR channcl, hut thcrc is also some inhihition of the closcd contormation. This inhihition IS voltagc-dependent, tlccreasing [.-fold per $34 mV dcpolarizntion. A siinilar inhihition is also prod~~cctl lrv .30 / I M H A 1004,;in :inaloguc of H-7 thnt tlocs not inhihit protcin kinasc activity'".
A potcnt r~ntcrgonist{ram Dclphiniuni sr~cds 09-51-03:Mcthyllycaconitine, a toxin from the seeds of Dclphinilrn? h r o w ~ i i , inhihits acctylcliolinc- ant! anatoxin-induced whole-cell currents in cultured foctal rat hippocampal neumncs, a t picomolar concentrations. This antagonism is specific, concentration-dcpcnctcnt, rcvcrsilrlc, ant1 voltagcindcpcndcnt. Mcthyllyelconitinc also inliilrits 7-1
The (19 rrccptor-chmnnrl shows nn unusunl pl1rrrnir~c.oIo,yicdprofile 09-51-04:T h c homo!ncric chnnncl produced hy expression of the cRNA in Xi,noprr.s oocytcs tlisplays novel patterns of c n c o d i n ~tlic rat 79 s1111~1nit response to cl;lssicaI nicotinic nntl rnuscnrinic agonists and ant;~gonists*'~. Thc classical cholincrgic agonists nicotine ;md muscarine rctlucc the currents induceti hy ACh with rC5,,values of 3 0 / / M and 75 ~ I Mrespectively. Thc 79 channcl is also lrlockcd hy thc nicotinic antagonist, (+)-tuhocurarine (ICSo O..i / I " ) 311d 1ry the mi~scarinicantagonist, atropine (IC;I1 = 1.311~1. /The homolnrric 78 channcl is also sensitive to :ltropinc, with an 1Cso of 0.4 /(dl7 - sce F r ~ i ~ i l i h r i di.s~o(.itr~ion ~~i~i C O ~ I S ( L I R I . 09-45.) T h e alkaloid strychnine, a I>l(lckcr of glycinc-gated chloride channels, is a potcnt -
-
antagonist of x9 channels, with an ICso of 0.02 /IM. The x9 channcls are sensitive to hoth r-Rgt and K-Rgt. Although ICso values tor these toxins have not hccn dcterrnined, ACh-induced currents through x9 channcls arc completely, hut rcversihly, hlnckcd hy 100 nM toxin2''.
~,~,~*,:~,~.~wu~*~c,:l~u~4,~
Database listings/primary sequence discussion 09-53-01: The relevant datobnse is indicated by the lower case prefix (e.g. gl~:),which shotlld not he typed (see lntrodr~ctionel lnyorrt o f entries. entry 02). Datnhase locus nmmes and accession nlimhers immediately follow the colon. Note thnt n comprehensive listing o f all available accession numbers is superflttous for location o f relevnnt sequences in GenRank" resources, which are now available with powerful in-built neighbouringt analysis rotitines (for description, see the Database 1isting.v field in the Introduction d lnyout o f entries, entry 02). For example, sequences o f cro,~s-spccicsvariants or related gene famil memher.~con he rendily accessed hy one or two rotinds o f nci,yhhnuring +ona1ysi.s (which are based on pre-computed alignments performed usins the R L A S T ~ a1,porithm by the N C R ~ ~This ) . feature is most use{ul {or retrieval o f sequence entries deposited in databases later than those 1i.qted below. Th~1.7,representative members o f known sequence homolo,yy groupin~sore listed to permit initial direct rctrievnls hy acce.ssion number, author/ reference or nomenclature. Follow'ng direct accessi~n, however, is .qtrongly rec:~~~nmendr!d to idcntily-n~;~lyreported nei,~hhour~nR~nnn~YY~iiv nnd rcliltcd sequences. -
Y
Nomenclature
Spccics, DNA source
Original isolatc
Accession
nAChR n
Calf skeletal musclc cDNA lihrary
4,77 + 20 aa cm: X02509
Mouse muscle cDNA lihrary
457 aa
T. cnlifornica electric organ cDNA lihrary T, murmorat(1 clcctric organ cDNA lihraiy Xenopt~slaevis devclnpmental stagc 17 c n N A lihrary Mouse myohlast cDNA lihrary
437 + 24 aa gh: JOW(x3
Scqucnccl discussion Nnda, Nnfure (198.3) 305: 81%
L3.
rr-la
nAChR
-
0
em: X039Rh
4.37 + 24 aa gh: M2589.3
457 aa
em: X17244
47R + 27 aa ~ h M145.37 :
Iscnhcrg, Nuclcic Acids Kes (1986) 14: S t 1 1 . Noda, Natl~rt J IOH2)299: 793-7. Dcvillers-Thicry, Adv E x p Med Bin1 (1984) 181: 17-29, Hartman, N ( I ~ I I ~ E (1989)343: 372-5. Ruonanno, Riol Chem (1986)261: 3645 1-8.
Nomenclature
nAChR h
Species, IINA sourcc
Original isol;~tc
Mousc gmomic DNA lihmrv
gh: 10461)O rntirc . j genc, incluiling introns 469 I 24 an gh: I00964
T. (.ill1(r)rr?ic.ii clrctric orRan cDNA library Chicken gcnomic 4Y7 DNA lihr;~nr
I
Accession
I8 ;la
I145 hp of em: X665.1l DNA sequence upstream of translation5tart T cnlifr)rnrr.ilelectric 501 t 21 an gh: 100965 organ cDNA lihranr C h ~ c k r ngrnomic 4112 t 22 na DNA lihran.
Rat gcnomic DNA lihran~
nAChR -y
M o u mvogrnlc ~ crll 497 t 22 an r m : X0.38IX llnr cDNA 11hran~ Mouse muscle cell Iinr cDNA library
nAChR
r
gh: M.30514
T crrlrlr)rnrcc~ c l r c t r ~ corgan cl>NA l~hrarv
490 t I6 aa gh. 100966
Mousc muscle cDNA lihrenr
4%1 na
em: X5571X
Mousc gcnom~q DNA lihran.
4'1.3 ;la
gh. 1046YX
Rat cDNA
493 an
em: X1.3252
Rat ~ c n o m i cDNA
Promoter gh: L19504 and exon 1 Goldfish [Carocrir~r 462 aa cm: XI4786 rrtinnl cirlr(~ttl
nAChR n2
Sequence/ discussion Ruonanno, I Rlol C1lc.m ( I9891 264: 761 1-16. Noda, Nilttrre (IV8.3I 301: 251-5. Nef, I'roc Not1 Aci~riSci 1ISA (I9841 81: 7975-9. Chah~nc, Drvr/r)p~t~rnt (I9921 115. ?I,?19
Nodn, N N ~ I I ~ C , (IYH.31 301: 251-5. Nrf, Proc Not1 Acclcl Sci I ISA (I9841 81: 7975-9. Yu, N~ICIC~IC Aci(1s I l i ~(1986l 14: ,35.39-5.5. Roulter, 1 Ncrlro~ci(1986) 16: ,3749. Rallivct, I'roc Nrrtl Acotl Sci 1ISA ( 19821 79: 4466-70. Gardncr, Nrlclric Acids Re%( 1 9901 18: 6714. Huonanno, I Hiol CI1i7rn ( 19891 264: 761 1 - 1 6 Criatlo, Nrlclric Acid< RPS (19881 16: 10920. C;oltlman, unpublished (I49~3l. Caulev, I Ccll Riol ( l981?) 108: 6.37-45. Tones, F E R S Lrtt ( 1990)269: 264-8. Sswruk, FMRO I (199019: 2671-7.
Nomenclature
Species, DNA source
Original isolate
Accession
Rat gcnomic DNA lihrary
exons 1-6, cncc~ling 511aa
gh: M20292- Wada, Science [19H8)240: 330-4. M20297 gb:LlOO77
Chicken gcnomic DNA library
496 aa
gh: M37.136
Goldfish (C. ~ u r a t u s )512 aa retinal cDNA lihrarv
em: X54051
Human T-cell cDNA 503 aa lihrary
gb: M.77981
Human neurohlastoma cell linc cDNA lihrary
502 aa
~ b :MKh3R3
474 aa (mature)
cm: X03440
Rat PC1 2 cell line cDNA lihrary
499 aa
gh: L3 1621
nAChR n6
Rat hrain cDNA l~hrary
466 + 30 aa gh: LO8227
nAChR n7
Chicken hrain cDNA lihrary
479
Human neurohlastoma cell linc cDNA lihrary Rat hrain cDNA lihrary
502 aa
Chickcn hrain cDNA library
4K1 aa [rnatl~rel
nAChR n3
nAChR nR
+ 23 aa" em: X70297
480 + 22 an gh: M8527.1
Sequence/ discussion
Cnuturlcr, 1 Hi01 Chcm (1990)265. 17560-7. H~chcr,N~icleic Acrdv Re$ (19901 18 529.3 Mlhnv~lnvlc,/ Fxp Nt~tiro1( 1 990) 111: 175-NO. Fornasari, Nt711roqciLett (19901 111: 3514. Roultcr, Nr~ture (19861319: 36874. Roultcr, rroc Nr~tl Acr~dSci USA (lYK71 14:77k7-7. Roultcr. unpuhlishcd (199.71. Couturier, Neuron ( 19901 5: 847-56. Pcng, Mol Phormt1cnl(1994) 45: 546-54. Si.gui.la, 1 Nr.urr)vci (199.1) 13: 596-604. Schoepfcr, 1 Ne~lro.sci( 1 990) 5: 35-48,
nAChR a9
Rat olfactory cpithcl~umcDNA lihrary
nAChR f l
Goldfish (C. allrrrtr~q) 460 aa [Cret~nalcDNA lihrary terminal fragment) Human foetal b r a ~ n 502 aa cDNa lihrary
nAChR fT3
457 t 22 aa
Elgoyhen, Cell {1994)79: 705-15. em: X54052
e r n X5,7 179
Goldfish (C. nurc~tusl 4%38t 28 aa gh: M29529 retinal cDNA lihrary
Caulcv, 1 N~urnsci( 1990) 10: 670-8.3.
cntry (19
!
---
Norncnclnt~~rc Spccics. 1)NA sourcc
nAChR /J4
Fillman atliilt hminatcm cllNA
Orlxinal ~sol:~tc
Accc*;.;it)n
42.3
em: X67il.3
;lilt'
Scqllcnccl discussion Willouxhln,, N ( * I I ~ ~l.($r < (r. I ( l 0 0 ~ 3 1155: 1.36
II I ~ r a w 4.34 I .ZO :In gh: 1046.76
I i n A l i!wary
Ilcncris, / /
Ilat gcno~nirllNA
I'rotnotcr gh: L2764h rcxiot~:~nd 5' cn(l
Hu, I
N~.rrroc.hr.ni02: ,3Y-S.
"The pt).;lt~on( > I clcavn~cof thc. Icntlcr scqilcncc t c ~Rcncrntc tlic N-tcrniinuc of the matilrc pr~lvpcptidccannot Re i ~ n a t n h t ~ i ~ o idcterrninctl. ~slv Thc numhcring shown i.i nccordinx to thc placclncnt of C:c~iitl~rlcr vr (11. M;ly 111, rncomplctc n t thc. 5' cntl. I,
Related sources & reviews
09-56-01: Major quotcd st~urccs~~'~.~"~*'.~"~"~;diversity of muscle and ncuronal n AChR channclsZ4.2~.Y".21.~. architecture of lfJX, 118. 1.3". 2'4. , activation of t h e nAChR ~ I i a n n c l s " ~ ~site-ciircctcd ;
surface charges and mutagcncsis of nAChR coding sctli~cnccs115~'-72; channel fu~~ction""; phosphorylation of channel s u h i ~ n i t s ' " ~ ~ ~snakc '"; venom toxins nntl n ~ ~ : h l i ' " ' ; r-Rgt-binding proteins"'. Sr.r~rrlso Ri.vorrrc.c E - 1017 (.h(lri11~1 l ~ o o kr i ~ f ~ ~ r l ~ riJrltrv ~ i - i , .60. ~,
Feedback Error-cnrrections, cnhoncement (ind extensions 09-57-01: Plcasc notify specific crrors, omissions, updates and c o m m e n t s o n this cntry hv contrih~sting to its e-mail feedback file (for dctmils, srr3 I. Srwr(.h Critrrirr (41 C S N Ilrnvc~lopml*r?r). For this cntry, scnd cKip.~ol~rc.c~ mail messages To: [email protected], indicating t h e appropriate paragraph hy cntcring its six-figure index n ~ ~ m h e(xx-yy-zz r o r othcr identifier) into the Subject: ficlrl of t h c message (c.g. Suhicct: OH-50-071. Please fccdhack cln only one specified paragraph or figure per message, normally hy sending a corrertc.~eplacernent according t o t h c guidelines in Frcdh(rck el CSN Ar,c.cb\c . Eti11;uicc.m~-ntsand extensions can also 1x2 s u ~ c s t c dby this route (i1jiri.l. Notified changcs will he indcxctl via 'liotlinks' from t h c C S N 'Home' pagc ( h t t p : / / ~ w w . I ~ . a c . ~ ~ k /from c s n / mid-1'+)6. )
Entry support
S ~ O I I J I .find F
c-ntriil ncwslettcrs
09-57-02: Authors w h o have expertise in o n c o r more ficlds of this cntry (and arc will in^ t o provide cditc~rinlo r othcr support for tlcvcloping its contents1 ciln join its support Rroup: In this case, scnd a mcssagc To: CSN09dle.ac.uk, (cntcring tlic words "support group" in thc Suhicct: field). In t h c tncssagc, plcasc inrlicatc principll interests (scc firlrlnnnic c.ritcpria in
the Introduction for coverage) together with any relevant http:l/www site links (established or proposed) and details of any other possihlc contributions. In due course, support group mcmhers will (optionally) rcccivc e-mail newsletters intended to co-ordinate and develop the prcscnt (tcxt-hascd) entry/ficldnamc frameworks into a 'library' of intcrlmkcd rcsources covcring Icln channel signalling. Other (more gcncral) ~nformation of intcrcst tn entry contributors may also he sent to the ahovc address tor group distribution and fcedhack.
' Noda, Nuture ( 1982) 299: 793-7. 'Devillcrs-Thiery, Proc Natl Acad Sci USA (1983)80: 2067-71. Natme (1983)301: 251-5. "oda, wads, Science (1988)240: 3,704. "ooultcr, Nature (1986)319: 368-74. " Galdrnan, Cell (1987)48: 965-73. Boultcr, 1 Riol Chem (1990)265: 4472-82. Sargent, Annu Rev Neurosci (1993) 16: 40,346. SCguCla, 1Neurosci (1993) 13: 596404. "Deneris, Neuron (19881 1: 45-54. " Deneris, 1 Riol Chem (1989) 264: 6268-72. l2 Duvoisin, Neuron (1989) 3: 487-96. 1S Iscnhcrg, 1 Neurochern (1989) 52: 988-91. l4 Ncf, EMRO 1(19RX)7: 595-601. '"Couturier, Rio1 Chcm (19901265: 17560-7. Ncr~ron(1990) 5: 354s. l"choepfer, Couturier, Neuron (1990) 5: 847-56. Nef, Proc Not1 Acad Sci USA (1984)81: 7975-9. lP N ~ d a Noture , (1983) 305: 81 8-21. "Formsari, Neurosci Lett (1990) 111: 351-6. 21 ~ h - ' Inl, !'roc Natl Acad Sci IlSA (19921 89: 1 5 7 2 4 . 22 Anand, Nrlclcic Acids Res (1990)18: 4272. '.' Hartman, Nature (1990)343: 372-5. 24 Steinhach, Annit Rev I'hysiol (1989) 51: 353-65. 2%teinhach, Trends Neurnsci (1989) 12: 3 4 . Ciha Found Symp (1990)152: 53-61. 2"tcinbach, 27 Fcrtuck, 1 Cell Rio1 (1976) 69: 144-58. " Loring, I hr~urosci ( 1987) 7: 21 5.3-62. Maconochic, 1 Physiol (1992) 454: 129-5.3. Lal, 1)roc Not1 Actld Soi USA (19931 90: 7280-84. Jasmin, Notnrc (1990) 344: 673-5. 2'. Frochncr, Cell Riol (1991)114: 1-7. .'* Froehner, Ner~ron(1990) 5: 403-10. ." Margiotta, Neurosci ( 1 987) 7: ,3612-22. .7.5 Noda, Notlire (1988)302: 528-,32. ." Rnllivct, l'rnc Natl Acrlrl Sci IISA ( 1982) 79: 4466-70.
' '
'"
"' "'
entry 09
"'Ruonanno, / Riol
C h e m (19861 261: 11452-5. ." Mishina, Notr~rc(1986)321: 406-1 1. 39 laramillo, Nntrlre (1988)335: 66-8. 40 Klarsfcld, Neuron ( 1 989) 2: 1229-.36. " Chahinc, Development (1992) 115: 213-19. " Tamaoki, Riochcm Riophja Rcs C o m m u n (19861 135: 397-402. u.'Pinsct, EMRO /(1991) 10: 241 1-18. " Dauhas, Nrr~mii( 1 990) 5: 49-60, 4" Matter, EMRO /(I9901 9: 1021-6. 46 Wang, Rroin Rcc (19761 114: 524-9. " Hichcr, 1 Nct~roc.hrm(I9921 58: 1009-15. " Fontainc, EMRO 1(1988\7: 60,1-9. " Wada, 1 Conip N C I I M(II 9891 284: .3 14-1 5 . 50 Corrivcau, I Ncurosci ( I 99.3) 13: 2662-71. " Stcinman, FASER ](1990)4: 272(L31. Schwirnhcck, / Clin Inrrcst (1989)84: 1 1 74-80. ""tefannson, Nrtv Engl I Mcd (19861 312: 221-5. R d o n c , Eur 1 Immunol ( l 9 9 l J21: L30.3-10. Neuron ( 1989) 3: 163-75. ""lucher, Mcrlic, 1 Cell Ijio1 ( 1 984) 99: ,3,12-5. Parkinson, Exp Rrtiin R1.s (I9881 73: 553-68. "' Rritto, Conip Nc11ro1( 1 992) 317: 325-40. 59 Sargcnt, I Ncurosci (19891 9 : 563-73. Vcmallis, Neuron (199,3)10: 45144. Cotti, Nrl~rosc.irnc.e (1992)50: 117-27. 62 Keyscr, Ncurosci (199.31 13: 442-54. ""iia~ara~havan, Nc~~rori (1992)8: 35.7-62. a Hcidmann, Scirncr (1986) 234: 866-8. "" Mishina, N(1furr (1984)307: 604-X. "' Kullhcrg, Proc Nut1 Acnr! Sci IJSA ( 1 990) 87: 2067-71. 67 Kurosaki, FERS Lrtt ( 1987) 214: 253-8. " Liu, / I'htlsiol (199<3) 470: ,349-6.3. Ncurocci (1985)5: 3386-92. 69 Rrccr, 70 Hankc, N(lt11rc (19X6)321: 171-4. " Marshall, EMHO /(I9901 9: 4,391-8. 72 COOPC~, N u t ~ l r(1991) ~ 350: 2115-8. 77 Anand, I Riol C h r m (1991 ) 266: 11 192-8. 74 Shihahara, Eur I Riochcm ( 1985) 146: 15-22. ,?MET) l(1989)8: 687-94. '"icttc, ''Wang, EMHCl l(1990)9: 78.3-90. " Crowdcr, Mo1 Cell Hio1 (19881 8: 5257-67. 7R Raldwin, Nnttire (1989)341: 716-20. 79 Mar, Proc Not1 Acod Sci IJSA (19R9185: 6404-8, '"Anderscn, Phj.siol Rcv ( 1 9921 72: SX9-SISR. " Raftcry, Science (19XO)208: 1454-7. R2 Whiting, Riochcn~istr>r (19861 25: 2082-9.3. Whiting, 1 Nrurosci (19881 7: 4005-16. " Whiting, Mo1 ljrl~inRPS (1991) 10: 61-70. Schoepfcr, Ncuron (19KU\ 1: 241-8.
"' "
"' "' "" "'
'" '"
-
'"Lindstrom, CfRA Foand Symp (1990)152: 23-52.
Schoepfer, Neuron (1990)5: 3548. Halvorsen, Neuroscience (1990)10: 1711-18. "Whiting, Proc Not1 Acad Sci USA (1987) 84: 895-9. '"biting,FEBS Lett (1987)213: 55-60. " Flores, Mol Pharmacol (1992) 41: 31-7. " Rlanton, Biochemistry (1992) 31: 37.38-50. 9.3 Giraudet, Riochemistrjl (1 985)24: 3121-7. 94 Toyoshima, Nature (1988) 336: 247-50. 9,5 Dani, Curr Opin Cell Rio1 ( 1989)1: 753-64. 9" Karlin, Trend.9 Pharmacol Sci (1986) 7:304-8. 97 Karlin, Horvcy Lcct (1989)85: 71-107. 9A Eisenrnan, Annrl Rev Hiopliys Bioplivs Chcm (1987) 16: 205-26. 99 Rlount, Neuron (1989)3: -349-57. ID" Kao, / Riol Chcm (1984) 259: 1 1662-5. Middleton, Biochemistry ( I991) 30: 6987-97. ln2 Tomaselli, Aiophys / (1991) 60: 721-7. ".' Oiki, Niophys 1 ( 1992)62: 28-<3O. Dennis, ISiochemistry ( 1988)27: L346-57. '""alzi, / Hi01 Chcm (1990)265: 10430-7. I m Galzi, FEHS Lett (1 991) 294: 198-202. 266: 22603-12. lo7 Czakowski, Riol Chem (1991) Karlin, Current Opin Neurohiol(l993)3:299-309. Gdind, I Memhr Riol(1992)129: 297-309. ''' Oleary, I Riol Chem (1992)267: 8360-5. Sumikawa, J Riol Chem (1992)267: 6286-90. ' 1 2 Mishina, Nature (1985) 313: 364-9. "" Conti-Tronconi, Riochernistry (1990) 29: 6221-30. 'I4 McLane, Riochernistry ( 1991)30:4925-34. "%anif Trends Neurosci (1989)12: 125-8. "%iraraudat, Proc Nntl Acnd Sci USA (1986)83: 2719-L3. If' Hucho, FEES Lett (1986) 205: 137-42. 'lR Galzi, Annu Rev Pharmacol (1991) 31: 37-72. Whitc, 1 Rinl Chem (1992) 267: 15770-8.3. 'M Lconard, Science (19881242: 1578-81. 12' Irnoto, Noture (1988) 335:645-8. ' 2 1 Green, Annu Rev Physiol (1991) 53: 341-59. '21 Dani, Gen Physic11 (1987) 89: 959-83. Charnet, Neuron (1990) 4: 87-95. Villarrocl, Proc R Soc Lond R Riol Sci (1991)243: 69-74. '21 Villarrnel, Riophys 1 (1992) 62: 196208. 324: 185-90. , 12' Fcrrerrnnntiel, FEBS Lett (1993) Rertrand, Proc Nntl Acod Sci IJSA (1993)90: 6971-5. 1 129 Revah, Noture (1991) 353: 846-9. '." Villarrocl, Proc R Soc Lond /Rial/ (1 992)249: .317-24. '"I Imoto, FEHS Lett (1991) 289: 193-200. Changcux, Trends Phorrnocol Sci (1 992)13: 299-301. '.',' Galzi, Nrlture ( 1 992)359: 500-5. 'MDani, Netrrosci (1989) 9: 884-92. R7 RR
'"
1
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'"
'"
'"'
1.75
Langosch, Niochim Ric~physAct(r (1991) 1063: 36-44,
'.'"Unwin, Cr11 (199,Z) 72: , 3 1 4 1 .
Mishina, Nnturr (1985) 313: 364-9. C;~strcsnnn,FEljS 1,t'tt (1992) 314: 1 71-5. Karlin, Hnrvc.\. 1,ct.l ( 199 I) 85: 7 1-1 07. Unwin, I Mol l(io1 (190.3) 229: 1101-24. Aknhns, Sc.icnc.cp( 19911 258: 307-1 0. j4' Mnntnl, FERS Lrtt (199.3) 320: 261-6. 14.7 Rutlcr, Piocllit71 /iioph\,.? Ac!(l ( 1 99.7) 1 150: 17-24. Ross, 1 Cvll Riol ( 1 99 11 113: (12.3-6. 14,s [;reen, Cc>ll( 199.31 74: 57-69, *"' Vcrr:~ll, Cc11 (I9911 08: ?.3-,3 I. Phillips, Scirnc.rp (191)11 251: 568-70. Butler, / / { i f ) C / / I V I I(l99?1 I 267: 621.3-1 X. "" Wagner, Ncrlron ( 190,Z1 10: 5 1 1-22. 150 Ihraghimov-Rcskrovnnya, Ntrt rlrcp(19'12) 355: 696-702. 151 Fcrrcr, I'roc hrnt1 Acclrl Sci IISA ( 1991 ) 88: 1021.7-17. Hugnnir, Nrltrrrc* (1986) 321: 774-6. Hopficltl, N(ltrrrcp (Ic)XXJ 336: 677-80. Miles, MoI N(~rlrol)iol(IOHX) 2: 91-1 24. Ross, I Hiol (711(pr11 ( 1 OX71 262: 14640-7. I"" Hugnnir, ('rit K r p v /Jioch~rllMol IJir~l( 19X9) 24: 18.3-215. 157 Huganir, Nrrrron ( 1YC)0)5 : 555-67. Mullc, 1)roc Nrrtl A(.oi? Sci rlSA (19881 8.5: 5728-.32. I S'J Himins, 1 (lc.11 Riol (19811) 107: 1 1 57-65. Wnllacc, I C1111 Hiol ( 19881 107: 267-X. Wallace, N1.uror1 (1991) 0: 860-78. '"* Pcng, N(,urr~r~ ( 1901) 6 : 2.3746. O'Lcnry, I Riol Cllrru (lVQ2)267: X.760-5. I"' Smith, I I'hr~siol(19901 432: .34,t-54. 16"u, I'111,siol (10911 436: 45-56. I"<, Vcrnino, Ncuron (1992) 8: 137-.74. "" Mnthic., I /)h,.siol (19901 427: 625-55. Ifunc, I l'hysiol (1992) 457: 14.7-65. Zort~rnski,A401 1)11(rrt~1(1~~01 ( I992) 41: 93 143. Im dc In (..'arm, N(.llros(.ir.~lcc, (1987) 23: XX7-9 I. 171 Fmnkc, Nr~trocc.iLr'tt ( 19971 140: 169-72. "* Kntz, I Ph\.siol (1957) 138: h
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entry 09
Cull-Candy, I Physiol [ I 988) 402: 255-78. MOSS,Neuron (1989)3: 597-607. Kuha, Pflu,yers Arch (1989)414: 105-12. '*' Heidman, Riochemi.~try( 1 98,3) 22: 3 1 12-27. Hcidman, Proc Nrrtl Acod Sci U S A (1994)81: 1897-1901. Rcvah, Proc Not1 Acnd Sci USA ( 1990) 87: 4675-9. Pcdcrscn, Ijiol Chem (1992)267: 10489-99. lvo Valcra, IJroroc Not1 Acnd Sci U S A (1992)89: 9949-53. Luctje, 1 Neurochcm (1990)55: 632-40. lV2 Alkondon, Mol Pharmncol(1992)41: 802-8. ".' Murrell, [Physiol (19911 437: 4 3 1 4 8 . Wachtcl, Ann NY Acad Sci (199I ) 625: 116-28. Rcrtmnd, I'roc Not1 Acnd Sci U S A ( 1990) 87: 1993-7. Mullc, Neuron (1992)8: 937-45. Couturicr, 1 Riol C h e m [1990)265: 17560-7. '91 Colyuhoun, Physiol 11985) 369: 501-57. lw Jackson, Physiol(1988)397: 555-83. Sine, Gen IJhysiol (1990)96: 395-437. Loring, 1 Toxicol-Toxin Ilev ( 1 99.3) 12: 105-5.3. Kecept Krs (1991)11: 1001-21. 20' Alkondon, FUC~S Prnc , R Soc (Lond) R (1992)248: 35-40. 2" Pinnock, Rrnin RES (1988)458: 45-52. 2wi Mclanc, Biochemistry (1993)32: 6988-94. Hung, Neuroscience ( 1 992) 48: 727-35. 20' Ahmmson, 1 Riol C h e m 11989) 264: 12666-72. MR Clarke, trend,^ l'harmacol Sci (1992)13: 407-I,?. Mcgroddy, Riophys 1 (1993)64: 325-38. Rana, Errr I'hnrmncnl ( 199.7) 250: 247-5 1. ~ ~ l w i Mol n , Pharrnocol ( 1992) 41: 908-13. 'I2 Rcuhl, Net~rophysiol(1992)68: 407-16. "I.' Mullc, 1 Neuro,~ci (1991)11: 2588-97. 2'4 Changcux, Q Rev Riophys (1992)25: 395-432. ""ole, Curr Opin Ne~trmci(1992)2: 254-62. Leidcnhcirncr, Trends Phnrmncol Sci ( I99 1 ) 12: 84-7. 2'7 C;crznnich, Mol Phormocol (1994)45: 21 2-20. 2'R Elgoyhen, Cell (19943 79: 705-15. Pcng, Mol Pharmaco1(1994)45: 546-54. Criado, Nucleic Acids Rcs (1988)16: 10920. - 22' Akondon, Phormocol Exp Ther (1993)265: 1455-73. I*.'
'*' '*' '*"
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Eclward C. Conley
Entry 10
Abstractlgeneral description 10-01-01: GARAA rcceptor-channels (GARAAR] arc meiliators of post-
inhihitiont in the hrain responding tn the rnaior neurotransmitter 7-aminob~ityricacid (GARA). <;ARA is n neutral a m i n t ~acid rclcascd from C.AI3Acrqlci ncuroncs, which form approximntcly d O X l of a11 central1 synapscsl. C.Al3A :~lso:lets at C. protein-linked rcccptors (CARAIl suhtypcsl which do not cont;lin i n t e ~ r ; ~ion l channcls hut arc couplet! to separate c;~lciilm ant1 potassiuni ch;inncl proteins (,st,(, Rrcrptor/trl~n.sdr~ccr intc.rclc.tions. 10-401. synaptic
10-01-02: Activation of ncuronal C;ARAA rcccptors increases inward chloride current through ;In integral channel which hyperpolarizes the post-synaptic cell and i n h ~ h i t ssynaptic activity (hrrt svr. nrst prrro,~rrrphl. Gcncrally, GA13AA rcccptors t~ndcrlicfast I P S P ~(inhibitory post-synaptic potentialsi) components following stimul:ltion c ~ fcxcitatory affcrcntst (with slow IPSP componcnts often hcing mctliatcd hv GARAl,, adrcnoccptc~r and 5-HT rcccptor responses\. CAR/\A-mcili;~tcilconiluctanccs inflilcncc impulse initiation nnil thc nmplitl~tlct ; ~ n dduration of cxcitatory post-synaptic potcnti;llst (EI'sI's~), cspccinlly thc expression of thc NMIIA-mediated component (F(T liLC; (:A 'r I ; L I I N M I I A , rntry O X ) . 10-01-03: All CNS ncurnncs, as wcll as gliali cclls, cxl~ihitGARAA responses, although in the latter arc characterized by an 'cxcitatory, dcpnlarizing' outwaril current response to GAIiA Lwc C e l l - ~ y p ccxprt>ssion indpx, 10-081. C.ARAA receptor-ch;~nnc.Is have nlso hccn charactcrizctl in the peripheral nervous system (c.g. uterus, ileum, lnctotrophst, retinal cclls, pancreas, pituitary intcrmctlintc lobe and ndrcnnl chromaffin cclls). A class of C.ARAA rcccptors that mcldul;~tcthe relcnsc of GAIM itsclf has hccn studied in prcsynaptic nerve fibres. These GARAA autorcccptnrst have n novcl agonlstt ;inti ;intngonistt profile. 10-01-04: Roth the <'.AKAA rcccptor and thc gl ycinc rcccptor (dcscrihcd in t hc cntry ELC; C:l C L Y , c'ntr!? 1 I ) arc hctcro-c~li~orncrict (pentameric) pmtcin cornplcxcs in thcir native1 statc, cornposcd of suhunits with molecular weights of -50-60 kDa. Multiple combinations of a largu set of distinct channel structural gene products undcrlics A large functional heterogeneity of <;AHAA channel complcxcs. T h e gcncs encoding C;ARAA rcccptorchannels f(?rm part of the txtmcullular lignnd-gntcd channel gcnc supcrf;unilvT. At least Ih diffcrcnt rcccptor suhctnit genus have hccn scpar:ltcd into thc sequence homolowi si~hfarnilicstalpha (zl-r,): hcta (/Il/i.,\; gamma (;.I-;.41; delta (dl, ;mil rho (p1, ,121 (scc 'r'trl,lr l ) . Thls large numhcr of distinct isofnrmst rcvcalcd t h r o u ~ h molt.cular cloningt has cnahlcd detailed subunit cn-expression studies to hugin. 10-01-05: Generally, GARAh subunit gcncs display differcntialt spatialt
expression and co-expression of multiple suhunit mRNAs within single cells is commonplace. 'Dynamic control' of GARAA rcccptor gene expression can potentially Icad to diversity in suhcellular locations, ligand affinities+ and ion channel gatingt properties. ~hosphorylationi of in trrlns gene regulatory factorst (coupled to extraccllular mcsscngcrt molcculc reception] may also regulate GABAA suhunit genc expression. In common with other ion channcls, post-transcriptional m@ificationst of CARAA protcin suhunits (principally by phosphorylation') are also important modulators of physiological function.
10-01-0kThc mechanism for assembling and 'targeting' of protein complcxcs with different isoform composition may he central to modulation of GARAn rcccptor distribution in ncuroncs. Assemhly of CARAARs from constituent subunits docs not proceed randomly to form all possihlc comhinations, hut certain suhunit comhinations arc 'preferred intermediates' during thc asscmhly proccss - e.g. transientlyt expressed GARAAR subunits in mousc L929 fihrohlast cells, an 'ordcrcd asscmhly process' appears to produce n 'preferred final form' of the receptor-channel (rlP1y2S) (.see Domtiin iirrongcmen!. 10-27). 10-01-07:In gcncml, there is a high degree of sequence conscrvationt within the putative transmembrane segments MI-M3 hctwcen the GARAARand the glycine receptor-channels (ClyR, scc EI,CI Cl GLY, entry I I ) , indicating thcir likely importance in chloride channel formation. GARAAR anti ClyR also display scvcral invariant rcsiducs at homologous positions and rclativcly high positive charge density within eight residues of thc ends of their tmnsmcmhranc domains on the extracellular sides. The existcncc of channel vestibulest containing thcsc net positive charges arc consistent with the anion-selective permeability properties of CA13AA receptor-channels and thcy may havc the property of attracting anions. This arrangement is in contrast to vcstihulcs of cation-sclcctivc rcccptor-channcls which possess 'rings' of nego t ivc1,v charged rcsidues [see ELC: CAT nAChK, entry 09). 10-01-08: GARAA rcccptor-channcls arc 'positively modulatcd' by scvcral clnsscs of clinically important d n ~ g s (such as the barbiturate CNS Jcprcssants, henzodiazepine anxiolytic/anticc~nvulsant/scdativc-hypnotics, and several anacsthctic compounds). (SARAA responses also partially mediate sensitivity to alcohol (ethanol) and a rangc of convulsantt compounds. The hinding affinityt as wcll as the modulatory efficacyt of thesc and other drugs change with receptor subunit composition and this factor offers clear potential for the dcvclopmcnt of more sclcctivc therapeutic compounds. A large numhcr of studies havc indicated that in mosr cases, modulatory drugs do not interact directly with GARA-hindinx sites, hut influence receptor-channel function hy hinding t c ~additional allosterict-modulatory sites on tho CARAA complex. 10-01-09: The availability of a rich diversity of cloned CARA* suhunit gcncs anrl pclwerful tcchnirlucs such as site-directed mutagcncsist, domain swnppinRt, photoaffinity lahclling~ and m i ~ r o s e ~ u c n c i n phas , ~ cnahlcd a proccss of 'mapping' key amino acid loci implicated in mtldulator hind in^ and function to hcgin. In addition, mechanisms undcrlying many diffcrcnt
cntry 10
- - - - - ..- .
'I
C;A13AA functions and properties are yielding to such analysts, including those involving or dctcr~nininglig:ind hinding/recognition, co-opcrativity~ for ;~gonist hintling, ch;~nncl conductance characteristics, modulator cffic;lcy, mechanisms of channcl hlock, rectification properties, allostcric m o d ~ ~ l a t i ohy n drugs, domain topography, suhunit arrangements, assemhly patterns, dose-response rclationsliips, phosphomotiulation properties, dcscnsitization kinetics, ctc. Such approaches will dcfinc in detail the functional criteria for the opcr;ition of the GAI%AAreceptor family in v i v o .
I
Category (sortcode)
10-02-01: ELC; CI C;A1tAA, i.c. cxtr:lccllul;ir ligand-gated chloride channels activated hv ganinin-aminohutyric acid. The s u ~ c s t c delectronic retrieval code (unique cmhcddcd identifier or UEII for 'tauing' ot new articles of rclcvancc to the contents of this cntry is UEI: CAINAsi or genes1 1. For ( I dircr~ssiono( thc. o f IEIs rlrlti guirir.1irlcc o n tilc7ir inlll1c3r??c~nl(ltir,n. scc thc sc>ction 0rirr~lnltrgi~s
rlrlt! 1(1\~out, cPntr!'02, flnti for furthcr dctrri1s. on I
0
r,
Channel designation 10-03-01: Cl(;,,,,,,
A;
CIAI%AAIl (-C;A13AA receptor); ionotropic GARA receptors.
Current designation 10-04-01: Usually designated as It;ARA.A or ((;A13A.Ali other designations used for C,ARAA currents include (typical, cxpresscd from ccrchral cortex niRNA in oocvtcsl' as distinct from (expressed from retinal mRNA in oocytcsl' ( F ~ Y ,C:c,ll-ty[~r r.ul?rrssion intirsx, 1 0 - O H / .
Gene family 10-05-01: T h e gcncs encoding GAHAA rcccptor-ch:?nnels form part of the cxtr;~cellularligand-gated channcl gcnc supcrfamilyi (dc,scrihrd undpr El,(: Krv /r~cts,cntry 041. For updates on the agreed nomenclatures, rcfcr t o the latest IUPHAR N(~mcnclaturcCommittee recommendations via the CSN t . 9 F ~ r~c d l ~ r ~ cc*,k CS N rrcc.css. cnt rrr 12).
GAHAn slthunit f ~ n ~ i l i rind e s suhf(1milies 10-05-02: Multiple types of suhunit associations underlie the great pharmacnlogical and binchemical diversity of the C;AflAA receptor-channcl family2 (si,c, IJrr.dictr~rlprott.in t o p o ~ q r ( ~ l dIll-,10, ~ y , rlnd I'rotcin intrractions, 10-31).
At lcast 16 diffcrcnt receptor suhunit gcncs have heen scparntcd into s ~ q ~ ~ e hornnlngy nte subfamiliesf. Thcrc is typically -70-80'X1 aminn acid sequence identity hctwccn mclnhcrs of a subfamily and -d0-40% hctwccn mcmhcrs of rliffcrcnt suhfamilics. Itasic distinguishing features nf the Rcncs encoding mcmhcrs of the suhunit families alpha ( r , - z , ] ; beta (P,-/l.,); delta (($1, and rho (p, and { I ? ) arc listed in Tahlc 1 . - gamma
Table 1. Distin~uishing chart~cteri.~ttcs o f genes enct~din~q suhunits of C:ANAA receptor-channels (From 10-05-02)
en cod in^
Suhunit Transcript size (khJ"
Rat: 3.0
Rat: 4.2
ORF"
Signal peptidc'
Rovine: 456 an; Rat: 455 aa; Mousc: 455 aa; Chickcn: 45.5 aa; Human: 456 aa"
Rat: 27 aa M, rat: 48.8 k ~ a 5' 1 kDa (rccombinantt by ~ e s t c r n ht l o t t i n ~
Rovinc: 451 aa; Rat: 451 aa; Mouse: 45 1 aa; Human: 45 1 aa"
Rat: 28 aa 53 kDa hy
Rat: 493 aa; Mouse 492 aa;
Rat: 2R aa 59 kDa by WR
WRJ
WR
Rovinc: 492 aa" Rovine: 4.0
Rat: 552 aa; Rovinc: 556 aa'.'
Rat: 35 aa Rat: 59 kDa by WR Revine: 59 kDa by WR
Rat: 2.8
Rat: 464 aa'
Rat: .3 1 sa
Rat: 3.ZP
Rat: 44,1 aa"
Rat: 19 aa 57 kDa by photoaffinityt lahclling with ['HI-~~154513
Rat: 12.0"
Human: 474 aa: Rovinc: 474 aa; Rat: 474 aa
Rat: 25 aa M,bovine: 55.4 kDa
Rat: 8.0",'
Human: 474 aa; Rat: 474 aa
Rnt: 24 aa 57 kDa by
Human: 473 aa Rat: 47.3 aa; Chicken: 476 aa
Rat: 25 aa 54-59 kDa by WR
WE
Rovinc: $4: 25 an Rovinc 134: 459 an Chickcn: 488 an (AS) Chickcn ~ 4 '463 : aa (AS1
Chickcn 14': 27 an
Rat: 2.0 + 3.0
Rat: 449 an Mousc: 449 aa
Rat: I6 aa 54 kDa hy WR
Rat: 3.8
Rat: 465 aa
Rat: 35 an
entry 10
Table 1.
Continlrcr/
Subunit
Transcript size (khJ"
72s
Ratlmousc
TT,:
4.2 t 2.8".
r l I;
721.
7t Pi
Pz
Rovinc: 3.9 t 4.8 -1 -7.1
Encoding
ORF"
Signal pcptide"
Rat -.?.s: 428 aa (maturcl See i l l ~ o Dotahnsc listrn~s.1053 13ovinc ? ? 442 aa; rat : 436 an (mature, AS"] SIY rrlso l?(i!rrIlmsr, lis!in,ys, 10-5.3
Rat - I ~ c 38 : ~a 4 3 4 7 kDa hy W R
Rat - ? < : .78 aa M, hovinc ??: -50.4 kDa
Knt: 467 fin; Mouse: 467 aa
Mouse: 17 aa
Human: 473 aa
Human: IS aa
Human: 465 an
For furthcr suliunit-specific chac~ctcristics,see niKNA dirtril~r~tion. 10-13, I'/~eriot,vp~(rxpre.~~~ir~n, 10-14, llroiciri i!r.stril~~~tion, 10-15, S~rl~reIl~~l(ir 1oco1io1i.c. 10-10, l'rot(vn n1olvc11l(1r tvcight ( p u r ~ i i ( ~10-22, ! ) , I)or~wir~ orr(~n~y(~ni(~r~t, 10-27, I>orncririconrrrvrIi ion, 10-2X. Ilon~rrinj~~nctions. 10-29, I'rc~tlictr~rl proiiyin topo,qrnphy. 10-30. l'rotrpin int~ror.tions,10-.31 , I'rotr~inphocphorvlotion, 10-.?2. rlmtrr. 10-41.Hlockcrs. 10Ac.tic.rrtron. 10-.33.I1nsr.-rrspons(~,10-.lh.Sin,qlc*-cl~rrnncl 4.7. (:hmnncl modt~lrrtinn,10-44. Erlltilll)r~un~ discocintion constmnt. 10-45. l(cccptor nntm,ponists. 10-,5 1. Ijot ril~crsc,liqt ings, 10-5.3 crnd Rel(rted sources cind re\~lCw,<, 10-50. "Shows transcript sizc as detcctcd hy ~ o r t h e r n tanalysis of total mRNA from the species shown. " ~ h o w sthe numhcr of ;~rninoacid rrsid~lcsin the spccifietl channel suhunit. For a s r ~I>mir~l~tr.sc~ , listings. 10-.5.7.(AS)indicates a product of more tictailcd I>rc;tk(lc~wn, alternative splicinRI. l plus 'Shows typic;~lcontributory Icnxth t c ~total O R F hy cleaved s i ~ n a pcptidc cxpcrimcntnlly dctcrminrd molcciilar masses for rccomhinant suhunits hv SIXI'A(;E~ nncl Western l,lottingl (WI31 procc~lurcs[predicted rnol. wt values ca" hc derived from scqurncc analysis lollowing retrieval using accession numhersi in I)cr/tr~~crs~~ I~
;,,
cntry 10
-
GARAn receptor gene nomenclat~~res 10-05-03: A scrics of gene names for those encoding GARAArcccptclr subunits arc a ISO in usc - gabral-gabrah, gabrbl-gabrb4, gabrd, gabrgl-~ahrg,?and, hy convcntion gabrr). Thcsc names also appear in un-italicizcJ uppercase within sequence databasc entries.
Functional co-expression studies of recombinant subunits 10-05-04: The largc number of distinct subunit isoformst rcvcalctl through molecular cloningt has enahled dctailcd co-expression studies to begin. Thcsc studies ultimately seek to relate clcctro~hysiolo~ical and phar~nacological paramctcrs mcasurcd in situ on nativei cclls to contril>utions of individual protein suhunits. Elcctrophysiological and pharmacolo~ical cxpcrimcnts havc suggested a remarkable heterogeneity of GARAA rcccptor complcxcs within individual cells (reviewed in rc/:'].
Theoretical and actual numbers of suhunit combinations 10-05-05: From thc largc numher of combinatorial possibilities (the 1 5 known suhunits would give -151 887 suhunit comhinations with 1-5 suhunits co11.scrl in native cclls is cxprcsscd) thc total numher of comhinations uct~(111j~ unknown. Ry means of RT-PCR~ techniques, single ncuronal cclls havc hccn rcportcd to co-express up to 14 distinct GAI
-
Subtype classifications Rntionolizution o f subtype classifications incorporating both molecular and phnrmncoIo,gical characteristics 10-06-01: As dcscrihed in the PHARMACOLOGY section of this entry, multiplc distinct drug-binding and othcr modulatory sites of GARAA rcccptors havc hccn charactcrizetl hy pharmacologists. The multiplicity of thcsc sites, and the large diversity of genes encoding CARAA rcccptors (set' Tr~hlc1 rrnrlcr Gene /rrrniIy, 1 0 - 0 5 ) havc resulted in significant prohfcrns for thnsc involvcrl in proposing agreed schemes of receptor nomenclature. At the timc of going to press, there is no 'universally accepted' nomcnclaturc for GARAAR. For updates on the agreed nomcnclaturcs, rcfcr to thc latcst IUPHAR Nomenclature Committee recommendations via the CSN (see rccdhnck d C S N ncccss, entry 12).
Existing classifications based on functional properties 10-06-02: Fcaturcs of a provisional GARAA receptor subtype classificatinn [GARAAl, GARAA2, GARAA,< hascd on thc func.tiona1 proper tit:^ c ~ fthcir
I
allosteric rnodtrlatory centres have bccn summarizcd"" (scr: rllsn Rlocker.~. 10-4,1, Chnnnol ~nodnlation.10-44, Rcccptor rr,~onist.s,10-50, rlnd Receptor nntnjy~nists.10-51). Distinctions hetween thc GARAA-related 'central'-type hcnzotiiazcpinc receptors (as descrihcd in this cntryl and thc C.ARAA, unrelated 'peripheral1-type benzodiazepinu receptors arc outlincd in thc - footnote to Tahlc 6 under Lj,~ands,10-47.
cntry 10
Novel C:ARA receptor suhtvpcs ntcdi(itinqq"cxcit(11 ory, dcpolnrizin,q' rcsponscs 10-06-03: Distinct rcccptor suhtypcs may he rcsponsihlc for 'excitatoryt, depolarizingtt responses of some GARAA receptors7. Thcsc novel suhtypcs may he associatc(1 with voltagedependent anion channels which arc capahlc of regulating chloride flux through the C.ARAAintegral channel (and vice vcrsa). ~xcitatoryt,dcpolarizingl CAI!AA suhtypcs may depend on co-cxprcssion of specific types of ion transportcr proteins also he involved in mediation of neurotrophicT responses (luring dcvclopmcnt /sc.r Cc~II-t,vpr rq~rrssioninllc~x. 1 0-08, I > ~ ~ ~ ~ r l o ~ rc~,yul(~tion, ~ r n c ~ r ~ t10( ~1l 1 , (~ri(l f'hc*notvp~c cx;)r(ls,sion,10- 14).
0
Trivial names 10-07-01: The GARAA rcccptor; the GARA receptor-channel; the A rcccptor; the hicucullinc-hlockcd GARA rcccptor.
)ptmQij -
Cell-type expression index GARA-inhihitory rc.sponscx clrc t ~ l ~ i q t t i t oin t ~ snervous tissuc 10-08-01: <;AKA is consirlcrcd to he the major ncurotransmittcr mediating inhihitory'i ne~~rotransmissiontin higher hrain functions, although the wide distribution of C;lyR suhtypcs in the CNS (.FCC ELG CI C:l,Y. cntry 1 1 ) may also silggcst para11c1 rt?les for glycinergic+ transmission. AII CNS ncuroncs, as well ;IS gliall cclls, exhihit GARAA r c s p c ~ n ~ e s ~The .~. cstahlishcd suhunit tlivursity (src T(il71r 1 ) and the multiplicity of sites for pharmacological morli~lationdemonstrate the consiticmhlc heterogeneity of GARAA complexes throi~ghout the ncrvoils system. Patterns oi C.ARAA expression in hrain h;~vchccn rcvicwcrl'".". 10-08-02: The wc*ll-c.hrrnlcti,rizi,(i ncuronal preparations expressing GAHAA suhtypcs qilotcd within this rntry arc as follows:
L
ci~lturcdrnt ccrchcllar ncuroncs tlissociatcd r:lt symp:~thcticncuroncs chick ccrchr;~lcortical ncuroncs porcine pituitary intcrmctli:~tclohc mouse spinal cord ncuroncs ccrchellar Purkinic cclls cultured hippr>campal pyramidal cells culturcci spinal cord ncuroncs adi~lt/foct;~l dorsal root ganglion ncuroncs synaptoncurosomal prepamtion from rat ccrchral cortcx cmhryonic kidney line (hctcrologo~~sl superior ccrvical g;mglion ncuroncs turtlc cone photorcccptclrs rat/cat dorsal root ganglion ncuroncs hovinc actrenal medulla chromnffin cells loc(~tions,10-16) prc-synaptic pcptidcrgic nerve terminals ( c c S~thcclItil(~t
cntry 10
-
Retinal GARA,, channels 10-08-03: RNA extracted from bovine retina expresses GARA rcsponscs composcd of two pharmacologically distinct C1 currents, one mcdiatcd by and the othcr hy 'atypical' GARA receptors' that GARAA receptors (lC.Arrt) arc resistant to hicuculline and are not activated by haclofen (IG.RR)(see Blockers. 10-43).
Excitatory, depolarizin~responses o f GARAA-likechannels in vertebrate gliul cells 10-08-04: Astrocytes and oligodcndrocytcs arc depolarized by GARA, ant1 pharmacological cxpcriments indicate that the channels underlying thesc responses are related to neuronal C.ARAA receptor-channels (for rcvirw, scc ref."). The 'glial' and 'neuronal' subtypes are distinguishahlc hy the actions of the henzodiazepine inverse aRonistt DMCM (sre the field Receptor inverse agonists, 10-52). Inverse agonistst such as DMCM augment thc GARA response in astrocytcs, whereas in ncurones (and cclls of the oligodendmcytc lineage) the response is decreased ({or discussion o f poasihlc rrndcrlyin,y mechnnisms, scc Protein interactions. 1031 and refs1*-'"). Notes: 1 . As judgcd by in situ hyhridizationt, the re subunit has hcen reported'" to hc highly expressed in the Rergmann glial cell layer. 2. The property of chloride cfflux (rom ~ 1 i a lGAlIA-c~c.tivr~tctI channcls may scrve to maintain or 'buffer' cxtraccllular chloridc for inllr~xthrorl~hncrrronal GABAA channcls at thc synaptic clcftt (Or cliscnssion. sce ref.'*). 3. Glialneuronal interactions involving GARAA ant1 glutamate rcccptor-channel activities may mediate trophict responses andlor synaptic plasticityt phenotypes hoth during dcvclopment and in the adult (srr re(.'* crncl E I X CAT CLU NMDA. entry 08).
GARAA channels in the PNS 10-08-05: GARA receptor-channels are also expressed in the peripheral nervous systemf 1e.g. in the uterus, ileum, lactotrophst, retinal cellsl pituitary intcrmcdiatc lobe and adrenal chromaffin cells]. Cultured melanotrophsT from frog pituitary can he maintained as a 'purc' population of endocrine cells cnrichcd with GARAA rcceptorsl'.
Pancreatic GARAn responses 10-08-06: GABA has a distinct role in pancreas (whcre it is co-secreted with insulin from beta cells) mediating part of thc inhihitoryt action of glucose on glucagon secretion (LC.hy activation of GARAA receptor-channelslR).
Other novel functional classes of GARAA receptor
u
10-08-07: A class of GARAh receptors that modulatc the rclcasc of GARA itself has hcen studied in pre-synaptic nerve fibres. Thcst. GABAA autoreceptors have a novel agcjnistl and antagnnistt profile ( s c ~Receptor agor~i.qla,10-50,and Rrceplor nntn,ponists. 10-57).
Specification o f subtype co-expression 10-08-08: Thc relationship hctween heterogeneity of nativct receptor propertics and specification of suhtypc co-expression is largcly unknown and the suhiect of ongoing investigation.
cntry 10
I
~
Channel density
GAHAn channel loss w i t h neurodegeneration 10-09-01: The density of benzodiazepine type 1t receptors in the suhstantia nigra pars rcticulata is rctluccd hy ,3470 following h-hydroxydopamincinduced degeneration of dopamincrgic pars compacta neuroncs (see ~ l s o IJrotein distrihution. 10-15, nnd Stthcellulnr 1ocntion.s. 10-16). Notc: Significant increases in GARAA rcccptor cxprcssion in thc lower CNS may scrvc a 'compensatory' function for losses of glycinergicf receptor function in the mouse mutant spnstic (see Phenotypic exprcsion under ELG Cl GLY, 11-14).
Cloning resource
Cell-line m o d e l s for developmental expression o f CA8An receptors 10-10-01: lmrnnrtalizcdt hyhrid clones that express characteristics of diffcrentiatedbcurnncs derived from thc cerehcllar and hrainstcm regions havc hccn cstahlishcd hy somatic cell fusion hetween a hypoxanthinc phosphoribosyltransfcrasei (HPRTI-deficient neurohlastoma, N18TC2, and ncwhorn mouse cerchcllar/hrainstem ncuroncs. ~mmortalizedthypothalamic (GT1-7) ncuroncs cxprcssing functional GARAA rcccptors have also heen cstablishcd in culturcf9. Note: Such immortalized cellst express a range of developmentally specific ncuronal protcins and are likely to prove important for studics of the molecular basis of neuronal differentiation+ and degeneration.
Developmental regulation Control o f GARAn receptor subunit expression at t h e transcriptional and post-trunscriptionfll levels 10-11-01: In common with othcr inn channcl gcnc families, cell type-selective developmental expression of specific GARAA genet isoformsi is r c ~ l a t c dhy tissue-specific and ontogcnctict factors (c.g. transcription factors/ morphogens) that are likely to act directly on sequences in cisT to the GARAA structuralTgenes. 'Dynamic control' of gcnc expression can potentially lead to diversity in subcellular locations, ligand affinitiesT and ion channel gatingt properties. Phosphorylationi of in trans gcnc regulatory factorst (coupled to cxtraccllular mcsscngcrT molcculc rcccption) may also rcgulatc C;ARAA suhunit gent expression (in addition to the well-chnrncterized mechnnlsms for post-tmnscriptionnl modulatinn o f the protein structures themselves - see Pmtein phosphonrlation. 10-32,ond EL(; Key facts, entry 04). Notahly, ccrtain GARAAreceptors (excitatoryt depolarizingt suhtypes, see Subtype ~ 1 0 s sificntions. 10-06, Cell-type expression index. 10-08. and Protein internctions. 10-.?I) appear to mediate trophict responses to GARA during developmentz". GARA agonistst can regulate transcriptional activation of CARAn suhunit gcncsf in hoth cell culture and animal m o d ~ l s ~ ' * * ~ . Developmental 'routing' o f multiple suhunit expression 10-11-02: The mechanism for assembling and 'targeting' protein complexcs of different isoform composition may hc central to modulation of GABAA
receptor clistrihution in neurones2.' (see Suhcellulrir Iocfltions, 10-16). Placement of receptors at specific Iocntions is predicted to depend (at Icast in part) on the suhunit composition of the complex if different suhunits hnvc specific routing properties2.'. Ncuronal phenotype (i.c. plasticityt) may hc directed hy expression-control of specific isoformst, which in turn could hc influenced hy dcvclopmcntal differcntiaticln~26~25 and the 'excitatory drive' that increases cxprcssion of transcription factorst2".
Stereotypicnl order o f ion channel gene expression in developitt~ spinal cord 10-1 1-03: In developing populations of cclls from scvcral regions of cmhryonic rat spinal cord, functional sodium channels appear prior to GABA* receptors, which in turn cmcrgc prior to kainate-activated glutamate receptors. This stereotypical pattern of sequential channel development occurs individually on most cclls in each region".
Zinc ion sensitivity dependent o n developmental stage 10-11-04: GARAn receptors of the rat superior ccrvical ganglion cxhihit a developmental increase in resistance to hlockadc by zinc ions". z n 2 + antagonism of GAI
Develnpmentnl delay symptoms in ~ e n e t i cdisorders linked tr, GARAn ij,?suhunit gene deletion 10-11-05: Many paticnts with the maternally derived genetic disorder Angelman syndrome^ (AS] carry chromosomal riclotionst r n a p p i n ~to the samc regrnn as the gene encoding the GARAA /?? subunit (for detriil.s, scc Phenotypic expression. 10-14). AS paticnts arc characterized by developmental delryt, seizurest, inappropriate laughter and ataxict movements. Dclctions in thc samc chromosomal rcgion arc common in suhiccts affcctcd Ily Prader-Willi syndromet (PWS)who displny infantile hypotoniai, childhood hyperphagial/ohesity, developmental delay and hypergnnadismt.
mRNA distribution Co-expression o f GARAn suhunit genes 10-13-01: Generally, GARAA suhunit genes display differentialt spatiall expression29-." and cxprcssion of multiple suhunit mRNAs within single cells is commonplace (.~ec exnmples in Tnhle 2). The mRNAs encoding the rl, and ;'L subunits arc co-localized in many areas of the CNS - c.g. ccrchcllar Purkinic cclls, hippocampal pyramidal cclls and mitral cells of thc olfactory hulh (see (11.~o I'rcdicted protein topo,~rophv,10-301.
n
Phenotypic expression GARAn rcccptat-nssocinted behnviouml phenotypes 10-14-01: Inhrcd strains of mice differ markedly in hchaviour thought to hc related t o C.ARAA receptors, including sensitivity to convulsantst and
Tahle 2.
Nr,lrrt ivc ( l i s t r ~ l ~ l ~ t i ol o n(v: A R A A rrac'rBptorrilKNAs lFrorn 10-13-011
Fcaturc/suhunit Characteristics
Refs
Cn-expression of multiple suhunit typcs
Co-cxprcssion of multiple suhunit typcs is common ( c . rat ~ curchrcllar granulc cclls which cxprcss rrl, ma, n(,,PI, P2, jj7, 7 2 and h; olfactory hulh mitrnl cclls cxprcss nl, PI, jj?, fit and -y2 IIIRNAS; hippocnmpal dcntatc granulc cclls cxprcss a11 subunit niRNAs with thc possihlc cxccption of m,l. In general, the cxprcssion lcvcl of an inRNA cncodinq n givcn suhunit is v n r i ~ h l chctwccn cell types
lo
rr, mRNA is ahuntlantly cxprcsscrl in ccrchcllar granule cells, hut is also dctcctcd throughout t h r forchrain, including hippocnmpal dcntatc granulc cclls. rrl hut not rrz mUNA is expressed in the pars rcticulatn, hut not in the pars cclmpncta of the rat sul7st;in tia nigr:~(srv* r ~ l s oClirrnni*l r / i . t ~ s i t y 10-00, . l J r o ! ( ~(~l ir. ~ri1111t i ~ ( I I I ,10-1.5, orid , S ~ i h c ~ e l l ~ i l r ~ r lor.r~tion.^. 10-10). (t2 rnltNA is of high ;ihuntl;incc in the hippoc:itnpus, ccrchcllum and Rcrgnian glia. (1%mRNA is mostly rcstrictctl to the forebrain, associated with dcntatc gr;inulc cclls 1,ut :~hscntfrom ccrehclluni. rr.8 mRNA is prcscnt in high conccntr;~tionswithin the hippocampirs. n ,niRNA is uniquely confined to the ccrchcllar granulc cclls of the ccrchcllum. (Scc o l s o (1 rr,~ricluo f n , , ni rrnd n , distriljrition in r ~ f : ~ ' 1
""
suhunit inRNA in the hrain has n siiliilnr distrihution pnttcrn to the nrc;ls of GARA r;irliolignnd binding which is not ;issocintecl with hrnzorlinzcpinc I7inding. (9 suh~rnitmRNA is p:irtici~lnrlv prominent in rat ccrchcllar granule cclls. It h;is ;~lsoIwcn suggested th;it +, suhunits arc ;~ssoci;rtctlwith high-;iffinity GAI%AAreceptors as they follow the distrihution of I'HJ-muscimol hintIing. Gcncrally, the 1oc;ition of b and 72 suhunit mcssaRc appears distinct
.3 4
71mIiNA is prcilomin;~ntin ncuronal cell
3.5
2
subunit-data --
p~
~
r5 subunit-tliitn p
-
b
pr)pi~lationswithin the limbic system (amygdnln, s c p t t ~ m nntI ) in the hypothalamus. y2 mRNA tfistrihimtion rcsemhlcs that of C.ARA,/hcnzodiazapinc rcccptors lahcllcd with I 'HI-flunitrazcpnm. y 3 rnRNA distribution is simil;lr to 7 2 hut is less ahuntl:lnt. Generally, the 1oc;ition of y2 nnil (4 suhunit message apptars drstinct
"
cntry 10
-
Table 2.
Continued
Fcaturc/suhunit Characteristics p
subunit data . .
Absolute mRNA transcript numhcrs
Human pl and p2 mRNA is expressed (and prohably rcstricted to) retinal tissue (see references under rho subunits in Datohase listings. 10-531 Determination of absolute amounts of mRNA (using compctitivc P C R ~tcmplatcs to nativc transcriptsf encoding 14 distinct suhunits of thc GARAAR)in primary culturcs of rat ccrchcllar granule neuroncs and ccrchcllnr astrocytcs show the latter to contain two orders of magnitude lowcr mRNA than the former. Granule cclls express 14 [different suhunit mRNAs, while astroglial cultures dn not express dctcctahlc amounts of a, and thc 7 2 message. ~ In direct comparison by competitive cDNA amplif i c a t i ~ n " ~nl, , as and a(,mRNAs arc prominent in culturcd granule cclls, the n l and n2 mRNAs arc abundant in cultured astrocytcs, whilc PI and P1 mRNAs arc abundantly cxprcsscd in both culturcs. The 72sand -y2~, mRNAs constitute the great majority o f ? suhunit mRNAs in ncumncs, while thc 71 suhunit mRNA is the most ahunrlant 7 suhunit mRNA in astrocytcs (see also Chnnnel modulmtion,
Rcfs
""."' "
10-44)
anticonvulsantst, alcohol and diazepam. Dysfunctional GARAA rcccptor proteins (or the exprcssicm-controlt of thcir gcncs) may play a role in CNS 'hypcrcxcitahility' statcs such as epilcpsyt and anxiety disorders, althougll direct cvidcncc of thcir involvcmcnt is lacking. 10-14-02: GARAA rcccptor dysfunction may hc rclatcd to somc forms of myoclonust (quick involuntary shock-likc movcmcnt disordcrs; rcvicwcd in ref.""). 13lockacie of CARAA receptors or GARA synthesis regularly cvokcs cnnvulsive seizurest, hut administration of many CARA aRnnistsf and snmc GARA uptake blockers par(~doxicnlljr may cvnkc myoclonus (i.e. cffects arc receptor type-dependent). Enhancement of CARA function is cffcctive as an anticnnvulsantf stratem in several animal scizureT modcls4" and receptor subtype-selective drugs may aid the dcvclopmcnt of effic~cinuscompounds for thc various types of c p i l ~ ~ s ~ t .
Incrensed expression o f CARAn receptors in the mouse spa mutnnis 10-14-03: Thc mutant mousc spastic (spa] acquires a scvcrc motor disease r~sricmicc display a f~inrlnmcntaldisturhnncc ahout 2 weeks after hirth4'. in the 'balance' of cxcitator$ and inhihitoryt irnpulsrs, which is likely to hc duc to aherrant regulation of normal glycine receptor4hannel gene switching from 'foctal/nconatal' to 'adult' isoforms {for dctnils, scc next p n m ~ m p hand Phenotypic expression under ELG CI GLY, 11-14). Notahly,
entry 10
-
facilitationt of GARAA responses also alleviates symptoms of affcctcd micc (s1.r nrxl pnrl~,yrc~l~ll\.
Allcvicltion o f spa svmptoms is .specifically medirlied h y C:AfJAA receptors 10-14-04: The developmental transitiont from a 'foctal/nconatal' glycine rcccptor ( C I V R ~tIo the 'arlult' GlyK complex (ClyRAl is pcrturhcrl in the spastic micc (scc prcviolls pnrflqrilph ilnd re/.''^. Changes in function or structure of the GlyR protcins a p p a r unaffcctciE in .vprlstic micc, factors which provide evidence for a rcgl~latoryrather than a structuml cffcct of the spc~sric.mutation4'. A significant increase in C,ARAA receptors in the Iowcr CNS may scrvc as a compensatory function in .spczstic tnicc, counteracting (in pnrtl losses in glycincrgic- function. Ph:~rmacc~lo~ical facilitation' of C;ARAA responses, for example hy administration c ~ fhenzodiazepine agonistsi or aminooxyacetic acid (which reduces rlegmtlation of cnrlogcno~lsC;At{A hy inhihiting GABA transaminasel also allcviatcs symptoms c ~ affcctcd f micc (sec I'llcnot,vpic cx~~rc.s.sion 11r1d~r EL(; C1 G L Y . 1 1-14).
C:AHAA chnnncl nctivotion h y cth(ino1 10-14-05: Application of ethanol I~zcili!rrtcsGABA responses in oocytcs injected with rnRNA from ' ~ o n g - s l e e ~micc l' hut rln!ogclnizus rcspclnscs in oocytcs injectctl with rnRNA from 'short-sleep' rnicc4'. Ethano! inhihits baroreflex bradycardial through C;ABAA ; ~ n d(;Af3Al, rcccptor-dependent potcntiationi of the actions of cndogcnc~usGARA in thc dorsal vagal c ~ t n ~ l c For x ~ ~an. illustration showing the relative effects on GAIIAA-mcdiatcd CI flux and the dpprrssion of NMIIA-, kainatc- and voltagc-gated ~ a "flux, scc Fi,y. 5 under Chilnnc] modtrlmtion. 10-441.A rcvicw .on alcohol intoxicationi and GARAArcccptclr activation is given in ref.'".
C:A BAA c h n n n r l nc.tivnt ion h y volfliilc nnnesthetics 10-14-06: CAHAA rcccptor complexes scrvc a s c o ~ n m o n'molecular target' sites for ;I v:lricty of structurally diverse anaesthetic molecules4/'. T h e major vol:~tilc:~n:lcsthctics isoflurane, halothane and cnfl~uaneopcn anionsclcctivc channcls in r;lt hippocampal ncuroncs w h ~ c h:Ire sensitive t o C;ARAA antagonists4".J7. Like several other ~nolcculcs with gcncrnl anacsthctic propcrtics (such ;IS barbiturates, steroids anti etomidatel, thcsc volatile an;~csthcticshavc a G ~ ~ ~ - r n i m e cffcct t i c ~ on vrrtchratc central t 'GARAncuroncs in culture. Notc: D n ~ g swhich dlrcct ' G ~ R ~ - a g r , n i s t 'and modulatory' properties will tend to produce ovcrall depression of the C N S hy increasing inhibitory' synaptic infl~lcnccand hy direct hyperpolarization1 of n c ~ ~ r o n e s ~ ~ .
u
Mcdintion o f cxcitr~toryrcspnnsc.c hv C:AHAAR 10-14-07: In addjtion to a majc~r inhihitor-yt role of post-synaptic cell hvperpolarizationi, C.ABAA receptors havc hccn shown to mcdiatc r~.uc.itr~lr,r!.rcsponscs in morphologically identified interneurnnest in thc prcscncc of 4-aminopyridine or following thc clcvatinn of extracellular K' concentrations. In thcsc cells, GARA can function t c ~synchronize firing of inhihitory intcrncurones. T h e 'enhanced output' of the intcmcuronci
-
population gives rise to giant IPSPS~in the principal cells7, consistent with the rolc of GARA acting as an inhihitoryt neurotransmittcr.
Transformation o f inhibitory to excit d o r y CARA response,$ 10-14-08: GABAergict inhibitoryt responses havc also heen shown to undergo 'long-term transformation' into excitator-yt responses following pairing of exogenous CARA with post-synaptic dcpolarizationl or following pairing of prc- and post-synaptic stimulation. This synaptic transformation is due to a shift from a net increase of conductance to a net dccreasc of conductance in response to GARA". Compare with responses of the NMDA reccptorchannel with concomitant dcpolarizing stimuli - scu I'h~notvpic~ x p r ~ s s i o n under ELC: CAT C ; L I l NMDA, 08-14,
Excitatory transients in astrocytcs 10-14-09: Stimulation of both GARAA and G A R A ~receptors ~ cvokcs c a " transicnts within type 1 astrocytes in rat cortical astroglial primary culturcs4". ca" responses arc (most commonly) in a singlc-phasc curve or arc hiphasic (i.c. havc an initial rise that persists at the maximal or suhmaximal level). Both types of c a 2 +responses appear with some latcncyf following activation of ~ a "channels andlor via relcasc from intcrnnl ~ a " mobilizing sites (stores). GARA-evoked rcsponscs arc rctluccd following incubation with GARA uptake hlockcrs4".
Phenotypic stzldies relating to specific GARAn receptor stll7units (summnry) 10-14-10: r suhunit data: Ccrchcllar motnr control+ and drug-induced behrvioural impairment has hccn linker1 to point mutations in 2, suhunitcontaining CAI\AA rcccptor suhtypcsmO, 'AIcohol-non-tolerant' rats arc highly susccptihl~.to impairment of postural reflextlst induccd hy hcnzodiazcpinc-site agonistst such as diazepam5". It has hccn found that thc m gcnc of 'alcohol non-tolcrant' rats is cxpresscd at wild-typct levels hut carrics a point mutation generating an arginine (R)-to-glutamine (Q) substitution at position 100. In conscrlucncc, rl,(~lOO)/!,y,rccrptors show diazepam-mcdiatcrl potcntiationt of GARA-activated currcnts and diazepam-scnsitivc hinding of I,'H]-R~ 1545 13.
u
10-14-11: subunit data: -. Deletions of thc Inng armt of human chromosome 1.5 (hands 15rll lq1,31 arc tc~unti in the mniority of paticnts with two distinct gcnctic disordcrs - Angelman syndromei (AS, matorn:~lly derived dclctionsl and ~rader-willit syndrome (PWS, paternally-derived dclctionsl. Dclction of the gcnc for GAIW,, rcccpzor suhunit /I,, (GARRR.3), which mapst to tho AS/PWS rcgion, was found in AS and TJWS suhiucts with intcrstitialt cytogcnctict dt.lctionsis'. (For ~Ie~cscriptionso f 1ypicn1 symptoms o f the Angelman and I'mdcr-Willi synrlrnmcs, scp ~ ~ h e n o t v l ~ i c exprcssion. 10-14.)
Contrihntion o f GA:ARAl recep.tors jn poin reception 10-14-12: Antinociception (1.c. ~nhih~tlcln of pain receptors1 produced hy microinicction of L-glntamate into the vcntrt~nicrlial mudulla of t h r rat
entry 10
C -
---. -.
(nuclcus rcticular~s gigantc~ccllularis pars alpha1 is antagnnizedt by hicuculline and cnhnncctl by diazepam. Thcsc obscrvatinns support thc hypothesis that the rcsponsc is ~nctlintcdin port hy an action of GARA at GARAA rcccptors in the spinal cclrd".
Protein distrihution Systematic studies o f GARAA distribution in suhstnntin nigrn 10-15-01: T h e regional, cellular and suhccllular distrihution of GARAA/bcnzodiazcpinc rcccptors has bccn systematically invcstigatcd hy light and electron microscopy in the rat suhstantia niRra".?. Thcsc studies employed a comhinntion of quantitative radioligand autoradiagraphyt, immunohistochemical{ localization anti in situ hybridization usinx antisenset probes. Moderate to high dcnsitics of C;A1lAA/l~cnzodiazcpincreceptors arc present throughout thc full cxtcnt c ~ fthe suhstantia nigra pars reticulatat with n very low density of rcccptors in thc suhstantia nigra pars cornpactaT. T h c pars rcticulata displays mainly ccntral I~cnzodiazcpinc typc l i rcccptors with the highest rlcnsity of rcccptors being in the caudal p:lrs rcticulata with lower dcnsitics of rcccptors in tlic middlc and rostra1 lcvcls of the pars rcticulata. C:ARAA [ j ! / f l , immunorcactivity is ohsctvcd as a punctatet distrihutian on dcndritcs and ncuronal ccll hntlies in the pars rcticulata.'" (.src3 r11o1C:hi~nnr?ldrnsity, 10-OY,clnrl S u l ~ c r l l l ~ lIocotions. r~r 10-101.
Hcnzodjnzepine immunoretlct ivc sitcs in hrnin 10-15-02: ~ o n t > c ~ o n anntihotlics lt against bcnzt,tlinzcpincs (scc Chonncl morlt~ltrtion,10-44) show c n d o ~ c n o u s'immunorcactivity' in rat hrain in an ortlcr compatihtc with thc regional rlistrihution of GARAA rcccptors in the brain [medial septum :.amygtlala ,-. hippocampus ,-. ccrchral cortex ,-. ccrchcllurn].
-
10-15-03: r s~thunittinta: T h e r-vnriant GARAA proteins ?re differentially expressedt - in t h c CNS and can he photoaffinityi-Iahcllcd with l,erizotli:~zcpincst.
1
SuhceIlular locations l'ost-synnptic <:A BAA receptor-channels
10-16-01: Thc majority nf C.ARAA rcccptnrs (located post-synaptically) arc apposed to transmitter-release sitcs and effect post-synaptic ccll hypcrpolnrizntiont (cf. sympathetic ganglion ncuroncs, which drpolarizr in s o cr.11 r c s p o n s e tlndrr Ccll-type exprc'.ssion rcsponsc to C;AI
Different GARAn responses due to r e c e p t o r het~rogcneityrlnd suhcellulrlr locations
10-16-02: Multiple types of GARAA rcsponscs recorded in rat hippocampal pyramirlal cells arc consistent with hetcrogcncous receptor suhunit tornpositinn and locations nf expression. At the cell somat, rcsponscs arc
entry 10
-
hypcrpolarizinRt and enhanced hy diazepam, whereas responses on dendrites are dcpolarizingt, pentobarhital-enhanced, and show hlgher sensittvlty t o picrotoxinin and hicuculi~nc.
Secretory control filnc tions h y pre-synaptic GARAn receptorchannels 10-16-03: Act~vationof GARAA rcccptnr-channcls in the membranes of small pre-synaptic peptidergic nerve terminals of the posterior pituitary weakly depolarizcst thc ncrvc terminal membrane and hlocks actlon potentials. In this way, GARA limits secretion by retarding thc spacat! of cxcitstion into thc tcrminal arhonza tiont5". C;ARAA autoreceptors (we Receptor a,ynni~t.s, 10-50,ond Receptor nnta~onisrs,10-51 ) arc locatcd prc-synaptically.
ImmunocytocJ~emicnIdistribution studies 10-16-04: Using immunocytochcrnical clctection, a diffcrcntialt subcellulat distribution has hecn dctcrmincd for thc r h versus thc rl and p7/P, GARAA suhunits in granulc cclls of thc rat ccrchcllar cortex: r6 suhunit are dctcctahle only at synaptic sites while rl and thc p l / / J , subunits are located at hoth synaptic and extrasynaptic sites". 10-16-05: GARAA Pz//13 immunorcactivity is ohscrved associated with thc prc- and post-synaptic mcmhrancs of axodendritic synaptic complexes along the length of small- to large-sized smooth dendrites in the pars rcticulata. Approximately 80% of 'immunopositivc synapscs' show equol staining of pre- and post-synaptic membranes and arc assnciatcd with small axon terminals [ < I .O l ~ r n ] .Approx~matcly 20Y0 (elf 'immunopasitivc synapses' dtsplny immunoreactivc thickening of the post-synaptic membrane and arc assoc~atcd w ~ t hlarge axon terminals [ > I .OlrmJ which arc in synaptic contact wlth largc mainstcm dendriter;t-'.' (see o1.m Channel density, 10-09, and I'rotern disirihution, 111-151.
Non-uniform (clustered) dist rihution
of GAHAn channels 10-16-06: Bascd on the distribution nf single channels in nutsidc-out+
mcmhranc patchcs of cul twcd rat cortical neurones, GARAA-act~vatcd channcls cxist almost cxclusivcly in clusters. This non-uniform distribution is present hoth in tnnervatcdt and un-~nncrvatedtneurnnesS5.
Sorting and subcellular targeting of GARAA channe1.s 10-16-07: Assembly patterns and spcc~ficatmnsof GARAA receptor suhunits ~letcrrnine sorting and lncalination of protein complexes in ?larizedt cells2', ~ o n f o c a l tmgcroscopy and suhun~t-spccificimmunohlot analyses of cpithclial cclls (tmnsfcctcd with the cDNAs encoding the a , and /{ GARAA subunits) show that the $1 subunit is targeted to the basalateral surface, and that the fi, subunit is sorted to the apicalt membrane. In cclls isoforms are co-expressed, asscrnhly of the P , with the z l where and /I1 suhunit 're-routes' the x l suhunit to the apical surface2'. For implicat~onsof 'suhunit rout~ng' in control of ncuronnl phcnotypc, see Developrn~ntnl rcgrrlnl~on,10-1 1 .
1
entry 10
Transcript size 10-17-01: S ~ Tnllle P I under Ckne foniih7, 10-0.5
For likcly hrnctionnl conrrihrrrions o f singlc subunits in homomrrict receptors ond those in C O - C X P ~ C S S C I ~hct~romcrictcon~plrxcs.sce r . P, (5. ; arid srrlwnit-specific dntn. The symhol Il'I>TM/ dcnotes an illustrated fcotrrrc or] tlic channel protcin dornoin topograpl~ymo(lr1. (Fi,c.I )
,)
Chromosomal location Relo red GARAn genes (Ire loco tell on different chromosomes 10-18-01: Three isofrirrns of the GARAA r suhunit arc located on different chromosomes5". Cotn~larmtir~cnotc: Di17cr.~~ chromosomal locations of related channcl gene fnmilyf members appears to he common (for cxamplc, S C P Chromonmr~lIncr~tion undpr VLG K Kr.2-Shak. 48-28) . In general, gene separation can occur hy Renc duplicationt and recornhinational transfcri or via rctroviral or retrotransposont excision and insertion events within the cukaryotic gcnomc"'.
Chrornosr~rn~~ linkcIge.qt to ,scgrc,rotedt diserrse markerst 10-18-02: For possihlc linkagc of C.ARAA gene function to chromosome deletions asst~ciatctl with thc Angelman syndrome and Prader-Willi syndrome, S C E Phcntltypic exprcssion, 10-Id.
rl I
Encoding
10-19-01: Supplcmcntary Ti7hle I under Gene fnrnilv. 10-05. Open reading framest c ~ cDNA f scqucnccs encoding GARAA suhunits predict polypeptide lengths between 42,3 and 521 amino acids with an average of -450 amino acitls.
Gene organization Vrrriahlr untranslated re,pions
10-20-01: Indcpcndcntly isolated GARAA receptor cDNAs can cxhihit
variahle lengths of untranslated (non-translated) sequences, suggesting expression contrtllt can operate at thc lcvel of translationt.
Homologous isoforms 10-21-01: Amino acid setluences of GARAA subunits arc generally wellconservedt across vcrtchrstc species. For detailed analyses, compare sequences rctricvahlc by the accession numbers listed under Datahase l i s t i n ~ s 10-53. ,
entry 10
Protein molecular weight (purified) 10-22-01: 1 suhunit data: The GARAA 'a! subunit' was originally affinitytpurified as a 50-53 kDa protein (see also Tflhlc 6 under Li~nnds,10-47). fi suhunit -- data: - The 'original' affinity-purified P subunit (55-57 kDaJ migrates at a similar position to the recomhinantt a3 suhunit on SDS-PAGE gclstSH. Estimates of the nativet molecular mass of the soluhilizcd GARAA receptor-channel range hctwecn 220 k ~ a " and 355 k~a'". Note: The molecular weight of nativcf (functional) receptor-channels dcpcnds upon thc precise subunit stoichiometry~ (see individtml suhunit doto). Supplementarjr note: For the predicted pentameric arrangement of suhunits (see Predicted protein topography. 10-,701, the molecular weight of native channels would he of the order -250 kDa. -~
Protein molecular weight (calc.) 10-23-01: Calculated molecular weights derived from summated amino acids in CARAA suhunits can he derived from analysis of scquenccs via the refcrcnces/acccssion numhcrs givcn under Dotahosc Iistin~s,10-S,?.
Sequence motifs Invuricint features o f the extraccllulnr Ii
For likely functionl~l contributions of single suhnnits in homomcricf receptors ond those in co-expressed hcteromerict complexes, see a, p, 6, y and p subunit-specific data. The symbol (PDTM] denotes an illnstmted fcnture on the clinnnel protein dornrlin tnpnjirmphy model. (Fig.I )
Domain arrangement Common domoin structures 10-27-01: All of the GARAA suhunit suhtypcs arc predicted to have a similar overall domain structure (see IPDTMI, Fi,y. I ) . This includes a signal
(sip) v Clarvad algnal peptlda sequence (19 0).
(a.
"
(pha-65)
"2-
c
*Invarlmt* eyrtalne (c) -flanked protein loop
2
-y
U
~ 3 ' U2
Raqlon of hlgh 'hydraphoblclfy cantraa1' maaoc~atod~ ~ zinc t h Ion blndlng 8118 (rw Blockerr)
r.,,
ll?
Y3
Y
(b) Pentamerlc arrangement (putative)
y2)13 ~ ~
1
' ~ 2
Y1
M4
Extracellular
~onomeric domains -
a . 225-245
Where the (al) aubtypo amino 8cid 101 is Gly, fhls confers 621-type-blndlng. Where the (a2. 03 or nS) mubtype arnlno acid to posltlon
KEY
-
0. 250-271
aa 283-305
401-423
rThcfi I/ I I
I
*The arnlno 8cid nurnbom shown apply to M e 'mafun' proleln (i.8. following slgnal psptlde cleavage). The poaltlons were allocaled by reference to fhm original papor (cited above) and not from a sequence database entry.
ApprOXlm8t8 po.~~ons of con~onaus ~q~ycosytmt~on 11t.s (K~UI pomWona 12, 10% 122- s r r Srquence moclh). Po8ltlonl of con8ensua aIte8 lor proteln kln8.e C (actual poaltlons 313. 356). POSltlOn of con8on8u8 sit8 lor protein kinas8 A (actual position 395).
.
80
NOTE: All relatlw positions of motifs, domaln shapes and slzes are dlagrammatlc and are subject to re-lnterpretstlon.
(c) Ollgorneric structure (putative) See
also rsctlon o n 'preferred' n l p2v2 comblnatlona under Pndlcted proteln topography
CI A
GAB&
Channst rymbol
ELO V
Figure 1. Gmenilrzed mononirrrr protcin dornoin topoqrph\v model [PDTMI for the GARAl receptor-chonnrl suhumt ~erjrrn . r and - R F C I ~ nI I~Ci m h e rand ~ motlf porltlonq applv to the rot GABAAa 6 subunit (see ref.'J7) (From 10-27-01)
C
scqucncet (see [lDTM], Fig. I ) , a large N-terminal [putatively cxtraccllular] domain (see IPDTM], Fig. I), thrce putative transmembrane domains (M1M3J( s e e /PII)TMl. Frg. I ) , a large variable sequencc (putatively cytoplasmic) M3-M4 loop (.FCC /PDTMj.F ~ R 1. ) and a fourth transmembrane domain IM4) ncar the C-terminus (see [PDTM], FIR. I ) (see also 13omn1nfirncrrr~ns.10-29).
Preferential arrnnKemen ts of su hunit co-nssem hly 10-27-02:Assembly of CARAA receptors from constitucnt subunits docs not proceed randomly to form all possible comhinations, but certain suhunit comhinations are preferred intermediates during thc assembly process"2: For example, when varying comhinations of a,, /II1and ;'IS subunits are transicnt lyt cxprcsscd in mouse L929 tibmhlast cells, predominant assembly of functlc~nnlalPj and al/l,yzFGAAAAR forms occur. Notahly, fllnct~onalrly7, and /J1;.2F GARAARs are not detected", GARAhRs co-expressed with and withnut the yz5 subunit differ in their GABA and diazepam pharmacology, and singlc-channel rccord~ngssuggest that the two prcdom~nanttypes of GARAARs [ r 1 P l and B ~ P ~ ; ' ~have , ) diffcrcnt conductancet and p t i n g t prnpcrtlcs. 10-27-03:Further co-expression studies to those dcscrihcd ahovc havc alsr~ shnwn that assemhly of subunits into mature C.ARAARs can arisc from an 'ordered process' to produce a 'preferred form' of thc rcccptorxhsnncl [a1jY1Y25)"3. ~ n t n n s i c t k ~ n e t i cproperties of a t & and aljItyzq assernhlies provide further evidence that y l C l GARAARs arc rarely [ i f ever) formed upon co-rxprcx~innof 011 thrre qtrhtlnitq. This fcaturc is apparently cnnscrvcd across several channel types which are asscmhlcd from many poss~hIe discrete subunitsh7 (FCC r 1 1 w Aciivntron. 10-3,7, and S i n ~ l e chonnci r l m t r ~ , 10-47). Cnmpr~rot~vc note: An 'ordered' asscmhly process has also hecn observed for n~cntinic ACh receptors (see F L G CAT
nAChK. entry 09).
Domain conservation Anionic seIectivtty determrnnnts rn the M2 dnmnin 10-28-01: In gcncral, there is a high dcgrcc of sequence con.iervat~ontnf transmembrane segments MI-M3 hctwetn thc GABAARand thc gTycine receptnrchannel4 [GlyR), indicating their likely importance in chloride channel f o r m a t ~ o n ' ~ ' Introduct~on ~. of three conserved amino acids present tn the M2 scgmcnt of GARAA anti glycinc reccptars intc~thc M2 scgmcnt nf a n 27 nicotin~c rcceptor subunit is sufficicnt to convcrt this cation-selective channcl type intcl an anion-selective channel gatcd hy a ~ c t ~ l c l ~ o l i n( fci r" ~ (urthrr d e t r ~ i l s ,see Domnin filnctzons 11nrler ELL: C1 GLY. 11-29),
Conserved anionic receptor-channel amino ocid sequence motifs 10-28-02:Both GARAA (subunits r, I$,7 , 5, and p ) and glycinc rcceptorchanncls display: [ i )an invariant prnline residue at mid-position In thc M1 domain; (ii) a cnmmon hydmxy-rich sequence Thr-Thr-Val-Lcu-Thr-MctThr [Sur) and a total c ~ ciglit f Scr (IT Thr In cach M 2 domain; (iiil n proline - residue n t the fourth pclsItlcm prcccdud hy a phenylnlnn~ncreslduc In the
entry 10
M4 domain; (ivl relatively high positive charge density within eight residues c ~ ft h r ends of the tmnsmcmhranc domains on the cxtracc~llr~lc~r sides (hy contrast, cation-sclcctivc EL(; channels display ncgativcly charged residues in addition to positive charges hortlcring ~ 2 " ' ~ ' l ; (v) potential to form a Ploopt hctwcen Cysl.39 ant1 CyslS.3, with 8 of IS positions heing identical or hiqhlv conserved in all suhunits6". Notc: A conserved G-TTVLTM-T motifi has nitlcd further isolation of multiple suhunit types in crosshvhridizntion 1 screens.
Conscrvilt ion with inr.crtehrrltc proteins 10-28-03: T h e zeta polypeptide from the freshwater mollusc Lymnr~cn strr,ynrrlis cxhihits hctwccn .10 ant1 40?0 identity t o vcrtchratc GARAA and glvcinc rcccptor suhunit scqurnccs7". Furthermore, locations of six out of seven intronsi occur at similar relative positions a s those found in vcrtchrntc GARA* rcccptor genes. T h e zcta polypeptide mRNA is cxprcsscd in the ndult nervous svsteln hut it can also he detcctcd in peripheral tissues7".
Domain functions (predicted) N - C ~ l ~ ~ c o s y l t iGARA-hind tcd in,^ cxtrrrccllulrir
donltiins 10-29-01: T h e N-terminal [ p u t j ~ t ~ v c lcxtmccllular) v domain of GARAAR contains three N-glvcosylationi sites (2rr /IJDTM/,Fig. I ) and two Cys residlles which help form ;I protein loop' (st7r[PDTMI. Fig. 1 ) may function in hinding C;AI3A and othcr ligands7'.
I'orr-lining domnin M2 10-29-02: Ry comparison with nicotinic receptor-channels (srr ELC; CAT nAChK, rntrt7 091, the M2 domain (sccp/I'I>TM/.Fi,p. I ) is thought to line the channel pore (see also pariigraph on conversion o f cation- t o anionselcct~vcch;~nncls"" in Ilon~c~iri c.ori.sc~rr~c~tion. 10-2x1. l'oint rnuttitions \within Drosophila GARAAA M2 tlomtlins circ u,ssocicited wjt11 jns~c'tici(1~ ~CS~S~RRCC 10-29-03: The cyclodiene insecticides and picrotoxinin (PTX) arc GARAA receptor antagonists which competitively tlisplacc each othcr from thc same hinding site. The field-isolated Dro~opl~iln mutant Rdl ('Resistant to tiieldrin'l is insensitive to hoth PTX and cyclodicnes anci carries a resistance-associated point mutation (alaninc t o scrincl within the M2 porclining domain. The widespread occurrence of this mutation in I>m.sopl~iln populiit~ons promotes resistance t o PTX/cyclodienes and could underlie -. (lOn;, of rcportctl cases of insecticide resistance7' (st7(.nlso rht~Dcltnl7r~sc li,st~ri,y,v t,rhlt* /'or CiARA,, /I .s111)11riit.s/. 'C11jstC ~ I ~ I oS f' p l ~ o s p l ~ o r ~ion ~ l t isltcs t In thc M3-M4 loop 10-29-04: T h e large (putat~vclv)cvtoplasmic loop hetwcen M,3 and M4 frcqurnt lv c o n t d ~ n sconsensust phosphorvlation sites I5r.r /PDTM/, FIT. 1 c~nrlI'rotl,~r~ pho\pliort~lr~t~ori. 1 0 ,12).
-
Positive char~esin the chnnnel vestibule 10-29-05: The cxistcncc of channel vestihn~est containing net positive charges arc consistent with anion-selective permeability propcrtics of C;AI\AA receptor-channels and they may have the property of attracting anions7.' (compnrr with v c s t i ~ ~ r ~ lofe . ~the cation-~rlccfivr nA ChK Dommin functions. rlndar ELG CAT nAChR, 09-29).
Evidencc for henzodinzepine anta~oni.stsit c s on GA \
Subunit contribution to henzodiazepine type it (2nd typc 1lt pharmacology 10-29-07: The suhtypc of r suhunit cxprcsscd in combination with othcr subunits affects GARA affinity and other pharmacological propcrtics: GA13AA 'r suhtypc functions' include (i] an association of a , subunits with benzodiazepine type IT receptor pharmacology with (ii] an association of a5 s u h u n ~ t scontrihuting to positive co-operativityt of agonist binding and benzodiazepine type I I ~ rcccptor pharmacolo~y with low affinity for zolpidcm ( s c ~also iollowing prrra,qrnphs).
Relationships o f recomhinantt suhtlnit comhinntion c~ntldisplny of I.]ZIor HZ2-typepharmacoIo,yy 10-29-OU: Renzodiazcpinc typc lt (RZI]pharrnacthom is associated with highaffinity 1 hinding of ethyl-fi-carholine-,l-carboxylatc mcthyl cstcr (P-CCM, scc T(1171~ 5 11nd~rCh(tnnc1 modolntion, 10-44)and CL21R872. In hetcrolonous 1' expression systems, GARAA alPlyz suhunit combinatinns cxhihit RZl-type pharmacology (i.c. producing C;ARAA rcccptors displaying high-affinity hind in^ for central henzodiazepine receptor l i g a n d ~ ~ The ~ ) . suhunit cnmhinntions a2P,y2 and a3Ply, cxhihit hindinp, characteristics of hcnzodiazcpinc typc I I i (RZJ receptors. A summary of key amino acid loci implicatcrl in modulator hindinpl [as dctcrmincd hy chimacrict cDNA constructions, sitc-directed mutagcncsist and photclnffinity-lah~lingt/microscqucncingf proccduresl arc shown in Tahlc 3 (sue 01.50 foo[notc to Tl~llIc6 under Lixr~nds,10-47).
'Domain swclpt' experiments dctcrrnininl: Ioci o f henzodiazepine hindin,?
U
10-29-09: Exchange of nuclcotidct scqucnccs hctwccn thc GARAhR variants a , and a(, shows that a portion nf the large cxtraccllular domain dctcrrnincs sensitivity toward henzodiazepine (RZ) ligands7', A single histidine in the x l variant is essential for RZ agnnisti binding of C:A1
entry 10
Lnrgc G A R A n curripnt rcsponsrs require o s n l ~ u n i tt!xpn~,ssion 10-29-10: From inicction of rccomhinantt C;ARAA sul,unit rnRNAs in Xcnclpus oocytcs, the prcscncc of (lr ir.r;st onru(I subunit in the ctimhination is required for intluction of 'large' currents (I,,:,, . ;iOOO nAJ. Howcvor,
Table 3. Amino clciri loci impliccltrrl in nlorlrilmtor I)inrlin*qwithin C:AIjAA rcccptr~r-r-hr~nncl cc~rnpir,xc.s(From III-29-08) Constructicln/sitcdirected mutant/ photonffinity 1;lhcl
Functional implication
~ h i r n a c r a s twith mixed domains of , r and r k 3 suhtypcs
Whcrc t h r ( , t i ) suhtypc ainino acid 201 is CIv, this confers 1E-tvpc-binding (/l'DTAf/. Fi,q. 1). Whcrc thc (it2,(1 4 or ( h i ) suhtypc amino acid I~rjrnologc~us t o pcisiticin rll-201 is Glu, this confcrs Liz7-type binrling
Refs
''
75 Mutation c ~ thc f or, sithunit cDNA (Argl(l0His)confurs ability t($ hind I . 3 ~ ] fli~nitrazcp:~rn. { N o t ( , :Niitivc (b(, rcccptors do swopst' - ,YCC c l l v t ~ 10-29- not hind the l i p n d , and His100 is fount! in h ( ~ m o l o ~pnsitions o ~ ~ s in o,,[I?, (1.3 and i t s ] 09)
s t ~ h u n i cDNAs t cocxprcsscd with o(, and 7 subunits - ('Domaintrl
Microsctlucncin~of c y a ~ ~ o g chrolnidci n clu;~vciIpuptidc fragments hillowing 1;lhcllin~wit11 ['HI-flunitrazePam
In scparatc stutlics, sitcs of attachment of 7nn77 the I,'~]-flunitr~zup;rnm photolnbclT has hccn loc;~lizcdto rcsiducs 59-148 of thc I,nvinc (11 ~ u h t ~ nnrl ~ c to ' ~rusiducs 8-2517 (with S. lrrrrerrs V X ~ ~nr~rose~~~cncin~'I has rcvc;~lctlp l ~ o t r ) l ~ h cnttachincnt lt ~t the i ~ r o ~ ~ l arcsirli~cs tic Pllc22h and Tyr2d 1 , closc t o thc hcginning of tht. first transineinbranc doni;~inrcgicln'" (JIJ13TM/.F?y. I )
78 ~hr,toaffinityt-l:~l~elli~ of Lnhclling was first local izcrl to a Pht65 in affinity+-purified chyrnotryptic suhstratc (hcginning at Thrhl ] conscrvctI in hovinc and rat r l !-(I,? Ix~vinc( 1 1 with !'HImuscimnl and (I< of mt, hut not in any , I subunit. T h e residue carrying thc lahcl was locnlizcil hy microscqucncinK1 t o Phc6.5 on which is conscrvcd amtinr: :jlf C,ARAA rcccptor (1, ?? nnil ;I subunits ( / I ' I l T M / , Fix. I ) 74 Phch4Lcu mutants exhihit m:~rkcd Site-directed mtttntionst of PheG4Leu in the rat (11 rlccrcascs in agonist anrl antagonist affinity when co-cxprcsscd with , I ! nntl 72 suhunits suhunit in X ~ n o p u soncytcs, consistent with thc hovinc 13hch4 homologuct irlcntificrl by pliotc~nffinityInbelling (si-t. [hi.TM/, e~ ~~~) ~ F1.y. 1 )
I
cntry 10
-
oocytc cxprcssion in the absence of r suhunit mRNAs confirms that they are not absolutely required for channel formation or GARA-dependent gating in
vitro.
Role o f GARAn i j subunits in receptor nssembly nnd chflnnel electrophysiolo,~icalproperties suhunit -data: Generally, GARA* $ suhunits are required for 10-29-11: efficient assembly of cnmplexes, and thc presence of different P suhunit - --
-
types in rccomhinant complexes influences GARA affinity, channel conductanceR"and modulator efficacyt (c.g. t o bcnzotlinzcpincs, harhiturates and ~ t c r o i d s ~ ' ~Although ~ ~ 1 . photo-lahcllingt studies implicate P suhunits in GARA hinding, channels composcrl exclnsively of r, P or 5 suhunits can he gatccl hy GAIIA, implying that the agonist-hinding site is not unique to the /i suhunit. GARA-activated currents in cclls hctcrolWouslyt cxpressinp GARA receptors incorporating the b2 subunit show faster desensitizationi and greater outward rectificationt (see Current-volta,pe relation, 10-.?5).P2containing complexes also cxhihit a shorter mean open timet than receptors composed of rl y2 subunitsRn.
GARAA 7 subunit functions 10-29-12: ;.-suhunit .. .- ...data: GARAA
7 subunits inlluenct. henzodiazepine binding, although the 1 stthunits dctcrmine whether the pharmacology matches 'classical' henzodiazepine type 11 or type 111 pharmacolow - see nhove. g subunits appear absolutely required fnr allostcric modulation of C.ARAArcccptor-channels: yl subunits contribute 'atypicaltl henzt,diatepine responses; y 2 ~subunits contrihutc typicalt henzodiazcpinc sensitivity and zn2+-insensitivity.When the ;. suhunit is present in rccomhinant CARAA cornplcxcs expressed in hctcrologclust cclls, thc GARA-activated channels generally have a larger conductancetH"(see n1.w Channel modulation, 10-441.
GARAA p suhunit expression associated with non-GARAA/GARAH responses in retinal hipolar cells 10-29-13: p suhunit data: The cloned GARAA pl (rho-1) subunit is highly expressed in tlic retina"" and, with thc exception of its high picrotoxinin sensitivity (ICSo i 200 n ~ p), hnmornultimcrt, channcls display an csscntially similar pharmacological profilc to that nf the novel GARA receptor-channels expressed in retinal hipolar cclls 1i.c. non-GARAA/ GARAR, insensitive to hicucullinc, harhituratcs, henzodiazcpincs and haclofcn - see Rlocker.~, 10-43IR3.Expression of the GARA 111 receptor suhunit cDNA in Xenop~isoocytes yiclds a pharmacologic profile charac- teristic of the 'GARAcPresponses (fordiscu.~sion,see
1
Predicted protein topography
GARAA receptor suhunits for functional hetero-oligomeric complexes 10-30-01: Functional diversity of native GARA receptors i s likely t o arise through formation of diffcrent hetero-oligomeric (pentameric) arrangcmcnts from suhsets of the 16 (prescntly known) C;AI%AAgcnc Isnformst (rl-rhi
4
entry 10
;.I-;',3,(5, and [ I , , p,). Although thcrc is littlc dircut information on the subunit composition and/or topography of nativr receptor-channels, a maior (prcfcrrcd) suhtypc may he 11P2yL since the mRNAs encoding thcsc suhunits arc often co-localized in the CNS.'~( S C F bclow, hut nlso set The slope of the concentration-response curve is steeper for the 'preferred' T~ /1,;~, combination compsrcti with thc z I or T I;.? sul~unitcomhinationsH". (For furthrr drtails on experimrnts rstmhIi.~hing'prr~erentirrl'clrmngcmcnts of sllhtypcc, scc Domrrin rrrrnn,qen7cntp 10-27.)
Recomhinan t s u l ~ ~ i nassem it hlies resernhlin!: nnr ive C;ARAA receptors 10-30-02: ~ c c o m b i n a n t tGARAn rlP?;.l subunit complexes exhibit pharmacr,logical and clcctrophysiolo~ical properties rcscmhling thosc of native+ C.ARAA receptors. The combination a5b2y2 also prnduccs 'consensus' propcrtics of the vcrtehratc CARA,-, receptor-channel, including thosc for co-operativity of gating, drug modulation ant1 Kc,'''. Notc: Suhstitutic~nof rl or 71 in GARA, complcxcs appears to have littlc cffcct on thc channel propcrt ics.
I'rol~ertics o f rccoml~innnthon~omultirnericGARAn reccptorchann~ls 10-30-03: Single-subunit channels (hamomultimcrst of any onc of the subtypes) have hcen reported tcl display GABA-dependent p t i n g t , barbiturate potentiatinnt, hicuclllline and picrotoxinin blockade, multiple conductance states and desensitizationt (but .~ecre/."). Notc: Recausc of their relative instability, homt~multimcrict GARAA ruccptc~r-channels arc ~~nlikcl\. to form native[ receptor complcxcs in vivo.
Invcrtcbrrltc C:AI{A,q protein topo,qrophv ond dornclin urrongcment 10-30-04: /! suhunit- data: Molluscan /I suhunits arc capable of rrplncing vcrtehratc suhunits in h c t c r o ~ o ~ n u scell t co-cxprussion cxpcrimcnts with the hclvinc GARAn rcccptor rl suhunit, st~wcsting that invcrtel?rrrtc GARA, rcccntors also exist as hctcro-olitromcrici comnlcxcs in vivoH".
Protein interactions Subunit protein intcrrtctionx d i s p l f ~ y i selectjvc n~ phutmncology 10-31-01: Co-expression of certain rccomhinantt 'a with 8' and ' a with y' suhunits can yield complexes with the pharmacological properties of G A R A A benzodiazepine type 1t or type I I ~receptors (see rc/.~"'." and Chrrnnrl modul(rtion, 10-44). Compnrrrtivc note: Ilcccptors cxpresscd in heterologoust cells which include the GARAA a , variant generally display the known propcrtics of benzodiazepine type 1t rcccptclrs. Receptors containing thc GARA* a2, ax or a, variants yenrrirlly have much in common with the GARA,, benzodiazepine type 111 rcccptors.
Hornogc'ncous clectrol~hvsrolo,~rcal prr)pcrtre
SIY
dlfic>rc,ntGARAA s u h u n ~ t ~qoformshave heen
entry 10
-
shown t o result in an apparently 'homogeneous population' of GARAAR ion channels, indicating that the protein asscmhl y process selects 'appropriate suhunits' to build functionat channelsn'.
A requirement for rrl//jl ,subunit interactions for fzlnctional protein assembly in insect cells 10-31-03: In S f 9 (insect] ccll hosts for rccomhinantt haculoviruscst drivingt expression c ~ f human CIARAA 21 and PI suhunit cDNAs, fluoresccntly lahcllcd monoclonalt antibodies to rl suhunits show fluorcsccncc of the plasma mcmhranc only in cclls co-infected with both rl and PI recombinant virus constructsRR.
Relative stmhility of co-expressed GARAA subunit complexes in 00cytE.S
10-31-04: 3 .- subunit . data: Co-inicction of bovine al and P1 subunit mRNAs produce GARAA channels that are more stable than suhunit mRNAs supporting cxprcssion of homomultimersT. Together, r l and PI replicate many properties of nativct GARAA rcccptors (ser above). Quantitative i m m i ~ n o p r c c i ~ i t a t i ostutlics d in a rangc of cell types suggest that nativci receptors can contain cithcr a single r variant or multiple r variants. 10-31-05: 7 suhunit data: 'Typical' benzodiazepine potentiationt following cocxpression'f o f G ~ R ~ and h a /I1~ suhunits requires the ndditional prcsence of one or morc 7 suhunits (e.g. 31 pl y 2 ) . Substitution of 6 for ;.has hecn shown to aho1i.~hhenzodi:~zcpinchinding.
Complex inieracfions of excitatory and inhibitory receptor-channcl proteins - on exnmple 10-31-0(,: The post-synaptic currcntt (PSC]in the outer molecular layer of the adult rat dentate gyms prt~duccsa dcpolarizing post-synaptic potential1 ( D P S P ~ ]in granule cells and is pnrt-rncdiatcd hy GARAA, NMDA and AMPA rcccptor proteins in the ratio 1 : 0.1 : 0.2 (as cstimatcrl hy pcakl The pcak currcnt ratio1 is essentially constant over a rangc of stimulus intcnsitics that produce compound PSC amplitudes of 80400 Sirnultanco~~sstimulation of prc-synaptic fibres from hoth the perforant path and interneurones results in a large tlcpolarizing Gc;AnA.A componcnt that inhihits thc granulc ccll hy 'shunting' the excitatory PSCs. The C;cAr3A.A componcnt tlccrcascs the pcak nPSP ampliturict hy ~ 3 5 % ~ shunts 50% of the charge transfcrrcd to the soma hy the excitatory PSC, and complctcly inhihits the NMDA receptor-mediated componcnt of the DPSP. Similar kinetics of the GARA* and NMDA PSCs effectively inhihit the NMDA PSC. The (morc rapid) AMPA PSC is less affected hy the C:c.AnA.Al s o that granule ccll excitation under simtlltaneous stimulnrion is primarily due to AMPA rcccptor activationRP.
Binding o f voltage-dependent anion chmnnel (VDAC) protein hy henzorlinzepine l i ~ n n d s 10-31-07: A .3h kT)a polypcptidc, ori~inallypurified on a hcnzodinzcpinr nlfiinity crdurnnt has shown channel-fnrming activity in lipid hilayer mcmhrancs that is 'virtunlly identical' to thc VDAC (voltage-dependent
-
anion channel, isolated from niitochontlria of vnrioi~s~ ( ~ i ~ r c e-~ SC*C ' " ' MIT (rnitochondrirl), entry 37). Notably, this distribution is consistent with the VDAC hcing ctlt~ivalcntt c ~the 'peripheral-type' benzodiazepine receptor, tlcscrihctl in tlw f(~otnotcunder 7';thlc 6 rin(lr1r Li,yr117tl~.10-47. Rat hippocnmpal ~ I I N Aclnnts ~ isnlatcd using prc~hcs synthesized following rnicro~ct~ucncingtc ~ f the 36 k13a pc~lypcptidc show -24"L amino ;~cid identity to vcnst VIIAC, anrl ovcr -70'XI itlcntity to the human 1% lymphocyte VDAC. Antiscra to the 36 k1)n polypcptitlc is ahlc t c ~ precipitate [3~]-muscimol-binrlingactivity, indicating a 'tight' association with thc GAIIAA rcccptor protcin in ~~itro"".
Dcpolnrizing actions of C:A NA in cylinlcells cxpliiinc~d cxprr.s.sion o f I riznsport crs
c l i f f ~ rt if11 ~n
10-31-08: In contrast to thc Ilypcrpol:~rizing :actions of GARA in most ncuroncs, astrocytes end oIigodcndrocytcs arc dcpolarized hy GARA (although the i~ncicrlying receptor-channel appears to he rclatcci t c ~ 'ncuronnl' GARAA rcccptnrs) (src C r / / - t y p ccxl~rcssionindex, 10-OX). In oligodcndrocytcs, thcrc is a markrrl increase in intraccllular C1 concentration owing to the functional cxprcssic~nof ;I h~lmetanide-sensitiveNa+/K'/Cl transport system. In astrocytcs, incrc;lscd intr;lccllular CI involves coexpression ol a Na'/K'/2Ct -co-transporter and C1 /HCOl -exchangerY'. Thcsc diffcrcnccs in transpnrter protein cxprcssion can :iccount for C;ARAgated CI channcls giving risc to CI rfflux from glial cells. F(1r thc rlcpcnctcncc r ~ f current dircctinn on elcctrochcmical driving forccT, srr Cnrrrnt typr, 10-.74, rrnti Voltr~.qr~ ( ~ r ~ x i ! i v10-42. jty.
Protein phosphorylation GARAn pmrein pho.sphorylnrion in vitro 10-32-01: Thc GAIIA* rcccptor is pliosphorylntcd in vitro by protein kinase A (PKA), protein kinasc C (PKC)and an ~lnidentificdreceptor-associated kinase ( r ~ v i r ~ w ~in* r l~ l " ' ) . PKC gcncrally 'dawn-modulates' different rccomhinant GARA* channclsy3(sr~r,1 ~ ~ ~ 1 Conipclrcltivr, 0 ~ ) . not(': Like thc nAChR (cntry 09), the C;ARAA rcccptor ~ l o c s not appc:ar to he phosphorylntcrl by calmoti~~lin kinnsc I! on any of its cc~nstitucntsi~hunits.
Fl~n(-tiont;l chonseqtrencr!so f C:AnAAR p ~ i o ? ; ~ ~ J ~ o r ~- lprotcin (~tion kinttsc A 10-32-02: In general, phosph(~rylation of C,ARAA subunits will hnvc diffcrcntial cffccts depending (In the receptor complex subunit composition and the number and loci of phosphorylation sites within thcse suhunits ( w ~~r r l h ~ l r ~ i t - ~ pd~r,rcti~fhi~~l.o w )111 . I ~ ~ V OCAMP-dependent , phosphorylation may incrcnsc thc ratc of CAR/\, rcceptnr desensitizatinni (in c l i ~ c kcerchral cortical ncuroncsl or may cnhancc receptor function hy phosphorylation follow in^ 1,-ndrenoccptnr stimulatic~n (in ccrcbcllnr Purkinic cells). PKA inhibits GARA-evokcd CI currcnts (in mouse spinal cord ncuroncs) both in whole-cell and singlc-chnnncl patch preparations. PKA dccrcnscs ircqilcncyi of channel 0pcnin.g withoi~taffecting either channel conductancct o r 1nc:ln chnnncl opcn timei I(:/. I'rotrin p h o s ~ ~ ~ l o r ~ ~ ~rlndrr n t i o r EL2<; l CAT
nA(:hK. 00-.?21.
entry 10
-
Amino acid residues sensitive to phosphorylntion by the endogenous protein kinase C of oocytes 103243:The functional effects of protein kinase C-mediated 'down-regulation' of GARAAcurrents have heen studicd hy mutations altering serine or thrconine residues of consensus phosphorylation sitcs for PKC in the large intraccllular (MA-M4) domain of suhunits r l , P,, and ;'zsY4:Following co-expression with wild-typet suhunits (to yield rl p2 combinations) 14 individual mutations did not affect the level of expression of GABA current. However two mutations, D2 (Ser410) and y 2 (Ser327) ~ result in significant reductions of the 'down-regulatory' effect of the I'KC activator 4 /I-phorbol 12-myristate 13. combinations of co-expressed suhunits sumcst that phosacetate ( I 0 n ~ )Other phorylation of hoth sites is required for a 'full, PKC-mediated clown-regulation' of GARAh currcntsY4. Note: In these experiments, PKC-dependent phosphorylation appeared to 'preferentially inactivate' a non-desensitized form or state of the GARAAreceptorY".
;.,,
Presence o f phosphorylation consensus sites in GARAA subunit sequences (summary) 10-32-04: x subunit data: Phos/PKC: a5 - .3 protein kinase C sites. ah - 2 protein kinasc C sitcs. PhoslPKA: 1 protein kinasc A site. 0 suhunit data: I'hoslPKA: 1 protein kinase A site. The intraccllular domain of t h e T , suhunit is phosphorylated on Scr409 by hoth PKA and PKC~".In vitro phosphorylation of P subunit isolated from porcine and rat brain has been demonstrated. Note: The loci of these consensust sites (and those described below) can he derived from prirnaryt sequence data retricvahlc by the accession numbers listed under Dotnl?ose listings, 10-53.
Extra phosphorylation sites present in alternatively spliced variants o f GARAn receptor proteins 10-32-05: p suhunit data: There arc two alternatively splicedt forms of the y2 subunit (;.2S/;.2L).The 'long form' (-,yl.) incorporates an additional exont of eight amino acids (LLRMFSFK) inserted in the first portion of the M3-M4 intracell~llarloop (see /I'llTMI, Fig. I ) . This cxon contains a 'new' phosphorylation site for protein kinase C (Scr.343) in addition to the PKC site at Scr,327 (on ;.ZS and ;.2L)9'eY7. Note: Phosphopeptide fragments of ;-?L can hc phosphorylated in vitro hy protein kinase C. Regulation of this splicing? evcnt may he under developmental control and could account for cell typeselective patterns of phosphorylation. Note: Roth the ;.I and ?; suhunit sequences havc a consensus tyrosine kinase phosphorylation sitc2"."'. 10-32-06: 1) suhunit data: 11 suhunit scclucnccs possess ?, 'consens~~s' protein kinase C sitcs.
Links between GARAA phosphorylated splice variants and ethanol sensitivity 10-32-07: Alternative splicingt of the GARA* subunit YPL can result in 'ethanol-sensitive' or 'ethanol-insensitive' forms of GARAA complexe!: (when comhincd with sc and /{ subunits)". Site-dircctcd rnutagenesist of thc ;'IL protein kinasc C consensus1 phosphorylation site (sre ~ h o v c )has
entry 10
I
shown that it is critical for modulation hy ethanol hut not hcnzodiazcpincs. Inhihition of phosphorylating enzyme activity in c~ocytcs cxprcssing s r I{,;*?,, can also prevent ethanol enhancement. Thcsc data have I ~ c c ntaken to infer that phosphorylation/dephosphorylation status of spccific sites on the CARAAR c;in act as a 'control nicchanism' for ncuronal responses to Ill-JJ). alcohol cxposurc" ( s c , ~rllso Chrlrl11l.1 rnodr~lr~tion.
)r wxwr WY
-
~ O I K O ~
Activation (:A RAAKs displny multiplc opcn stc~tc,closcd strrtc, rrnd 'bursting' hehaviour 10-33-01: Main contiuctancc statest of the C.ARAARsin cultured mousc spinal cord ncuroncs opcn singly and in burstsTof openings. T h e channcl opens into at least three open states in this prcparatic~n: 01 (T 1.0 0.2 ms), 0 2 ( s 3.7 f 0.4 ms), 0 3 ( 7 11.,3 + 0.5 msl, which do not vary ovcr t h e rangc 0.5-5 ~ I Mc,ARA'"". T h e transition 0 1 (shortest) -- 0 2 / 0 3 shifts in rclativc frcqucncyi to give increases in long opcn tirncst with increasing agonist concentration. T h e channel has scvcral closed states, the two shortest time constants hcing concentration-intlcpcndcnt, with thrcc longer time constants, each drrcrr.asrn,y with incrrrlsing agclnist concentration1'" (see m1.w ref.'"). The hi-liganded rerepto! state (A2R) is then convcrtcd to an activated statet (A2R0)Icarlinx to burstsi (-= 15-40 nls, with 2-3 interruptions per burst]. Shortlived hursts 1-1-2 msl arc also present, which :irc consistent wit11 thc opcnings, possihly due to a mono-liganded (AR) state of thc rcccptor.
-
-
+
+
Kinc~ticpropcttiip,sol C:A NA A channcl act ivntion dcpcnd on stlht~nit uoml?o,sition 10-33-02: Recombinant a l p l and a /?1172s hctcron~ultimcrst transicntlyt expressed in mouse L929 fihrohl:~st cclls differ in their opcn statct and hursti properties. On avcraRc, a l p I,p,s GARAARs opcn for almost thrcc times the duration o f the alpl cntnl>ination, cARAA!ls (6.0versus 2.3 ms, respectively1 :ind havc thrcc opcnings per hursti (scc also prcviorrs pelrc~srclph).r l / l l cARAARs opcn predominantly as single opening hutstsi"". Thcsc kinetic propcrtics havc hccn used to dctcrminc assembly patterns of hctcromultirncric' CARAhRs (,ccc I>ornc~incJrrcln,ycrtlc.n!. 10-27).
1
Typiccll condtlctflnce rla1uc.s o f multiple snhstmtes
-
10-33-03: The main conductance statet ohscrvcd in mammalian ncurones varies hetween tissues - c.g. 1.20 pS in hippocampal neumnes and -+10 pS for spinal cord neurones (the former is similar to that ohscrvcd for 7 2 containing: rcconihinant complcxcsR"l. Wholc-cell currcntt propcrtics, such as desensitizationi and the slope of the concentration-response curvc for ;.?,containing complexes arc similar to the nativct C.ARA,\ receptors of hovinc ~ d r c n a lchromatfin cells and pancreatic islet cells. .~_suhunitdata: Multiple subconductance statest exist for homcllncrict a subunits, pri%cipally 19 pS ~ havc hcen ant1 2s pS. /i suhuni: tl:it:i: Multiple s u h c o n d ~ c t n n cstntcsi shown for hon%~iicricl /lsubunits, mainly at I8 pS and 27 pS.
entry 10
Dose-dependent activation and desensitization propcrties 10-33-04: Typically, GARAA reccptor-channel (in mouse spinal cord ncuroncs) openings are agonistt-cnncentratinn dependent in thc range 0.55 /IM GARA. However, application of GARA to cultured neocortical ncuroncs from rat produces a desensitizingt response, i.c. one that declines over several scconds, even in the continued presence of agonistl"*. There are two activation and desensitization phases to the GARA-induced chloride current of frog scnsory ncuroncs at high agonist conccntrations ) to only one at lowcr agonist conccntrations. Notc: This (-30 p ~comparcd hchaviour may reflect receptor heterogeneity at thc single-cell level ( s c ~ Suhcellular locations, 10-16, and Inactivation, 10371.
Pharmacological modulation o f open duration and frequency 10-33-05: GARAA receptor 'positive modulators' such as pentobarbital (a henzodiazepinc-site agonistt) increase average channel open durationt without increasing opening frequency, whereas picrotoxinin (see Rlockers, 10-4.7) sli htly reduce average channel open durationT and rcduce opening frequency .
7
Activation by single molecules o f GARA 10-33-06: The henzodiazepinet chlnrdiazepnxide (CDPX)modulates CARAAR s o that one molecule of hound GARA hecomes sufficient to open thc ~hanncl'",~.This effect may he explained hy a property of CDPX yielding a closcd statct which can result in channel opening mediated hy a s i n ~ l e CARA-hinding site (see cil~oDose-response, 10-,?(1). Alternatively, CDPX may act at one of the channel opening hinding sites without a postulated, sccond closcd conformational statct'"'.
Vesiculnr and non-vesicular GABA agonist release 10-33-07: Thc majority of endogenous GARA rclcase occurs from vesiclest (whosc rcleasc can hc cvokcd cxperimcntally hy K+-induced drpolarizationt). A sccond type of rclcasc (endogenous glutamatc-dependent releasel is nonvesiculart'". Thc glutamate-sensitive rcleasc is mainly mediatcd by NMDA receptor-channels and consists of a singlc, sustained phase. This phase is insensitive to nocodaznle, partly inhihited hy verapamil and can he blocked by SKF 89976A. The blocking action of c o 2 + ions has also hecn attributed to a hlock of NMDA-associated ion channels"" (see ELG CAT - GLIJ NMDA, entry 08).
r
Current type General dependence o f GARAn current direction and magnitude on drivin,~force 10-34-01: Activation of CARAA receptors leads to either inwardly or outwardly directed CI ion movcmcnt (see Chrmnel modulation, 10-44), dcpcnding on thc electrochemical driving force! (i.c. determined hy the resting potential and the C1 gradicnt - .see Current-voltmgc relntion. 1035). D u r i n ~the activation of excitntoryt receptors, GARAA channcls act to
stahilize the cell resting potcntialt. A striking c x ; ~ n ~ pof l c current 'direction' hcing influenced by d r i v i n ~force is the cxcitatoryt, dcpr,larizingt responses to GARA seen in glial cells (dcscril7cri rlndcr Crll-type cxprcssion indcx. 10-08). hJotr:T h e r~~rlprlitrltlr of the current also depends on the clcctrochemical driving force' (,sr.c. Fig. 3 undpr Voltrigc sensitivity. 10-42).
I
Current-voltage relation liectificrltion propi?rt~ C ' So f n(lt i v GARAn ~ R ch(lnne1s
10-35-01: Current-volt;~gc (I-VJ relations! of C;ARA-activated currcnts ohtainctl from whole-cell mc;lsurcmcnts in some preparations (e.g. mouse culturcri spinal ncilroncs, 145 mM C1 intraccllul;~rly ant1 cxtraccllularly) display outward rcctificatiani. In v o l t a g e - / u m p ~ c p c r i m c n t s , the 'instantaneous'. I-V telatiansi arc lincnrt, whilc thc steady-statet I-V relations- arc outwartily rcctifyinRr indicating that thc fistinKi of GARAnR channels is voltage-sensitive in this prcparatir)nT7 (scc Vol!rr,yc sensitivity. 10-42).
-
Dose-response (:AHA
, clinnncl open time depc?nds on nxcmisr cancentrotion
10-36-01: In gcncral, open-time! frequency liistclgramst arc shiftcd to Iongcr times as GARA concentration is incrcasuti from 0.5 to 5 { I M . GARA
cnnccntration-response curvcs arc sigmoidnl and havc Hill cocfficicntsi of ahout 2 in scvcral prcparaticlns (sr:r Hill corfiiiuient, 10-40).
1,ow-conccntrntion C:AIjA rc7sponscx nrc enhonced chlordinzc~poxidt.
hv
10-30-02: The I>cnzodi;~zcpinc chlnrdiazepoxidc (CDPX) shows an cnhancclncnt of C.ARAA channel opening (In-fold ;at -0.3 I I M GARA) which dccrc;~scs with increasing GARA conccntmtion. At maximnl aRonistT responses JGARA concentrations ---, 1000 ,:MI, no cnh:3nccmcnt hy CDPX occurs'"'. Typic;~lly,thc half-rcsponsc concentration is rcduced from 8 0 t o 50 ~ I Mwith CDPX. In thc prcscncc of CDPX, channel opcning occurs with only one bauntl GARA molecale, whereas in its ahscncc, channcl opening with two hound GABA molcci~lcsappears much more favc~urahle(src also
Actirrntion. 10-,?.?I. Subunit d c p c n h ~ n c co f c~,pnni.stoffinityt r~ntIco-operntive pztingt 10-36-03: 2 suhunit data: The GARA,, a5 suhunit appcars to he important for high GARA a t t i n i t J : ~ n dco-opcrativity~of (;AHA ptinRH1./lsuhunit data: In m l < N A co-inicction studies in oocytcs, the prcscncc of PI or P2 subunits arc not rcquirc~lfor cxprcssion of GARA-gatcd ion currents tlisplnyin~agonist coopcrat~vityiX'.;. suhunit data: T h e GAR/\/, subunit affccts CARA gatinRt of channels cxpres
Dose-response o f GARA,, receptor inactivation properties 10-36-04: ~esensitizationtof native1 GARAA receptors expressed in cultured rat hippocampal neurones is agonist dose-dependent (higher concentrations of GARA induces both larger and faster desensitization). Desensitization phcnotypcs may vary with the 'passage number' of the cultured cells. Note: GARAA receptors are not desensitized without first heing acti~ated"'~.
Inactivation Modulation o f desensitization kinetics 10-37-01: In common with other extracellular ligand-gatcd rcccptor~hanncls, CARAA receptors desensitizet in the continued presence of agonist. CARAbinding sitcs spccific for thc initiation of desensitizationt are distinct from thosc mediating the opening of the channel (see Channel modulation. 10-441. CARAA channel modulators such as bicuculline slow dcsensitizationt while agents such as diazepam enhance the ratc of desensitizationt. In cultured cortical neurones, singlc-channel responses often 'fail to recover' after only a fcw exposures to agonistt. Functional note: Desensitizationt of GARA,, rcsponscs may help r c ~ u l a t ecortical inhihitiont, especially under conditions of intcnsc cxcitatoryt and inhihitoryf synaptic activationfo2.
2
Kinetic models Modelling o f multiple o en and closed states - an example 10-38-01: For recnml~inant x, I, CARAA channels in chincsc hamster ovary (CHO) cells", open duration frequency distributionst are fitted hcst with the sum of two exponential functionsf, suggesting that thc r l P I GARAA receptor-channel has at lcast two open statest. Distrihutions of closed durationst hetween main conductancc levclt openings arc fittcd hcst with multiple exponential functionsi, suggesting that the rl/ll GARAA rcccptorchanncl also has several closed statest. Cr,rnpamtive note: ~ a t i v c tGARAh main conductancet and suhconductancet levels arc charactcriscd hy longcr opcnings and display at least three open statcs (see Singlr-chnnnel dmta, 10-41). Kinctic modcls for GARAAR cquilihrium single-channel gating+ propcrtics havc hcen reviewed".
i
Rundown Phosphorylrtion o f GARAA receptor may he required to maintain responsiveness (examples) 10-39-01: The GARAh receptor has hccn shown to undergo 'rundown't in cultured hippocampal pyramidal cclls and cultured spinal cord ncuroncs (.FCC also pnm
entry 10
clicitcd by GARA dccrcascs (runs down) with time of cell registrationt, with a timc constant of 7.,Z minJn7, while 'residual' responsiveness is maintained thereafter. T h e kinase (or another associated protein) involved in 'maintaining' the GARAA channcl activity has not been charactcrizcd.
An ATP-receptor site a,ssociated with CARAARrundown phenomena 10-39-02: Rased on the action of ATP and specific ATP analogues, it has hccn shown that the GARAARs native1 to rlissociatcrl nucleus tractus solitarii (NTS) neurones possess an intracellular ATP-sensitive binding site (i.c. an ATP receptor1 which can mndulatc the channel1"': Following fast ;ipplic;~tinnof 2 mM ATP, thc aml>litudc of IC.! clicitcd hy 10 ' M GAHA docs not show any time-dcpcndcnt decrease (i.c. apparent rundown) over 60 min. In the ohscncc of intraccllular ATI', the a~nplitutlcof GARA-induced I,:I dccrcascs with timc. Note: Removal o f intracellular ME2+ induces nlndown, even in the prcscncc of ATP"" (scc r11.s.oncxt p r ~ r o g r t ~ p h ) .
Effective and non-cffcctivca,yents in prevention o f rundown
-
10-39-03: In ncuroncs of the nucleus tractus solitarii, activation or inhibition of dephosphorylation processes (hy alkaline phnsphatasc and the phosphatasc inhibitor okadaic acid rcspcctivcly) docs not affect thc GARA rcsponsc in the prcscncc of 2. rnM ATP and 3 m M Mg.". Rundown can also hu prcvcntcd by 2 mm ADP plus ME2' or M ~ plus ~ ATPyS + (adcnosinc-5'-0-.3-thiotriphosphatc], however application of M ~ ' ' plus 1 mM :idcnosinc, AMP, cyclic AMP, AMPPNP (arlcnylimiclo-diphosphatc) or ADP/IS (adcnosinc-5'-r)-2-thindiphosphatcl can nor prevent rundown in this prcPnrationJ".
Selectivity C:AHAA rcceptor-channels are chloride-,~clcctjve 10-40-01: E,,., in rcsponsc to GARA is given hy thc chloride equilibrium p t e n t i a l t : When thc cxtcmnl C1 concentration is rcrluccd, ~ , : ~ is, ~ ~ i shiftcrl in the c ~ c p o l a r i z i n direction ~~ by -51.5 mV per 10-fold change in external (11 , which is close to thc shift predicted by the Ncmst cquationi for ;I selective increase in CI1 condi~ct;incc'"". Detcrlnination of E,,,! in native preparations of moilsc spinal cord ncuroncs with diffcrcnt anions has derived the pcrlncahility ratios' for a variety of anic~nswith respect to chloride (scc Fig. 2I. ~ ' c r m c a h i l i t ~falls / with increasing ionic diameter 1i.c. the hydrated anion sizet or Stokes diametert), reaching zero at ahout 5.6 A ( S C C Fig. 2). Comptrrotivr nrltc: These data indicate thc internal diameter of the GARAA channel to hc slightly smaller than that of the nAChR jcstitnatctl at 7.4 A, scc E L ( ; (:AT I I A C ~ KCIntr:, . 091.
-
Conrlllc-tnncc vrr\us pcrmcal?lIltv scqnenccy for C:ARAA receptor$ rrvdcnce for anlonlc lvndrn,y sites 10-40-02: T h e conductance sequencei ohtalncd from s~nglc-channelcurrent mca\ilrcmcnts for the C,ARAAK In mouse splnal cord neurones 1s Cl > lir I SCN -,F whlch I \ almost In r c v c m c ordrr to the selectivityt Rr ,-C1 -.F (scc FIR 2). Thls (permeabilityf) sequence SCN 1 I 'reversal' lndlcatcs that thc rate of Ion transport 1s l ~ m l t c dhv anion binding
-
-
-
10.0
SCN
I
pJ
4' 0.1 -
2.0
3.0
4.0
5.0
6.0
Sfokes diameter (hl
Figure 2. Ionic permeohilitvt through notivc GARAA receptor-channels. I'crmcahility f n l l with increrlsing ionic diameter; the hydrated anion size1 or Stokes diameter+, rcclching z c m nt ahnut fi.h/q. (Kcprnhlccti with prrmission from IJormnnn (1987)J Physiol Lond 38.5: 24.7-8h.) (From 10-40-01) inside the channel. This, and other cxperimcnts indicate that GARAA channels contain at least two anionic binding sites per pore7,?.Comparntive note: These properties arc shared with glycine receptor-channels (for n direct compnrison ol the pcrmcohility proj~rrtirso f the C A R A A rlnri glycinc receptor-channrls. .we Se1cr:tivity un~hlrELG Cl G L Y , 1 1-40).
Permeability sequence for large polyatomic anions 10-40-03: The permeability sequencet for large polyatomic anions through CARAA channels in mouse spinal cord ncurones has hccn determined as formate > hicarhonate > acetate > phosphate > propionate. Compllrntive note: Clycinc receptor-channels in the same preparation (see ELG C1 GLY. cntrv 11) arc not measurahly pcrmcant to phosphate and propionatcT3.
Similarity o f GARAnR and GlyK ionic selectivity filters 10-40-04: Note: CARA and glycine agonists activate channels with almost itlcntical permeation properties in cultured hippocampal ncurones"". Comparative features of the permeation pathway for a range of neurotransmitter-gated ion channels have hccn revi~wed'~'.For the molecular determinants of anionic selectivity, see Domnin conscrvntion, 10-28, c~nd
Ilomc~inluncrions, 10-29.
1
Single-channel data
Multiple condrictance levels o f native GARAn receptor-channels exclmples
-
10-41-01: Patch-clampt studies of single channels with permcantt chloride ions show prominent multiple conductance levels: Thc typical rnngr of GARAA sin&-channel conductances 1145 mM svmrnctricall C1 I in mouse
entry 1 0
as 27-30 pS (rnnint conductance spinal cord ncuroncs h;lvc heen Icvcl, rcsponsihb for > 95% of tho cttrrcnt through thc channcl) with a sul,ctmd~ict;~nccTPcvcl rango of 17-19 pS ant! an infrequent 11-12 pS Icvcl. With thiocyanate n s the pcrmcant iont, the lmain conductancu lcvclf ~ and thiocyanatc reduces to 22.5 pS. Nntc: With mixtrrrcs c ~ cf h l o r ~ d(H4'XI) ions (lh0A,J,main cr,nductancc states reduce trl -12 pS. T h i s annmalous mole fraction? cffcct has hccn cxplainctl Ily invoking two in!rrr~r:ting hintling sitcs in cnch chnnncl.
Sin~lc-chr~ttncl r.onrJt~cinncr. st uclies of
11r1t ivr'
chr~nncl.~ - uxrnrnplu.~
10-41-02: GARA-activatct! ion channels dctcrmincd hy power spectraf incnsurcmcnts in culturcd mt ccrcl,cllar ncuroncs d~splavsin~lc-channel conductnnccst of -22.7 pS [which arc ~n!'-insensitive) nnrl 14 2 pS (which arc picrotoxinin-inscnsitivc). With high GAR,A concentrations (- 10-
-
+
2000 I I M ) , tlrc main state conductancct ot nativci C;ARAAR in supcrir~r cervical ~ a n ~ l i oncurclnus n is -30 pS with other (less froqt~cntlyohscrvcd) lcvcls at 15-18 pS, 22-23 pS, tc~gcthcrwith (Icss wcll-dcfincd) lcvcls nf 3336 pS and 7-9 p ~ 1 ' 2 .
' D j i 1 ~ 1 7 ic u ~' t.~in~qIt~-c.hi~nnel conduct r~ttctp1r~vcl.cof rrt:omhinrtnt channels 10-41-03: T h e tnain ct~ndiictnncclcvcl of C;AIIAAK chnnncls has hccrl sltown to vary with subunit type oclmp{tsition. For cx;unplc, alpl nntl a l f l l y z , hctcromultimcrsl trnnsicntl yi cxprcsscd in rnourc L92'1 fihrt>hlasti cells tlisplay singlc-chnnncl c~pcnings to hot11 main cctntluctnncci (15 and 29 pS rcspcctively] and s~thcontiuctanccl lcvcls (10 and 21 pS rospcctivcly), with greater than clOx of thu tclt$l current thmugh the main contluctsncc lcvcl opcninRs'"'. Thcsc kinotici properties havc hccn used t c ~ tlctcrminc assembly patterns of hctcromul timcrict GARAARs (scc Drmmin , ~ r r t r r ~ . y r ~ n ~10-27). r ~ n r . Sin~lc-channelanalyses for rccolnl>innnt a l i l I C;ARAA channcls in chinrsc hamster clv:~ry(CHCh cclls"'" 11;lvc s l ~ o w nthc kinctict ~wolwrtiosof the r l P l main c t m d ~ ~ c t a n clovcl o to ciific~rfrom thosc of thc n;~tivcispinal ctrrrl ncuronc 27 pS lnain conrluctnncct lcvcl and the 19 pS suhconductancc 1 Icvcl 'IM (srlr,olcr, Kinrt ir. ~ l l r ~ d tIll-.7RJ. ~l.
Sicroid rnodulnfion of s i n ~ l rC:ANAA channel properties 10-41-04: Ncurosternid replatinn of GARAA rcccptor single-channel kinctic prrqwrtics havc hccn systematically studioti (see Cllnnnel mndrr![ifion,10-44).
Cnmporison with 'refinul-type' GARA-mctjvuf cd chonnels 10-41-05: T h e novel GARA rcccptor-channcls cxprosscd in retinal hipolar crIlsN' (GARA, or 'non-GARAA/GARAnJ-srrJ Rlr,ckcrs. 10-43) display a singlc-chnnncl conductance1 of -7.4 pS with an npcn timct c ~ f-150 m s ( r : ~ n ~120c 1 XI) ~ n s lin syinrnctricalt U1 cr,nccntr;~tic~ns of 145 InM.
Voltage sensitivity Sensiiivity to ionic driving force 10-42-01: GARAA currcnts depend mainly on the electmchemical driving fnrcet, i.c. on the resting potcntialt and the CI (sce Fi,r. (7 rrnrl
entry 10
Current type, 10-34).In rat locus cneruleus (LC)neurnnes, the time constantt of GABA current decay is decreased by membrane hyperpolarizationt , possihly due to a voltage-dcpendentt change in receptor or channc!
-90
2P
A
L
60 rns
Figure 3. Dependcncr n{ GARAn receptor-chnnnc/ current on rleclrochrmicnl drivrn~ir~rce.(Hcproducud with pcrmissron from Rorrnnnn ( 1 9111 7) 1 PhysinF Lond 385: 24336.1 (From 10-42-01)
entry 10
1
kinetics1". GARA exerts a stronger inhihitory cffcct on LC ncurones at dcpolarizedt than at hyperpolarizedt mcmhranc potentials, which could serve as a negative feedback mechanism to control neuronal An cxatnplc oi current 'direction' hcing influenced hy driving force is the excitatoryi, depolarizingt responses to GARA seen in glial cells (descrilwd undcr Cell-typc cxprcssion intlcx. 10-081.
-
Note: A comprehen.sir~etreotnient o f GARAA receptor pharmacology is difficult for scvercll reasons. The lcirge volume o f available information cnnnot hc r~dccllrnt~lyc o v e r ~ d in ( I recr.sonahle .space, leading to prohlcms o f choice. ovcr ~ 1 1 7 ( 1 t to include and omit. This section thcrc~forcreports on overvicrv, with citation o f specialist reviews. More cmphasis hrrs hcen plmccd on thr molecular aspects o f GARAA and less on behavioural effects, c11t hough thesr* (Ire out lined. Compo~rndnames arr n1.so listed in IIcsonrcc C - Compor1nd.s ~ n proteins. d entry 58. For a dicrgrmmrnatic sr~mmrrryo f the in~portantpharmacological modulatory 'sites' includin,q those c1ctin.q (1s ionic pore blockers. see Channel mod~llntion, 10-44.
Blockers 10-43-01: For a summary of GARAA receptor-channel hlockers, see T(lhle 4.
Presrnce o f a ,suhunit determines 2n7+-insensitivityo f r~comhinant CANAn complrxrs 10-43-02: ;. suhunit data: The presence of a y subunit appears to confer insensitivity to ~ n "ion block: Receptors formcd from 2 , and /I2 suhunits are concentration -0.56 / I M ) whereas thosc sensitive to ~ n ? [half-hlocking ' formcd from 7 1 , / j 2 trnrl subunits are in,sensitive to ~ n ' ' hlockade (10 / I M zn" for 2 min, plus 10 / I M GARA in the presence of 10 / l M ~n")'". This subunit-dependent pattern of ~ n "hlockadc is illustrated in Fig. 4. ;q2
Novel chnracteristics o f retinal bipolar cell CARA receptor-channels 10-43-03: GARA receptor-channels exprcsscd in rat retinal bipolar cells havc hcen characterized as inscnsitirrc to GARAA antagonists (e.g. hicuculline) and GARAR agonists (c.g. haclofen) hut can he sclectivcly activated hy the GARA analoguc cis-4-aminocrotonic acid (CACA). These novel channels havc a single-channel contfuctancci of 7 pS and an open timet of 150 ms. Note: 'GARA,.'-typc receptors havc also hccn characterized as bicuculline- and haclofen-insensitive GARA rcccptorsr2.'. The retinal hipolar cell channelsH' arc also not sensitive to modulation by flunitrazepam, pentoharhital and alphaxalone and are only 'slightly hlockcd' by picrotoxinin (for the association o f tlicsr 'non-GARAA/C;ARAN'with p , subunit expression. see (11,sof>ornciin functions. 10-291.
Figure 4. Presence o f a subunit determiner zn2+-insensitivityo f recombinant GABAA complexes. (a) Whole-cell currents ellcited by GARA (10 j i ~ ,indicated b y bar) in fibroblast cells expressing only GABAA a1 and P2 subunits display sensitivity to increasing micromolar concentrations o f Zn2+.(b-e) GABAA receptors reconstituted from subunit combinations which include a -y subunit become insensitive to 2n2+-block.For example, 0 1 - 1 (compare panels h and c) or nl .j1-,: (compare panels d and el. Note: The control traces in 17 and d show whole-cell inrvard currents recorded following application o f 10 /c,v GABA. The traces in c and e show currents recorded in the same cells following equilibration of the bathing solution with 10 /,.IT zn2* for 2 min and application o f 10 pnf GABA in the presence o f 10 /i:w zn2'. (Traces reproduced with permission from Draphn (1990) Neuron. 5: 781-8.) (From 10-43-02) -!
entry 10
Table 4.
Str n~nlnryof C;A 13AA rrceptor-chmnncml~~~~~~~krrs (From 10-4;1-03)
Rlocker
Description and examples
Refs
Bicuculline block
Ricuculline is a hlockcr of GARAA channels which reduces CARA I P S P S ~and in hchavioural studies, can cause convulsions1 in a dose-dcpcndcnt manner
Scr Rcccptor nntagonist.~. 10-51
Picrotoxinin Picrotoxinin can hoth hlock thc chloride rrccptor-channel and elicit outwartl currcnts block ( S P P (11.70 I)orn(iin hlnctions. 111-29,cind Ch11nnc.i modlllntion. 10-44).In hchaviouml stutlics, 'post-training hlockadc't of t h r GARAA-related chloridc chnnncl has hccn rcportcd to rnhancc memory
I I.?
Divalent ~ n t a ~ o n i s moft CARA-clicitcd chloride cation block currcnts in cluturcd rat ccrchcllnr ncurnncs t o by z n 2 + z n ? ' ions dcclincs hy SO'% -4K days after hirth. z n L +at 25-100 ,,hi ilocs not sffcct the singlc-channel conductance, nor rlocs it inilucc any rapid closurc/hl(~ckingcvcnts. A ntwcl GARAA zn2+-binding site has hccn proposed for some (hut not all1 C;ARAAR (sco /I)DTM],Fig. 1 ) . T h c binding sitc is predicted t o he within an area of 'high hydrophobicity contrast' (prohahly a t a sitc located (In thc extracellular part of the GARAA rcccptorchanncl complex). ~ n ? ions ' appear t o hind t o an 'un-ligandcd' or 'mono-ligsndcd' state of t h e C.ARAAR, which stahilizcs the channcl in t h c closed conformationi. F~.lrlction(rln o t ~ s : 1 . z n " ions arc good candid;ltcs for tnodulators of ncuronal rxcitahilityb - they arc present at h i ~ hconccntr:itions in hrain [cspcc,ially the synaptic vesicles of mossy tihrcsi in the hippocampus) and arc rclcnscd during nc~ironalactivity'". ' I " . 2. Roth proconvl~lsantTand CNS-deprcssantt actions of ~ n ? have ' hccn reported: Zinc is a potent non-compctitivct antagonist of NMLJA responses on c i ~turcd l hippocamp;il of ncurc~ncs"~.Thus, ~ n ? antagonism ' C;ARAA responses may underlie ohscrvrd proconvulsant activity whilc ~ n "antagonism of NMDA rcsponscs may untlcrlic their CNSdepressant properties. S I T ( 1 1 . ~ 0 E L ( ; CAT ( ; L I I NM13A. c,nt rL7 08. one! pnrr~~rcrpll10-4302
I Ih
entry 10
-
Tahle 4. Continued Rlockcr Divalent cation block by co2+, cd2*, ~ n and ~ i "(hut not c a L + , M ~and ~ ' 13.1-+ )
J
Description and examples
Some nativet cell preparations (e.g. turtle cone photoreccptors) exhibit GARA-activated currents which are hlocked hy co2+,c d 2 +and ~ i at " ~micromolar ' concentrations. These ions also block GARA-activated currents from channels reconstitutcd from purified nl and ;h_ suhunit proteins, hut in these cascs~n'' is morceffcctivc (seeahove a n d ref."'). The divalent caticlns ~ a " , ~ g and " I3a2' appear to have little c ~no r hlockingactivity at similar concentrations to those dcscrihed ahove Penicillin Thcrc is some evidence for the antibiotic penicillin G acting as an open-channcl hlockert at block the GARAAreceptor. Penicillin C; ruduccs average channel open-duration1 and increases average hurstt duration without altering single-channel conductance. Notes: 1. Pcnicillin is negatively charged at physiological pH, and may thcrcforc inturact with positively charged amino acitis within thc channel vcstihulc (see Domflin functions, 10-29).2. At high doses, penicillin G acts as a convulsant and can inhihit picmtoxinin hinding. 3 . Pcnicillin C. can also induce potentiation and hlwk of currcnts through the glycine receptor-channel (see Channel nlodrrlation under EL(: CI GLY. 11 -44) Ro54864 GARAA channels are blocked at micromolar block lcvcls hy the 4'-chloro-derivative of diazepam Ro54864 (which is also an agonist at nanomolar concentrations for non-C;ARAA'peripheraltypelt henzodiazepine receptors - ..;cca footnote to Tohle 6 under Li4yands, 10-47).On GARAA 'central-type' receptors (this entry) Ro54864 occurs at a site close to (hut not identical to) the picrotoxinin site (see Cliannel modulotion. 1044). In hehavinural studies, Ro54864 can causc convulsionst and reportedly, facilitate memoryformation. The effects of Ro54864 can he antagonizedt hy the isoquinolinc carhoxamidc PK11195, which is generally used as a specific 'pcriphcralt-type' GARA rcccptor antagonist. In addition to PK 1 1 19.5, the phenylquinolincs FK8165 and PK9084 appcar to modulatc the C;ARAAR hy hinding at the Ro54864 site AntiThe antisecretory factor [ASF)has hccn reported secretory to non-sclcctivcly hlock ncuronal chloride factor (ASF) channels including those activated hy GARA hlock
Refs 'la
See also Receptor antasonists. 10-51 .
119. I20
12'
'"
entry 10
e.g. GABA, chlordiazepoxide, muscimol, Isoguvacine, Zinc THIP, Piperidine-4-sulphonate ions Field refs: DA, PPT, A, Field refs; DReg DR. S, CM, EDC, L. RA
Barbiturates e.g. phenobarbital, pentobarbitone Field refs: PPT, CM, RA
Picrotoxinin -TBPS e.g. Picrotoxinin, TPBS, cyclodienes Field refs: DF, A, 6, CM
Steroids
Chloride channel e.g. Bicuculline. anionslcations, Field refs: PE,
Avermectin Field ref: CM
R054864
e.g. ethanol
e.g. androsterone. pregnanolone, androsterone, alphaxalone, THDOC (competes with TPBS site), DHEAS (antagonist)
Field ref: CM
Benzodiazepines e.g. Diazepam, flunitrazeparn, P-carbolines, flurnazenil, zolpidem, pentobarbital, endogenous benzodiazepines (DBI), U-78875, Partial inverse agonist: Ro154513 Field refs: PE, PO, DA, DF, PI, A. I.CM, RIA e.g. gamma-HCH Field ref: CM
Field refs: PE, PP. CM, RIA
Unsaturated fatty acids
Propofol
e.g. Arachidonate, oleate Field ref: CM
Field ref: CM
Volatile anaesthetics e.g. isoflurane, halothane, Field refs: PE, CM, 0 , RA
Figure 5. Principal binding sites for endogenous and pharmacological modulators o f GABAA receptor-channel function. Key to fieldname references: A , Activation, 10-33; B, Blockers, 10-43; CM, Channel modulation, 10-44; DA, Domain arrangement, 10-27; DF, Domain functions, 10-29; DR. Dose-response, 10-36; DReg, Developmental regulation, l & l l ; EDC, Equilibrium dissociation constant, 10-45; I, Inactivation, 10-37; L, Ligands, 10-47; 0 , Openers, 10-48; PD, Protein distribution, 10-15; PE, Phenotypic expression, 10-14; PI, Protein interactions, 1031; PP, Protein phosphorylation, 10-32; PPT, Predicted protein topography, 10-30; RA, Receptor agonists, 10-50; RAnt, Receptor antagonists, 10-51; RIA, Receptor inverse agonists, 10-52; S, Selectivity, 10-40. (From 10-44-01)
entry 1 0
r~r~t,l - n(,r(rlr~ot(,s,f'x(11?1ptc,5 Table 5. (;AljAtl r ( ~ ( ~ ( , \ ~ t o r - ( . / ~ ( rr~io(i~110tiorl ontl r c f ~ r c n c c s(scr301.vo Receptor agonists, 10-50 onri Receptor ;~ntagonists, 3 0-51 ). (Fronl 1 0 4 4 - 01 )
Mntiulator class
I
Rarhiturate mnd~rlation (gcncr;~lnotcsl
T h e barbiturate drugs, c.g. phenobarbital which a t -50 potentiates (cnhanccsl t h e ~nhihitclryaction clf GARA hy increasing avcraKe chnnncl open durationst for t h c suhunit cornhination n l , PI, r2,h c t c r o l o p l u s l y ~ exprrsscrl in ctnhrvonic kidney cell lines. Rnrhituratcs hoth ;Illostcricallyf cnhnncc G A R A binding ant1 c a n in clinical m i m i c G A R A in its ahscncc. I%nrhiti~ratcs use h ; ~ v csedative-hypnotic{, anaesthetic1 and anticonvulsantT properties. Pentobarhitone cnn directly activate C;ARAA CI channcls ; ~ thigh conccntrations ( > 50 , r ~ ) Note,<: 1. Relntcd non-h:~rhituratcss ~ r c has t h c nnncsthctics etazolate, etomidate and LY81067 also cnhnncc C;ARAA currents ns mensurcd I7y r;ldiotraccrt ion flux o r e l c c t r o p h y s i o l o g i ~ lmcthorls. 2. 'Activc' I>nrhitur;~tescan :~llostcricallv~inhibit t h e binding of antagonists [picrotoxinin/hcnzo(li;~zcpinc; ~ n t a ~ o t i i s t s J nntl c;ln ; ~ l s oc n h : ~ n c cbinding of c~tlicr; ~ g o n i s t s{hcnzotli;lzcpinc inverse ;~gonists/(;Al%A-likengonistsl
Rcnzodiazepinc modulation
Srct p t r n ~ ~ r o p l l10-JJ-OJ s rrnrl 10-44-05 rrnrl I(i-cr'ptor
'Neurosteroids' anti steroid anaesthetic modulation
T h e neurosternids, c o m m o n l y <3o-hydroxy ring A rcduccd r n c t a h o l ~ t c sof progesterone and dcoxycorticostcrone 1c.g. 3n-OH-DHP, IS,,-hydroxy-So-prcgnan-20o n e o r THDOC, .3o-,?I -d1hydroxy-S~,-prcgnan-20-onc'), compete with t h c high-;lttinityl hinditlg site of t h c convuls;lnt TPPS (tc.rt-hutylhicyclophospIior~~thi(~natc] t o potcntintc* (;AIM-nctiv:~tcil C1 currents in syn;lptonc.urosotn:~l prep;~r;~tion from r;It ccrehrnl cortex. Ncurostcroid m o d i ~ l ; ~ t i ooccurs n ; ~ nnnomolnr t conccntr;~tionsand can dircctlv elicit hicucullinc-sensitive C1 currents a t micromol;~rconcentrations (stpr' (rl.so p ~ r t ~ , q r ( ~IO-JJ-Or(I. ph T h e stcroidi anacsthctici alphaxalone (.3,,-hydroxy-S-,1-prc~n;1nc-l 1,20-tlioncl incrc;~scs:~niplitudc; ~ n ddurntion of C;ARAA chloride currcnt in cultured spinal cord ncilroncs nntl can tiircctly ; ~ c t i v : ~ct eh ; ~ n n c l ;s ~ high t conccntr:~tion.T h e nicclianisni of motlul;~tionhy prcgnane steroids ;Ippcnrs distinct from that of barbiturates1'-. I{y cmtrclpv/cnth;~lpyc;~lculntions, t h e anaesthetic CARAA cliannrl modulators alphaxalone, propofol ;lntl pcntoharhitone have I ~ c r n dctcrtnincd to intcr;~ctwith t h e (;AItAA rc-ccptor nt rliqtinct recoxnition s ~ t c s " ~
IIM
(~,yor~ists. 10-50
cntry 10
Table 5. Conttnued Modulator class
Picrotoxinin modulation
The non-cornpctitivct, non-sclcctivc GAnA R antagonist picrotoxinin (a non-nitrogenous convulsant of plant origin) rvduccs GARA I P S P S .~ Plcrotoxinin blnds preferentially to agnn~st-houndforms of the receptor a t a a t e d ~ s t ~ n from ct GABA and appears to stabilize a n agonist-hound shut state, enhancing the occurrence of a dcsensitizcdJ statc r)r an allosterlcallyt hlockcd state. At concentrations whlch s~gn~flcantly reduce thc amplitude of whole-cell GARA currents, plcrotox~nindoes not alter spectral trmc constants1 or s~nglc-channelconductance In dissociated rat sympathct~cncuroncs (3s cstirnstcd hy currcnt nolsc anal ys~qt)f2q. In hehavioural studies, picmtoxlnin can inducc convulsionst. Other drugs acting at thc plcrotoxmin s ~ t c include tutin (natural), the synthetic hicyclophosphate 'cage' compounds 1e.g. TRPS, see nbove],s y n t h c t ~ cpolycyclic convulsants (e.g. prntylentetrazale, PTZ) and 'convulsant hcnzodiazcp~nes'(c g. Ro5-3663and Ro5-4864, whlch arc nn! activc at the 'hcnzodlazcpinc s ~ t e ' )N, O ~ PP i c r n t o x ~ n ~ISnahlc to inducc complete block c ~ ~f ~ ~ ~ ctransrnissmn, r ~ l c and t can close GARAA channels that havc been opcncd by steroids, harb~tnratesor avcrmcctln. P~crotoxinln-hinding appears to requlre a multi-subunit complex and picrotoxlnin may act as a dnw channel hlnckert or t . 1 altcr ~ the intrinsic gatingt of thc channel oncc GAI1A is hound
0-C;lrboline modulation
Thc P-carhalines, c . ~ mcthyl. [.I-CCM),cthyl (,fiCCEJand pmpy l (.j-CCI') cstcrs of r~-carhot1nc-,7-~arhoxy3atc havc anxiagenict effects hy reduc~ngthe action of GARA agnnlsts. Scvcr:~l,I-carhnlines act as full or partial inverse1 agoni~tsat the bcnzodiazcp~nercceptnr sltc (for further rletarfs w e Iieccptnr rnver5e ngani~ts,18-52). Methyl h,7-d~methnxy-4ethyl-8ficarholine-3-~arhoxylatc (DMCM) 'ncgatlvcly modulates' chlorirlc currents c l ~ c ~ t chyd GAI1A In granule cells hut 'posit~vclymoduratcs' GARAh currcnts In astrocytcs. This differential modulation is also nhscrvcd w ~ t hrecombinant GARAA rcccptors contalnlng a 71 instcad of a 72 s u h u n ~ tQuantitat~ve . mKNA dctcrmlnat~onsin both cell types suggest key molecular dctcrminant rcspons~hlcfor DMCM-pos~tivemodulatory effects in astroglial nnttvc GARAARs1s the presence of the 71 subunit In the assurnhtcd rcccptor ~ o r n ~ l (for c x other ~ ~ cornparmtrve Irndlngc, sec r n R N A dxtrlhut~on,10-131
Volatile ga* anaesthetic modulation
GARA* rcccptor cclmplcxcs serve as common but ~ c n c r a l l y nnn-sclcctivc 'molecular target' sltcs for a vnrlcty of structurally diverw volatile anaesthetic molecules4" (e.g. isoflurane, halothane, and enfluranel. A hravf rlcscrlpt~r)nof GARAA currcnt c l i c ~ t a t ~ ofollnw~ng n admlnistratton of v o l ~ t ~anaesthetics lc nppcars ~tniicrI'hmotjp'~ rrxprcwion, .
P
1 0 - 14
Table 5. C o n t ~ n u e d Modulator claw Intravenous anaesthetic modulation (pmpainl)
The ~ntravenous eneral anaesthetic propofol (2,ci-diisoprnpylphennll may exert part of its effect through GARAAR: q t low dnses, propofol dose-dependently potentiatesT GARA-activated currents whrle at high doses i t is capable of directly actlvatlng bicucoll~ne-sensitive chlor~dcflux. Propofol may act at a site d~stinctfrom other sedat~ve-hypnoticsltes (harhiturate/henznd~azepine/ GARA) ({or RrleT revfew, we re{ 5 ,
Avermectin B,, modulation
The macrocyclic lactone avemectin B1,(AVM]isolated from Streprornyces nvermrtrl~\has potcnt anti-helminthic and insecticidalpropcrtics. A V M aqrl its stnlctural analohwcs increase chIoride ion permeability1 of vcrtcbratc and lnvertchratc ncrvc anrf rnqsclu mcmhrancs. Whcn applied ARA agonisml , A V M can act either as an as a synergist 1 . Hqh-afflnlty AVM-binding s ~ t e exhibit s a senes of ctjmplex allosteric interactinn7 with other (drstlnpishablel binding s i t c ~tor benzod~azep~nes, In ref.5, harhiturates anti picrotoxrmn-TRPS (hrrefiyrev~cwcd
or
Ethanol modujation
Ethanol mor\ulation of thc GARAAR (gcncrally a potcntiatlonT of GABA rcsponscs) may occur at some receptor suhtypes hut not at others. Ethanol modulation of GARAhR resernhles some properties typical of hgrhiturate and henzod~azep~ne mndulatron (a ticonvulsant7, anxiolyt~cTand sudativc functions F. For fiirther d e t n l l ~ , scc pnrii-grop11~10-44-05 to 10-44-15 and FIR. (1
f
Chlorinated hvdrocarbon modulation
Ccrtain chlorinated hydrocarbons such as hexachlorocyclohexanes arc potent inhl hltors of lC;.Artx (typ~calGAHAA currcnt cxprcsscd h r n ccrcbral cortex mKNA in oocytcsl; c.g. with 7-hcxachlorncyclohcxanc [lindane or 7-HCH) current suppressjon is detectable at cnnccntrattons as low as 50 n ~ ' .Ry contraqt, n-HCH and S-HCH induce clcar positive modulation of ic Acrx elicited hy low 1c.g. 10 ),MI conccntrations of GARA. Atyplcal GARA rcccptors (hlcuculline/ haclofcn-mscns~tivclG expressed by retinal RNA in oocytesl rescm hlc GARA,, reccptnrs In their scnattvlty to HCH hut arc largely Inscnsltlvc to rr-HCH and h-HCH'
-
-,-
Unsaturated fatty acid modulation fol!owing r t ~ by n phospholipase A2
Phospholipase A2 (PLA2)treatment of synaptosorna~t rncmhrancs, which causes the release of unsaturated fatty a c d s (see ILG K A A , entry 261, both i n h ~ h i t schloride ]on flux thrt~uh GARAAchannels m rcsponsc to activation hy agonists"' and affects llpand hlndinp to the receptor. Arachidanic acid and oleic acid has heen shown t o m ~ m i c t h c cftect of PEAz trcatmen t hy enhanc~ngflunitrazepam and muscimol binding hrrt ~ n h t h i t ~ nhgn d ~ n gof thu 'ncurostcroici' TBPS (t-hut lhicyclnpho~phort~thinnate) in a dose-dependent ~nanncr'Y-'. Note Fxternal applrcatron of a punf~ed(neurotcix~c) PLAz fmm the venom of the taipan snakc to chlck sensory neurones rcvcrs~hly~ n d u c c sa picmtoxin-scnsitiv~(DKIS-insens~tlvcl anlon currcnt In a vnlta~e-dcnendcnt"manner"'
cntry 10
Table 5. Continued Modulator class Clomethiazole modulation
The anxiolytic anticonvulsant and sedative-hypnotic c l o m e t h i a z o ~ e * ~ ~may ' ~ " "exert part of its effect a t the GARAAR complex. At low-doses, clomcthiazolc dosedcpcndently potentiatest GARA-activated currents while at high doses is capahle of directly activating hicucullincsensitive chloride flux. Clomethiazole may act a t a sitc distinct from othcr scdativc-hypnotic sitcs (harhituratcl benzodiazepinelGARA) (for hrief r e v i ~ w S. E E ref.5) For details of the GARAA-modulatory cffcct of thc henzoChlordiazepoxide diazepine chlordiazepoxide (where one molecule of hound modulation GARA becomes sufficient to open the channel1 see Dose-response. 103h, nnd 7 suhtrnit dntr~,thrs field Anion All GARAA modulatory sites (and their interactions) show modulation dependence on (and modulation hy) the anions C1 , Rr , I , NO,3 , SCN or C l o d (hricfly rcvicwcd in ref.') Excitatory amino Monosynaptically cvoked inhibitory post-synaptic acid receptor currents in hippocampal pyramidal sliccs diminish in thc antagonist presence of CNQX (an AMPA-rcccptor-sclcctivc (indirect antagonist, see ELC; CAT GLU AMIJA/KAIN,entry 07) modulation) and APV (an NMDA-receptor-sclcctivc antagonist, see ELG CAT C;LU NMDA, entry OH) following a train4 of action potentials. However, responses to GARA applicd hy iontophoresist do not change significantly1"" Post-synaptic Localized physiological changcs in past-synaptic intraintracellular cellular calcium (post-synapticspikcT firing) has hccn shown calcium (indirect to potcntly modulatc synaptic GARAAinputs [i.c. reduce modulation) responses toGARA)within CA1 cellsof thc hippocampus'M: Followings trainof action potentials, spontancous lPSPsT arc transiently supprcsscd, which can not he accounted for hy memhrane conductance changes following the train or activation of a rccurrcnt circuit. Amplitudcs of spontaneous GARAAinhihitory post-synaptic currcnts (IPSCsJarc also diminished following the action potcntial train. The ~ a " channel agonist BAY K 8644 enhances the suppression of IPSPS~, whilc huffcring ~ a changcs " in with EGTA or RAPTA prcvcnts s~ppression'~~" Other indirect Other indirect modulators of GARAA function includc modulation of glutamate (potentiating),intracellular ~ a "(in porcinc C;AIIAA pitu~taryintermediate lohc) (extrricellulrlr CA"-influx rcsponscs indcpcr~dcnt)"~', external Na' ~oncentration'.~'and somc purines and peptides
'"',
--
Note: Not all o f the sites listed in this tahlc nnd elsrwherc in this entry (Ira
nccussarily present on nny onc GAHAA con~plex.Thc lar,pc vrlriety o f .. and ohserverl 'cullcomponent GARAn slllwnits (scc Grnc f ( ~ m l l ~10-051 type-specific'pntterns o f modulation indicntc there is much scope for selective modrilr~tionof GAIIAA receptor-channels accordin,y to .~ri/~tinit uompmsition lit the sin,y1e-ccll l c r ~ l .
channel itself (possibly the picrotoxinin site, scc TnI~li,5) hut may include many other discrete sites. Features of a provisiclnal GARAA receptor subtype classification (CIARAA,, GARAA2, GARAk3J hriscrl on ~ h c i~~nctionrnlpropprtics o f their allosteric modulatory centresi has hccn s i ~ m r n a r i z c d ~Note: . Updates on IUPHAR Nomenclature Subcommittee recommendations on GARAA rcccptor-channels will appear via the 'homc page' of the Cell-Signalling Network (CSN) availahlc over the World Wide Web on the Intcrnct (for cicscription, scr I-ccdhrick c*) CSN r~cccss.Pntrv 12).
Potenticrtion o f c:AnA, currents
I ~ j rcthanol
10-44-05: Thc 'positive-modulatory' cffccts of ethanol on GARAA currents arc rcvcrsihlc (in chick ccrchral cortical ncuroncs\. In -(-OR, of ncuroncs, cthanol causes a pntentiationt of thc mcml~ranccurrent elicited hy GARA [thrcshnld conccntrfltion 1 mM, maximal cffcct at 10 m ~ )At. higher concentrations (4050 m ~ )cthanol , inhihits GARA-activated currcntsl.'" hut this effect may hc subunit-specific: In other synaptoncurclsomal preparation from rat cerebral cortex, cthanol stimulates CI flux to -250Y1, saturating st -50 m ~ ) . Ethanol sensitivity of C;ARAA rcccptors in Xcnoplls oncytcs requires eight *~. (sec Kcucptor amino acids contained in the y2,, s u h ~ ~ n i t ~Ro154513 invrrsc n~onists. 10-52) can ;~ntagonizc certain cffccts of cthanol. A commentary on ethanol modulation of cxtraccllular li~and-gatedruccptorchannels including the C;Al
Rocnmhinant C:A BAA rcccptor responses to hiph concentrationx o f cthnnol 10-44-06: Rcplaccmcnt of the ;*,, suhunit hy the alternatively spliced+ variant y ? ~ . in rccomhinantT C.ARAA channels in X~nopu.q oocytes significantly stiniulntcs the GARA response to 111,qh concrnrrr~tions of ethanol ( > (70 m~)"'. The rcsponsc to cthanol may also he tlcpcndcnt upon thc
precise sl~htypcsspecified :it different stages of developmental expression ( ~ ndi~lt/foetal n dorsnl root ganglion ncuronrs). Sclectjve .supprrssion o f C:A [{AA R suhzrnit transcripts h y continuous
cxposure t o ct hot101 10-44-07: Chronic ethanol exposure has hccn reported to reduce thc lcvc! af mRNAs cncoding the 1 1 , 12 and T~ (hut not ~~1 C.ARAA suhunits in hrain. Nr~rr: The tlccreascd rcsponscs to GARA following chronic cthanol treatment may play a role in the neuronal hyper-excitahilityj associatctl with ethanol withdrawal (c.itor1 in rci.'"~.
N~wrostc.roidsinurcasc C:AfJAA sin,plc-channel open times 10-44-08: Ncurosteroid~ regulation of GARAA rcccptor single-channc! kinctici proprrtirs hnvc hccn sturlicd in culturcd mousc spinal cord ncuroncs "'. ~ v c r a g c d f CARAA receptor currcnts arc incrcascd in thc prcscncc of the steroids (c.g. with 2 ~ I MCZARA plus androsterone (53andrc1stan-.3r-ol-17-onc 10 nM-10 / I M \ or pregnanolone (5/1-prcgnan-,?z-ol-20one, 100 n ~ - l O ~ I M I .GARAn rcccptor current amplitudest (main
cntry 10
conductancc lcvcl -28 PS, -96% of cvnked current plus suhconductance level of -20 pSj arc unchnnged by the steroids, hut average channel open durationst arc increased (due to an increased rclativc proportion of the two Inngc.cr opcn-duration t l r n t r c o n s t a n t ~ t ) ' * ~Avcragc . durations of channel burstst (groups nf openings scparatcd by closurcs > 5 ms) arc alst, incrcascd hy ncurostcroid spplicntmn (sce also next pnragmph).
VSCC
-80 -60
-100
NMDA
-
Figure 6. Compnrntrve mndulntory c f k c t s o f ethanol on innrc (lux through G A RAn receptor-channels (rhrs entry), voltage-sensitive cnlcirrm chnnnelv (see VLG Cn, entry 41), karnntc-selectivc glutamnie rec~ptor
I
44-051
entry 10
Neurosteroid enhnnccmcnt o f GA13AA channel suhconductrmces 10-44-09: Andrnsterone and pregnannlonc cnhancc averaged currcnts resulting from suhcnnductance levels of GARA, channels. T h e mechanism for 'prolnngation' of average open and hurstt durationsi is similar to that described for harhituratcs, although significant changes in channel-opening frequencyt has not hccn a prominent effect ohscrvcd fnr harbituratcs. Thcsc rcsults s u g c s t that steroids and harhiturates may rcgi~latc thc GABA rcccptor-channel through at least one common effectort
Subunit-selective tnodulntory rcspon.ses - r l suhunit determinnnts 10-44-10: -x -subunit-data: The 'exact' pharmacology of the henzodiazepine response [whcthcr it conveys n hcnzodiazcpinc typc I+ or typc IlT-llkc pharmncc~logy- see rcf.'*"l varies accnrcling to the a suhunit type cxprcsscd (rcviewc~iin rr,f."2: s w (11'io~ ~ ( i r ( ~ g r in ~ p Ilomnin ~i,v Irjr~ctionr.10-29). T h e binding sitc for the partial inverse1 hcnzodiazcpinc agonist Rn154513 (cthyl-X-azido-S,h-dihy~lro-5-mcthyl-(1-oxo-4H-imidazo~ 1 ,SriI[ 1,4]hcnzodiazcpine-.?-carhoxyl:~tcl is otherwise hcnzodiazcpinc-insensitive,and this hinding sitc appears to require the a, subunit. ~ c c o m h i n a n t treceptors composed of a,, P2 and y? suhunits display a pharmacology similar to that of nativcf rcccptors irnmunoprccipitatcd hy rr, suhunit sntiscra (i.c. high affinityt for the GABA structural analogue muscimol and ~o154513)'". Varying thc a suhunit type (with constant P and ;- suhunitsl in complcxcs also affects steroid modulation of GARA responses, hilt docs not apparently affect hsrhituratc, picrotoxinin or hicucullinc sensitivity.
Subunit-.selective modulatory responses - suhunit dctcrminonts 10-44-11: /1 suhunit data: Replncement of a P2 hy a PI subunit in co-expressed -
comhinatic)ns ot (;ARAAR in the Xcnop~jsoocytc expression system results in strongly incrcascd sensitivity to diazepamx1.T h e affinity of PI for CARA is usunllv xrcatcr than that for /I?. 11 suhunits (/Il or /I2\ arc not rccluired for expression of CARA-gated ion currents displaying diazcpam sensitivityw'. hut r i o t the /i? suhunit can form a picrotoxinin-sensitive, GARAT h c I{,, indcpcnrlcnt anion-selective channel, which can he suppressed by cocxprcssicln with any Y. suhunit, hut not hy thc ;.? suhunit (for {~irthcr injormrltion, set, ref."), N o ~ c :/I suhunits arc not c.~scntiolfor picrotoxinin I>lock (as the complex y l y r , ; . , is picrotoxinin-scnsitivc).
Snhunit-sclectir7e modulatory responses
-
h
suhtrnit rlererminants
10-44-12: 6 suhunit- data: Possihlv associated with insensitive' rcccptors. -
'hcnzodiazcpinc-
Subunit-selectir~emodulatorv responses
- 7 suhunit determinnnts 10-44-13: ;. suhunit ciata: ~ a t i v c 7i subunits are necessary for henzodiazepine rcccptor-channels 1i.c hinding and modulation - sce sensitivity of GARA rt*f:'). ~ e t c r o ~ o g o u s co-expressed ~y~ alP1 subunits lacking respond to GARA hut the response is only 'weakly and incons~stcntly'modified hy hcnzadiazcpincs. Thus, nlthough these small effects of henzodiazcpincs can he detected in the ahscncc o t ;.I suhunits in co-cxprcssion c x p e r ~ m e n t s ~ ' , the prcscncc nf the ;*? suhunit is essential for lnrgr effects (set, also DOSP--
; )
entry 10
-
response. 10-36).For control of ethanol sensitivity by ~ 2 , phosphorylation, . see Protein phosphorylotion. 10-32.
Interactions o f hretazenil and diazepam modulation 10-44-14: 7 suhunit data: In vivo, the modulatory efficacy of the anxiolytict drug bretazenil is generally much lower than that of diazepam. In cultured HEK-293 (human embryonic kidney] cells transfccted with y2 suhunits and various isoformst of Iand /? suhunits, brctazcnil efficacy is always lower than that of diazepam'4R. However, in cells transfectcd with yl or y3 suhunits and several isoformst of r and /l suhunits, the efficacy of hoth diazepam and hretazenil is lower and always of similar magnitude. When bretazenil and diazepam are applied together to GARAA y2 suhunit homomcrict receptor-channels, the action of diazepam is curtailed in a manner related to the dose of b r e t a ~ e n i l ~ ~ ~ .
Subunit-selective modulatory responses - 1) subunit determinants 10-44-15: p suhunit data: p suhunit expression is associated with GAR& ( ' ~ ~ ~ - G A R ~ / G responses A H A ~ ) in retinal bipolar cells and is insensitive to hicucullinc, barbiturates, and benzodiazepincs and haclofcn (see Domain functions, 10.29).
-
Equilibrium dissociation constant Sites and subunits affecting affinityfor GARA and its agonists 10-45-01: The GABA-binding site (GRS) on the GABAAR complex can be selcctivcly labelled with radioligandsf such as [.'HI-GARA or [,'HI-muscimol (see Ligands. 10-47). The CRS charactcristically display both 'low-affinity' labelling ( K d I c ~ ~ AI ~l Mrange) and 'high-affinity' Iahelling (K,llGAnAl nM range). The 'low-affinity' recognition site appears to be an 'antagonistpreferring site' (see ref.149)since it can also be sulectively labelled by specific antagonists such as (+)-bicucullineor SR95531 (scc Tahle h under Lj,qands, 1047 rind Tr~blcX under Iieccptor antn,ponists. 10-51). The 'high-affinity' and 'low-affinity' GBS may represent alternative forms of the same receptor, since (i)both sites show similar drug spccificitics and (ii)'positive modulators' such as pentobarbital (a henzodiazepine-site agonistj - see Activation, 1033) increase the number of 'high-affinity' sites while proportionally reducing the numhers of 'low-affinity' sites (/or a rcvicrv, see ref.""). The 'high-affinity' GRS may represent a desensitizedt form of the receptor.
-
-
10-45-02: /l suhunit data: In studies on cloned suhunits expressed in Xcnop~~s oocytes, replacement of the p2 suhunit by the /II suhunit in a given suhunit combination generally results in a decrease of KaIGARAI. Notr: For native GARAA receptors in ccrehcllar Purkinie cells, a half-maximal response is - elicited by -50 p~ GARA.
n
Hill coefficient 10-46-01: Hill coefficientst for hoth vcrtehratc and insect GARAA receptors arc generally greatcr than unity (e.g. 1.8 in nativei cerebellar Purkinic cells or -2 in cultured spinal cord ncuronc preparations), suaesting that at least
cntry 10
two CARA molcculcs arc rcqi~ircdfor activation. (Scc rrlxo thc mndrrlr~tory 10-30.) c f f c c t s r d chlortliuzrpoxitlc c~ntlcrIlosr~-rr~sl~ons1..
Ligands 10-47-01: Availahlc radioligandst for the C,ARAArcccptor arc summarizeti in Tahlc 6. Ligand interactinns at the GARAA rcccptor complcx havc hccn rc~icwcd'~~". ~
GARAA competitive site
Renzodiazepine rnadulatnry site (also known as -., site)
Othcr noncompetitive antagonist sitcs Steroid rnodl~latorvsite
,
[-'HI-GARA (cndogcnous agonist) [.'HI-~uscimol,as uscd for ph(ltoaffinityt lahclling of a GARAAR ' , I suhunit' of -56 kUa" ['HI-sR~ss.~ 1 ( ~ ~ ' ~ ~ - 2 - ( c a r h o x ~ - ~ 3 ~ - ~ r o ~ ~ l l - . 7 - a m i n t mcthoxyphcnyl-pyridazinium hromirlcl [ ' H I - ~ c t l i ~ l - h i c u c u ~ methyl linc chlorirlc (antagonist) [ ' k ~ l - M c t h ~ l d i ; ~ z c p(agonistl a~n [ ' H I - ~ c t l i ~ l f l u n i t r a z c p a(-2 ~ n nM, a ~ o n i s t Jas , usud for the o r i ~ i n a lphotoaffinity 1:lhclling of thc C,ARAAR in c n ~ d c hr;iin honiogcnatcs with a molecular wcight of -5 1 k ~ a " ~ ['HI-~lurnazcnil(0.9 n ~ l ; [ . ' ~ I - ~ - ~ c t h y l1-195 l ' ~(anta~onist, l sclcctivc at 'peripheral'i RZ receptors1' [ 'H]-',9-R01545 l,3 (photoaffinity ;~ntagonist,sclcctivc at ccntr;ilt rcccptors). I 'HI-Mcthyl-l
channelq in opcn ant;igonistl conforniat~on "F{hlowing photonffinity-l;il,clling ;lnd SDS-PACEI analysis, scvcrnl GARAAsclcctivc r;ldioligantls can discriminatc 'microhctcrogcncoi~s'protcin hanrls frc~mcrude tissuc preparations (according t o sol~~hilization and hinding continurd o n $44
entry 10
continued from p,W3 conditions). This type of result is consistent with the cxistcnce of multiple ligand-hinding suhunits being cncoded hy a large rclatcd gcnc family. Peripheral' henzodiazepine reccptors arc hinding sites that are localized to the outer mitochondria1 membrane of many tissues (including brain). 'Pcriphcral' bcnzodiazcpine reccptors are pharmacologically distinct from (and unrelated to) thc GARAA-associated 'central' typc hcnzodiazcpinc receptors, where the GARAA modulators (listcd in Channel modnlotion, 1044. Receptor (rgonist.~,10-50,nnd Receptor anta~onists.10-51 ) cxert their 'clinically rclcvant' cffccts. Notahly, thc 'pcriphcral typc' RZ rcccptors havc a similar tlistrihution to the VDAC (voltage-dependent anion channel) isolated from mitochondria of various sources"" (for further tlctails, scc I1rotein intrmctions, 10-31, and MIT (mitochondria),entry 37. Somc compounds listcd in this tahle can distinguish hctween periphcral and ccntral RZ receptor types in r a d i o ~ i ~ a n dhinding t assays (as indicated). 11,
High-affinityIigands for henzodiazepine receptors 10-47-02: Imidazo(1,S-~Jquinoxalin-4[5 HI-one, 3-(5-cyclopropyl-l,2,4oxadiazol-3-yl)-5-(I-mcthylethylJ or U-78875is one of a scrics of imidazoquinoxaline-derivative ligands which show high affinity for henzodiazcpinc - rcccptors. A stn~cturc-activity study for U-78875 has hecn published'"".
0 -
Openers 10-48-01:See Receptor n ~ o n i s t s10-50. For effects of volatile anaestheticst on elicitation of GARAA currcnts, see I'henotypic expression (al>ovcJ.
Receptor/transducer interactions Distinction o f GARAn and GARARreceptor suhtvpes 10-49-01:GARAA rcsponscs should not hc confused with those from GARAR rcccptors, which do not form integral ion channcls hut arc couplcd to ~ a or " K' channcls via C, protein tmnsduccrs (scc Rrsorjrce A - G protein-linked rcccptors, entry 56 rrnd refs.'"'C'"2 I. Activation of C.ARAF,rcccptor proteins can inhihit the function of GARAA receptors, for cxamplc in thc ~ c r c h c l l u r n ' ~ ~Note . also that 'GARAcl rcccptor-mcdiatctl rcsponscs (hicucullinc- and I>aclofcn-inscnsitivc GARA rcccptors) have hccn characterized in rctinal horizontal cclls and linked to GARA rho subunit cxprcssionx'. "-,' l M (see 11 /SO Rlockers, 10-43).
Neuromodnlntor receptor potentin tion o f retinal GARA,, responses 10-49-02:Vssoactivc intestinal polypeptide (VIPJplays a neuromoclulatory role in hipolar cclls and ganglion cclls of thc rat retina hy potentiatingt GARAA rcspons~s'"~.Whereas VIP alonc elicits no current response, VIP potcntiationt of CARAA chlorirlc currcnts controls thc cxcitahilityt of inner retinal ncuroncs and thus can modulate the efficacy of synaptic transmission in thc rct inn'" (see n1.w Ahstmctlgencral dcscript ion, 10-01).
Muscnrinic receptor stimulation increnses inhibitory post-synoptic potentials t 110-25 IIMI can be ~iscd - 10-49-03:The muscarinic rcccntor a ~ o n i s carbachol
entry 10
n
experimentally to increase GARA-mediated IPSP frequency and simultaneously hlock 'potentially confounding' K' conductances during analysis of GARAA receptor-mediated processes1"".
Receptor agonists Note: A diclgrammrrtic summarv o f the important pharmacological moduk(1torj7 'sites' includin~those c~ctingns receptor agonists is included under Channel modulation. 10-44.
Endogenous axonis? activotion o f GARAAR 10-50-01: Ry definition, GARAAR-chloride channel gating is regulated by the hinding of gammaaminobutyric acid (GARA, and its analogues) to the extracellular GARA receptor portion of the intact complex. Since only micromolar concentrations of GARA are necessary to activate the integral chloride channel1" it is likely that GARA cxcrts its physiological effects at the 'lowdissociation affinity' GARA hinding site (for description. see E~~uilibrium constmnt, 10-451. It has been c s t i m a t c d l " ~ h a tone 'inhibitory quantum' of GARA opens -12-20 chloride channels and that GAHA agonist molecules hind only once to post-synaptic rcccptors. 10-50-02: The principal synthetic and naturally occurring agonistst for the GARAA receptor are summarized in Tahlc 7. GARAAR-channel currents are generally enhanced by three classes of 'CNS depressant' drugs: the benzodiazepines, the barbiturates and the anaesthetic steroids ('neurostemids').The propcrtics of the hinding sites for these modulators arc indexed in Fig. 5 under Chnnncl morltrlntion, 10-44 and Tahlc 7.
Tahle 7. The principal synthetic and nl~turnlu,qonists for the GARAn receptor (Fmm 10-50-02)
u
Site or receptor ty
Agonist (notes)
GARA competitive site agonists"
Muscimol (a naturally occurring structural a n a l o p e of GARA from the mushroom Amnnitci muscaria; which induces -2-fold increases in open timest of GABAARchannels 1. Isoguvacine and muscimol have heen shown to prcfcrcntially activate suhconductancei states15". Isoguvacine (a synthetic GARA agonist; which induces -0.5-fold increase in open times't of GARAAR-channels). THIP (4,5,6,7-tctrahydroisoxazoln-[4,5-c]pyridin-3-tl, a rigid hicyclic synthetic analogue of muscimol/GARA, which generally induces -0.5-fold increase in open timest compared to GARA). Different agonists affect gating1 properties and do not alter the single-channel conductance sre olso THDOC in Tahle 5. Piperidine-4-sulphonate, a synthetic structural analogue of GARA
entry 10
-
Table 7. Continued
site Or receptor type
Agonist (notes)
Renzodiazepine modt~latorysite agonists"
Flunitrazepam -- see Chnnnel modtilation, 10-44 Zolpidem, a henzodiazepine type 1t-selective imidazopyr~dinehypnotic drug Abecarnil (a partial agonist] Pentobarbital --see Activation, 10-33 Phenobarbital - see Chnnnel modulation. 10-44.
Barbiturate site agonists 'Anaesthetic steroid receptor site' agonists
Alphaxalone -see Table 5. The development of alphaxalone asa 'steroid anaesthetic' was based on the ohserved sedativet effects of the naturally occurring steroid hormone progesterone. Notably, progesterone and its structural analogues, as wcll as metabolites of progesterone, testosterone and corticosterone (like alphaxalone) can enhance GARAA receptor function. These metabolites do not activate intraccllular transcription factori-like receptors, and so this group of molecules may act as cndo,penous phy.qiolo,yical modulators of GARAAreceptor-channclsn. This intcrpretation is consistent with several hehavioural observations following steroid drug use, namely variations in CNS pharmacology related to the sex of subjects, oestnlst and diurnalt rhythms and drug-dependence phenomena
GARA-binding agonists
Protopine-hydrochloride is a GARA-hinding alkaloid activator of GARA-gated channels. See nlso I'hcnotypic exprc.ssion, 10-14, for effects of volatile anaesthetics on elicitation of GARAA currents
GABAA autoreceptort agonists"
di-N-Naphthyl-GARA (pD2 7.4) L-Amino-laevulinic acid Progahide Fengahine
"Taurinc, glycinc and haclofen do not have detectable agonist activity at GARAA receptors; e.g. see characterizations in ratlcat dorsal root ganglion ncuronesl"' ant1 hovine adrenal medulla chromaffin c ~ l l s ' " ~ . 1, Distinctions hctwcen the GARAA-related'central1-type hcnzodiazcpinc receptors (as described in this entry) and the CA13AA-unrclatcd'peripheral'type hcnzodiazcpinc receptors arc outlined in footnote" to Tahlc 6. C;ARAA autorcccptors appear insensitive to ncurostcroid modulation and to the GA13AA antagonist SR955.31.
'Non-discrimination' o f inhibitory agonists b y mutant ~ l v c i n e receptor-channels
I
10-50-03: Gamma-aminohutyric acid and 17-scrinc 'cfficicntly' activate certain mutant g l y c i e receptor-channels, demonstrating a rcquircmcnt for aromatic hydroxyl groups in ligandi discriminationi at inhibitory1 amino under ELG C1 GLY. 11-29). acid r ~ c c p t o r s ' ~1~1. " Domain {~~nctions
entry 10
Receptor antagonists Norc: For r j dirn~r(jn7rnrrtic.-suiiiriirjry o f the important phfirrnlrcokr)~iL.NI mod~rlrltorv 'sitcs' itic-lr~din,qtliosra (rctiii,y ns receptor ontogonists, .see Channel modulotinn. 10-44. For o ~ c i i r s(rc+tin~ spl~c.ll~~.nIly or pcrrtirrlly t o block chlor~rir~ l1tl.u. srbr Blockers. 10-43. 10-51-01: T h e princip;ll synthetic and naturally occurring antaKonistst for the C;ARAA receptor arc su~nmarizerlin Tnhlc 8.
Table 8. i'rincil~(j1.s!~nthcric.rrnd 'rr(rtun11' rintrrqonists for thc7 C;AHA,q r(7(.t!ptor(Frort~10-5 1 - 0 1 )
Class or type of antagonist
1)cscription of ;lction or physiolugicnl cffcct
Competitive site Ricuculline, n convulsant alkaloidt of plant origin, is a potent compctitivct antagonist (pA2h.(l) of vertchratc antagonists C;ARAA receptors; often applicd as the mcthiodidc. Othcr ;intagonists acting nt the same sitc includc the quaternary nitrogcn analogues of hicucullinc, pitrazepin and the :~midincsteroid RU135". SR95531 (2-jcnrho~y-,3'-propyll-~3-arnino-h-p-mcthoxyphcnyl-l>yritIaziniumhromidc) is :I compctitivc antagonist which is a pvririnzinyl dcriv;itivc of GARA and thcrcforc shares cl(>scstr11ctur;il simil:~ritywith it (.FCC pr~nrgroph 10-5 1-03). SR42641. d-a-Hydrastine (isocorync, s n nlkalnidt which is ;~pproxitn;itclytwice as potcnt as hicucullinc] Rcnzodiazcpinc modnlatory site antagonists1'
Flumazenil (Rol.4 I7HHJ.Rchavioural studies (rcvicwdinl*') suwcst that flumazcnil may he antagonistic to n n t u r ~ l l y occ~trringhcnzodiazcpincs like .{-carbolinc-.7-carlmylatuo r v:~riouspcptidc lig;lnds of ccntr;llt hcnzodiazcpinc rcccptors; flumazcnil is nnxiogcnict : ~ high t doses
PicrotoxininTRPS site antagonists
Kcccptor sitcs for plant-derived convulsantt picmtoxinin (sotnctimcs called picrotoxin) arc closcly associated with the chloride ion channel of the GARAAR. Picr:>toxinin can act as a channel hlocker hy 'steric hindrancei' of agonistinduccd f l ~ ~ xanrl c s under some conditions can act to elicit chloride flux. For furtlli~rrlctoi1.s. scc Domrrin {t~nctions. 111-29, Rlockcrs. 10-4,3. (jnrl Cllcrnncl rnodr~lotion.10-44 TRPS (I-h~~tylhicyclopho~phorothion~tc) is ;I hicyclic 'cagert convtllsantt which (like picrotoxinin) docs not tlisl>l;~cchcnzodinzcpincs from thcir high-affinity hintling sitcs. High-nffinity TPRS hinding may he associated with the 'closed' conformation of the integral chloriilc channel. For { ~ r r O ~clrt~rils, (~r s ~ ~'1'0111(~ (> 5 r~ri(l(~r Ch(ri~r1~1 ti~o(lttl(rt ion. 10-44)
entry 10
-
Table 8. Contin~red Class or type of antagonist
Description of action or physiological effect
Steroid antagonists
DHEAS (dchydroisoandrosterone3-sulphatc), has hccn characterized as an antagonist in astrocytct (glial) ncuroncs'"'
Antibacterial/ non-steroidal antiinflammatory agent interactions
In vitro, the GARAA-antagonistic effects of enoxacin (a quinolone antibacterial agcnt) are potentiated+ -80-fold in thc presence of felhinac, a non-steroidal anti-inflammatory drug. This may provide a mechanism for known convulsant reactions following concomitant administration of these classes of drugs in vivol"'
Other antagonists with convulsant activities ('some non-selective compounds also hlock voltagesensitive Cl channels)
TETS (tetramethylenedisulphotetraamide (a noncompetitivcl GARA-gated CI channel antagonist; antiplatelet agent) Flucybene [an organoflunrate convulsant) Clofluhicyne (organofluorate convulsant) Propyhicyphat', Isobicyphat*, Mebicyphat*, Etbicyphat" (or~annphrisphorousnon-cornpctitive antagonist compounds which can induce cpileptiform seizures) Scvcral chemically distinct classes of insecticide are noncompetitive antagonists of GARAA receptors and include the trioxabicyclooctanes, dithianes, and cyclodienes Pentylcnetetraznle (PTZJ In addition to their principal target of Na' channels, (we VLC: Na. entry 5 5 ) neuroactive insccticidcs such as the pyrethroids and DDT can contrihutc t o hyperactivity hy suppressing GA13AA channcl complcxcs'""
'For prc-synaptic GARAA autoreceptorst, bicuculline is an order of magnitude more potent (PA? 8.0) as an antagonist than at the 'classical' postsynaptic receptors. A derivative of hicucullinc, WAY100359 acts as a more potent antagonist (pA? 9.14) in some preparations. isti tinct ions between the GARAA-related'central'-type hcnzodiazepine receptors (as dcscrihed in this entry) and the GARAA-unrelated 'peripheral'type henzodiazepine receptors arc outlined in footnote" to Tahlc h under Li,ynnds. 10-47.
Discrimination o f (SARAA from glycine inhibitory chloride rcceptorchannel
J
10-51-02: Strychnine can he used to distinguish the GARAn receptor from the glycinc receptor C1 channels (scc ELG Cl GLY, entry 1 I ) . pA2 values against the agonistst rnuscimol and glycine are 5.3 and 6.0 (neonatal) or 8.0 [adult) rcspectivcly. Note: Some antagonistic action of strychnine has hccn reported for the C A M A receptor1"".
entry 10
-
Mapping agonistlrlntr~goni,~t-l?inciin,q sites to ( 1 suhunits hv sitcdirccteci mutogenesis 10-51-03: 7 subunit.-data: GARA* a subunits arc generally associated with the agonistlantagonist-hindingsites of intact receptor-channel c o m p l c ~ c s An ~~. a1 Phc64 Leu suhstitut~on strongly decreases the apparent affinity for GARA-dependent channel gating /from 6 /IM to 1260 /(MI when the r suhunit is co-expressed with P2 and y2 suhunits7". Homologous mutations in a5 suhunits show similar phcnotypcs, hut homologous mutations in 82 anti y2 result in intcrmcd~atc anti small shifts in potcncyt ( E C ~ ~ , I J rcspcctivcly. Apparent affinities of hici~cullincmcthiodidc and SRY55.11 (SCC T N ~ I 81 I *:Ire riccrc?scil 60- to 200-fold by thcsc cmi~tationsin r suhunit C D N A ~cotling regions' . C o r n p l ~ n ~ tnotc: i ~ ~ c Agonist /;lntngonistt affinities1 arc largely unaffcct,ctl ilpon introduction of the homologoust mutationst in 12 and 72 cI)NAsr, or upon mutation of a 'nci~hhoiiring'rl aminn acid d d n t l ~ scc , Tr~hle?, under (Phe6S to ~ c u l ~( / o" r clr~rificrltion~ n .stlpp)rtin,q Domr~infttnctinns. 10-291.
GARAn rcc'clptor clntn~onisthindin
hlock 10-51-04: The binding kineticst : ~ n d relative affiniticst to hovinc hrain C;ARAA receptors havc hccn dctcrmincd for 25 CARA* antagonists, including four raciiolahcllcd c ~ m ~ o i i n d s '(src ~ ' I,i.q(~n& 10-47). The low association rate constantst for nil ligands ( < . 3 .: 1 0 7 M min at 25 C ] is consistent with a slow transition to a hlockcd receptor conformation upon hinrling of thcsc clianncl blockers. Thc 'association rate-controlled' affinities for the trioxahicyclooctanes and dithianes'" siiggcst an inducedfit model in which hinding of the ligand initiates a conforrnational chnngc in the rcccptnr complex to the hlnckcri ~tatc'.~'.
'
-
'
Receptor inverse agonists Notr: For ( I tlir1,qr(1rnn7(~1ic. stimnlnry o f the irnportnnt phnrinmco~o~icn~ nlochilnton. 'sites' inc+Itidin,qthosr (1ctir1,y11s recpptor inverse ogonists, ser Channel mndulatinn, 10-44.
,{-Cnrholincconlponnds clctin~0s GARAn inversct crRonistst 10-52-01: Several p-carholines not only antagonize the effects of GARA* agonists hy occupying the hcnznrliazepine receptor sites hut also induce pharmacological cffects that arc opposite those of the 'classical' benzodiazcpinc agonists 1i.c. they arc inverse1 agonistsl. For cxamplc, the /I-carholinc DMCM ( m e t h y l - 6 , 7 - d i m e t h o x ~ l - 4 - c t 1 1 y l - / i - c ~ - c r h o x y l arlecreascs tcl C;ARA-induced whole-cell currents althoi~ghit acts at the hcnzodiazcpinc modulatnry sitc. The inverse ngnnist Rn194603 and Ro1.54513 (ethyl-Xazido-S,6-dihyriro-S-mcthyl-6-o~o-JI~-imiinzo 1 ,Sr~[1,4]henzndiazcpinc-,?cnrhoxylatc, n partial invcrsct hcnzodinzepinc agonist 1 can nlso antagonizct certain effects of ethanol (for dctr1il.7 of cthonol mndtrlation, sce Chrrnncl rnodull~tioii. 10-44). Notr: Inverse agonists havc revealed striking differences hctwc.cn neuronal anrl glial (astrocytic) GARA rcccptor-channel rcsponscs (for drtnils, scc (211-r!fpr exprrssion index, 10-08).
-
A putntive peptide precursor for nn endotyenoust ligand acting nt GABAA benzodiazepine sites 10-52-02: DRI (diazepam-binding inhibitor) is a pcptide isolatcd from hrain tissuc and a precursor o f putative natural ligandst o f henzodiazepine recognition DRI has an action similar to DMCM (see porngrnph 10-52-01 and Fig. 7). 10 M GABA
-
10 pM GABA +10 pM 001
-
TT 1
"AL 2s
Figure 7. EJ(cct o f hmin-derived DRf (diazepnm-binding inhibitor) pcptide on native GARAA receptor-channel activity. (R~producedwith permission from Rormnnn ( 1 985) Regul Pept (Suppl.) 4: 33-81, (From 10-52-02)
) ~ a ~ x a ~ : q i b r ~ r s ~ a ~ Database listingslprimary sequence discussion 10-53-01: The relevnnt drrtnhnse is indicnted h v the lower cost prcfix ( c . ~ ~ h : )dcltohaac: ; clccession nnrnhers immediately follow the colon. Note tho1 (I comprehensive listing o f all nvnilr~hleaccession ntlml>ers is superfltrous for Iocntion of relevant sequences in CknRank" resources, which ore now ovailnhle with powerfill in-huilt neiRhbouringf analysis routines (for description, see the Datnbase listings firld in thr Introduction d lnyo~rto f entries, entry 02). For ~ x ~ m p sequences k, o f cross-species varinnt.s or relnted Krne f(1milyt members cnn he rendily ncccsscd hy one or two rounds o f neiShhntlrinRt r~nnlysis (which nrc hnsed on prc-computed rrli,pnmcnts per{orrned u.~in,ythe R L A S T ~nkorithm h~ the NCRII). This fe(~tureis niost useful for retrievnl o f suclucncr ctltrirs deposited in datrlboses lrrtcr than tho.^ listed helow. Thus, rcprcscntntivc memFcrs of known sequence homology grorlpings (Ire listed to permit initiol direct rctricvals ??y (tccession nrlmher, authorlreference or nomencloture. FoDowing-.direct nccession, however, neighhottringt analjaix-is.strongly - - ---rccommcnderl to identify tkwlv-rrported rc111tcdseqocnces. ---. -- r~nrJ . -
entry 10
C;ARAA
rb
suhr~nitdntrll?ust:lis!incys
Nomenclature
Spccicr, I)NA
SourCC
alpha -
-
354 ;1a (partial]
gh: M22868 gh: X1.3584
Garrett, Rtochcn~ Riophvs lies Commttn ( 1988) 156: 10.39-45.
Chicken
455 ns
sp: 19150 em: X54244 pir: CHCHAI prositc: PSO02<76
Ratcson, Mo1 Rrltin Res (I9911 9: ,3<3(3-9.
Mouse
455 aa
gh: M86566 prositc: 1500236
Wang, Mo1 Nettmsci (1992)3: 177-84.
Rovinc
456 aa
Mouse
455 aa
gh: X614,30
Kcir, Genomic.~ ( 1991) 90: 390-5.
Human
456 aa
sp: PI4867 em: X1.3584 cm: X 14766 pir: Ac31588 pir: S03.3,12 prositc: PS00236
Schoficld, FERS Lett (19891 244: 36 1-4. Garrett, K.M. ( 1988) Rioohcm Rioph y s Res Commun ( 1 98811 56: 103945.
CARA(AJ rcccptor alpha- l suhunit; precursor
alpha-1
-
C;ARA(A) reccptnr alpha- l suhunit; precursor
alpha-] -
Scqucncc/ ciiscussion
Human
C;ARA(AI receptor alphasuhunit
alpha-l
Accession O R F ~for original C ~ N A ~
GARA(A1
receptor alpha- l suhunit; precursor
alpha-1 C;AIIA(AJ receptor alpha-l suhunit; precursor
alpha-] C;AItA(A) rcccptor alpha- l suhunit; precursor
entry 10
GAHAA rr subunit database listings continued
O R F ~for original CDNA~
Accession
Sequence/ discussion
alpha-1 Rat GARAIAJ reccptnr alpha- 1 subunit; precursor
455 aa
sp: PI8504 gh: M86566 gh: Mti3436 pir: A39062 pir: SO3889 pir: JQ0158 prnsite: PS002.36
Lolait, FERS Lett 246: 145-8. Khrestchatisky, Neuron ( 1989) 3: 745-53. Wang, Mo1 Neurosci ( 1992) 3: 177-84. Kcir, Genomics (1991)9: ,390-5.
alpha-1 Rat GARAIA) receptor alpha- l subunit; precursor
455 aa
gh: LO8490
Seehurg, Cold Spring Harb Symp Qltant 13101 (1990) 55: 29-40. Draguhn, Neuron (1990)5: 781-8. Wisdcn, Curr Opin Neurohiol (1992) 2: 26.3-9. Wieland, 1 Riol Chem ( 1992) 267: 1426-9, Wisden, 1 Neurosci 12: 104(M12. Laurie, 1 Neurosci (1992) 12: 10k376.
Rat alpha2 GARA(A) receptorfienzodiazcpinc receptor alpha-2 chain precursor
451 aa
sp: EL3576 pir: JH0370 prosite: PS00236
Khrestchatisky, 1 Neurochem ( 1991J 56: 17 17-22.
alpha-2 Rovinc CARA(A) rcccptor alpha-2 suhunit; precursor
451 aa
em: X 12361 Lcvitan, Nnture pir: ( 1988) 335: 7(-9. ACROG2 prositc: 1' 1 0063
Nomenclature
Species, DNA source
O R F ~for original CPNA~
Accession
Scqucncc/ discussion
alpha-2 Rovinc GARA(A) rcccptor alphn-2 suhunit; precursor
451 aa
gh: XI2361 prosite: PSn02,Zh
Schoficld, Ndtrrrc ( I YK8) 335: 76-9.
M(lusc alpha-2 CARA(AJ rcccptor alph.7-2 suhunit; prccursnr
451 aa
gh: MHhSh7 sp: P2604X pr(lsitc: PS002,Z6
Wang, Mol Neuro.~ci(1992) 3: 1 77-84.
Rovinc alpha-3 GARA(A1 receptor alpha-.? suhuni t; precursor
492 aa
sp: PI0064 Lcvitan, Nflture em: X12
Rat alpha3 C;AIiA(A) rcccptor alpha-.3 suhunit; precursor
49.1 aa
gh: XS 1991 pir:A.341,70 sp: P202.36 prosi tc: PS002sZ6
Malherhc,FERS Lett. (19901260: 261-5.
alpha3 Rovinc C;AlIA(A) rcccptor alpha-.1 suhunit; precursor
492 aa
gh: X12.762 prositc: PS002,36
Schoficld, Notorc (19881335: 76-9.
Mouse alpha3 GARA(A1 rcccptor alpha-<3 suhunlt; prccursor
492 aa
sp: P26049 gh: MH6568 prositc: PS00L36
Wang, I Mol Nenroscr ( 1992) 3: 177-84.
13ovine alpha4 C;ARA(AI alpha4 sulwnit prccursor
556 aa
$11. P20237 em: X61456 plr: SO68.38 prowtc: I'S002.36 gh: YO74 IS
Ymcr, FEES Lett (1989)258: 119-
Nomcnclaturc
Species, DNA source
22.
C:ARAA rr .vu hunit database listings continued
ORE^ for original CDNA~
Accession
Scqucncel discussion
alpha-4 Rat GARA(A) rcccptor alpha-4 subunit; prccursor
552 aa
sp: P28471 pir: S17551 prosite: PS00236
Wistfcn, FERS Lett 11991 ) 289: 27730.
alpha5 Rat GARA(A) reccptor/hcnzodiazcpine receptor alpha5 chain precursor
464 aa
pir: K341.70 sp: P19969 pir: JQ0159 prositc: I'SOOL36
Khrestchatisky, Neuron (1989)3: 745-53.
Norncnclaturc
Species, DNA source
alpha-5 Rat C;ARA(A) rcccptor alpha-5 suhunit; suhunit precursor
alpha-6 Mousc C;AHA(AJ rcccptor alpha-6 suhunit; precursor
Malhcrhc, FERS Lett [I9901 260: 261-5.
44.3 aa
sp: PI 6305 Kato, Mo/ Riol em: XSF986 (1990)214: 619pis: SO8684 24. pir: S1 1396 pmsite: PS00236
Note: Thc listing has hecn madc by rcfcrcncc to namcs used in sctlucncc dntahasc cntrics; thc d c s i ~ a t i o n sgabral-gabra6 arc also in usc as namcs for genes encoding GABAAtr subunits.
Nomcnclntlirv
Specics, DNA
O R F ~for original
SOUTCC
C ~ N A ~
beta
Mollusc 499 Invcrtchratc (Lvrnnncr~) (Great pond snail) GARA[A) receptor l7ct;lsuhunit
beta-1 -
ail
Rov~ne
474 an
gh: X05718 plr: I327142 sp: PO8220 prclsi tc: I'S002tZ6
Schoficld, Nuturr (1987)328: 221-7.
Rat
474 na
em: X I 5466 sp: P154,ZI pir: SO4464 prosite: PS00236
Ymcr, E M R O (1989) 8: 1665-70.
Human
cxons I, 2 and ;i cxon 4 rxon 5 cxons 6, 7 and 8 cxon 9 (trans]: 474 aa l
gh: gh: gh: gh: gh:
Kirkncss, C:cnomrcs, ( 1 99 1 ) 10: 985-95.
Humnn
474 aa
gh: S67368
GARA(AJ rcccptor hcta-1 subunit; prccu rsor -
human gcnomic DNA cncorling thc hctn- 1 suhunit of the C.AR Aa rcccptor (GAI3RRI)
beta-2 GARA(A1
receptor hcta-2 suhunit tnRNA, complctt. coding sctlticncr
Hsrvcy, EMRO I (1991) 10: ,123945.
sp: P18505 Schoficld, FERS gh: M59212 Lctt (19891244: toM59216 3 6 1 4 . incl. Kirkness, cm: XI4767 C;comics(l991) pir: S0,3333 10: 985-95. mim: 1.37190 prosite: PS002.36
precursor
beta-1
cm: XS86,ZX sp: P26714 prositc: I'SOOZ-lh
474 an
GARA(A) rcccptor hcta-l suhun~t;
beta-1 -
Scqucnccl discussion
Human
C.ARA(A1 rcccptnr hctn-l subunit; precursor
beta-1 -
Accession
M59212 M5921,3 M59214 M59215 M59216
Hadingham, Mol Phnrmucol (1993) 44: 121 1-18.
GARAn Il suhunit datnhose listings continued
Nomenclature
O R F ~for original CDNA~
Accession
beta-2 Rat GARA(A) rcccptor hcta-2 subunit; precursor beta3 Human GARA(A) reccptor hcta-3 suhunit mRNA. complete coding sequence beta3 Chicken GARA(A) receptor bcta-3 suhunit precursor
474 aa
beta3 Rat GARA(A) reccptor bcta-3 suhunit; precursor
473 aa
beta-4 Chicken C.ARA(A)alternatively spliced bcta-4 and bcta4' subunits
488 aa
em: XI5467 Ymcr, EMRO sp: P154.32 (1989) 8: 1665-70, pir: SO4465 prositc: PS002&36 gb: M82919 Wagstaff, sp: 28472 G e n o m i c ~[I9911 11: 1071-8. MIM: 137192 prositc: PSOOW6 em: X54243 Rateson, Nucleic gh: X54243 Acids Res (1990) sp: PI9019 18: 5557. pir: S11440 pmsite: PS002.76 em: XI5468 Ymcr, EMRO J sp: P154.7.1 (1989) 11: 1665-70. pir: SO3890 pir: SO4466 prositc: PS00L36 sp: P240454 Ratcson, J gh: X56646 to Neurochem (1991) gb: X56648 56: 1487-40. inclusivc pir: JH0360 pir: JHO,l59 prosi tc: PSOOL36 gh: M69057 Ffrcnch-Constant, sp: P25 123 Proc Nntl Acad flyhasc: Scr USA (1991)88: 04244 7209- 13. prositc: PS002.36
beta suhunit precursor Invertebrate
(Drosophila) GARA(AJ receptor cyclndicnc resistance protein
Species, DNA source
473 aa
476 aa
Drosophiln 606 an (see also Domnin functions. 10-29)
Suqucncc/ discussion
Note: T h e listing has hecn madc hy rcfcrence to namcs uscd in scqucncu database entries; the designations gahrhl-gahrh4 arc also in usc as namcs for genes c n c o d i n ~GARAA subunits.
entry 10
Nomcnclaturc
Spccics, DNA
O R F ~for original CDNA~
Accession
delta - C,ARA/ Rat hcnzodiazcpinc receptor type A delta chain; elso complctc ccldlng scqucncc
449 aa
pir: A.34625 Shivcrs, Neumn pir: JQ0076 (1989) 3: ,727--37. sp: PI 8506 Zhao, Iqiochcm gh: M.35 162 Riophys Res prosi tc: Commun ( 19901 167: 174-82. PS002.7h Zhao, Ric~chern Riophys Res Commun ( 19901 168: 887.
delta -
Mousc
449 aa
gh: Mh0587 Sommcr, DNA gh: Mh058X Cell Riol (1990)9: gh: Mh0589 561-8. xh: M60591 gh: Mh0592 gh: M6059.7 gh: M60594 gh: M60595 gh: MA0596 sp: P229.7.7 pir: A,36,30,7 prositc: PSO0Z36
delta suhunit - Mousc
449 aa
hhs: 1 1 1421 Wang, Rtoin Res (Mcdlinc R1111 (1992) 29: identifier: 1 19-2,3.
SOU~CC
C;ARA(A) rcccptor dcltasubunit gene, exons 1 to 9 rcspcctivcly
CARA(A1 rcccptor delta s u h u n ~ mKNA, t cc~mplctc coding scqucncc
Sequence/ discussion
92.3 7045,1)
Notc': The listing has hcen made hy rcferencc to names used in scquence datahase entries; the designation gabtd is also in use as the gene name for those encoding GARAA b subunits.
entry 10
GARAA y suhunit datahase listings
Nomenclature
Species, DNA source
O R F ~for original CDNA~
Acccssion
gamma 1 -
Rat
465 aa
cm: X57514 Yrncr, EMRQ 1 sp: PL3574 (1990) 9: 3261-7. pir: S12056 prosite: PS002.36
gamma 2 Mouse GARA(A) receptor gamma-2 chain altcmativcly spliced precursor
pir: 474 aa; gb: 466 aa (apparent conflict)
sp: P227L3 gh: M86572 pir:JH0.317 pmsite: PS00236
gamma 2 C;ARA(A) receptor gamma-2 subunit
474 aa
em: X54944 Glencorsc, sp: P21548 Nuclcic Acid.$ Res pir: S1,7086 (1990) 18: 7157. prosi te: PS00236
gamma 2 Bovine C;AR A(A ) rcccptor gamma-2 suhunit mRNA, complctc coding scqucncc
475 aa
gh: M5556.3 sp: P2Z700 pir: A39272 pir: R19272 prosite: PS00L36
gamma 2 GARA(A] receptor gamma-2 suhunit precursor
Human
467 aa
sp: P18.507 Pritchett, Nature em: X15376 (1989)338, 582-5. pir: SO3905 MIM: 1,37164 prosi te: PS00L36
gamma 2 C;ARA(A) receptor gamma-2 subunit precursor
Rat
466 aa
gh: LO8497 sp: P18508 pir: A37164 pir: JQ0077 prositc: PS002%36
CARA(A)
receptor gamma-1 suhunit mRNA, complete coding sequcncc
Chicken
Scqucncc/ discussion
Kofuji, 1 Neurochem ( 1991) 56:713-15.
Whiting, IJtoc
Natl Acad Sci lJSA (1990)87: 9966-70.
Shivers, Neuron ( 1 989) 3: ,327-37. Malhcrhc, Neurosci (1990) 10: L330-7.
entry 10
RA A 7 snhunit rlrrtohrzse list i n ~ continued s
Norncnclnturc
Spcclcs, DNA
C > R F for ~ ong~nn!
Acccssinn
Scqucncc/ discussion
1
S~LITCC'
Scchurg, Cold Spring Hnrh Svmp Qunnt Rim] (1990) 55: 2 4 4 0 . i)raguhn, Neuron (IYSCll 5: 7x1-X. Wisticn, Curt Clpln Neurohiol ( 1992) 2: 263-9. Wieland, B i d
C l ~ e m(1992)2h7: 1426-9. Wisdcn, I N r u r n ~ c12: ~ 1040-62. Laurie, Neorn~cr (1992)12: 106*'476. gamma 2
-
Mousc
474 aa
gh: M62.774 Waffnrd, Netrmn ( 199 1 ) 7: 27-3.3. Sikcla, listed as unpuhlishcd.
Rat
467 aa
gh: M8 1 142 gh: Xk3.724 pir: S19317 sp: P2847,3
Mousc
467 aa
Wilson-Shaw, sp: P276R1 cm: X59,700 FERS Lett (1991) pir: S154hY 284: 21 1-1 5 . pir: Sl(i915 prosite: I'S00236
C;ARA(AJ
rcccptor gamma-2 suhunit, complctc
cod in^ scqucncc
gamma-*3 suhunit
gamma 3 C;ARA(A]
rcccptor gamma-.? suhunit; precursor
Hcrh, l'roc Notl Acrrd Sci USA ( 1992) 89: 1433-7. Knotlsch, F E n S Lett (1991 ) 293: 1914.
N o ~ tThc . ttst~nghas been made by rcfcrcncc to namcs used in sequence datahaw cntrics; the dcsignat~nnsgahrgl-gabrg3 are alm in usc a s names for Rcnrs encoding GARAh suhun~ts.
-,
GARAA p subunit database listings
Species, DNA source
O R F ~for original
rho 1 GARA(A) rcceptor rho- 1 suhunit, complete coding sequence
Human
473 aa
gb: Mh2400 Cutting, Proc Nut1 sp: P24046 Acad Sci USA gh: M62'323 ( 1 991 ) 88: 2673-7. pir: Ad8627
rho 2 -
Human
465 aa
gb: M86868 Cutting, sp: P284Jh Genomics 11992) MIM: 133162 12: 8 0 1 4 . prosite:
Nomenclature
,
GARA(AJ reccptor rho-2 subunit, complete coding sequence
Accession
Sequence/ discussion
CDNA~
PS00236
Note: The listing has hccn made hy rcfcrencc to names used in sequence datahasc entries; cnnventionally, the designations gahrrll-gabrr2 could be used as names for genes encoding GARAA p subunits.
Official nomenclatures of GARAR receptor-chnnnel genes, subunits nnd native receptors 10-53-02: Updates on lUPHAR Nomenclature Subcommittee recomrcccptor-channcls will appear via the 'home page' nf thc Ccll-Si,ynalling Netwnrk (CSN) available over the World Wide Wch on the Internet (for description, see Fcedhock d CSN ncccss, entry 12). mendations on GARA,,
Related sources & reviews (mainly >1988)
10-56-01: GARAA receptor properties and suhtypc reviews (major source^)"-^-^; rnokcular biology of GARAA r e c ~ ~ t o r s 'GARAA ~~; clcctrophysiology'"; GABAA phosphorylation92~'74; GARAA modulation hy cthanol and endngenous henzodiazep~nes14'~'75;GARAA structure-function overvicwJ7'; r e l a t ~ m st o psychiatric i~lness"'; meeting reports/GARAA pharmacology12h, 142. I7R-IRO. , definition of 'consensus' GARAAproperties and a full datasct for co-expression of X I , n 3 , 15, Pi, /I2 and ;'z s ~ h u n i t s ' * ~ ~ ' ~ ~ " ~ ' ; properties of insect GARA properties of 'high-affinity' and 'Inw-affinity' GARA-hinding s i t ~ s ' ~ligand ~; ~ntcractions at thc GARAA receptor ~ o r n ~ l c xbcnzt~diazcpine '~~ i n t c r a ~ t i o n s ' ~ ~ - 'comparisons ~~; of ~; rcccptors as GABAA rcccptors with othcr ELG c h n n n e l ~ " ~ . " GARAA GAHAAR in molecular target5 of insecticides and othcr toxicantsf"- "'."lr; vcrtchratr: gl~al ccllsf2; suhtnmlly nomcnclaturc re~icws"."~; subunitscqucncc and function d i s c ~ s s i o n s * ~ ' ~GARAn '; rcscarch r ~ t r o s ~ e c t ~ v c ~ ~ ~ GARA,., suhunit mRNA quantitatlon r n e t h o d ~ " ~ ~ ' ~ ~steroid ~'~";
entry I n
1
m ~ d u l a t i o n ~ ~ ' " ' ~rclation ; to pharmacologically ilcfinctl ~ u h t ~ p c s " ' ~ ' ~ ' ; permcation p a t h w ~ y sof ncurotmnsmittur-gntct1 ion channels"'; GARA* ovcrvicw and gui11u t o carlicr litcmturcEY~'.Ruvivws on aspccts of spccific CARA, suhunits inclutlc: z subunits - function and pharrnacdnw~'2 and suhunit-spccific data"; /E suhunits - GARA rotcin phosphr~rylationv2~19d; 5 suhunit-spccific d~ta,'; subunit-spccific data"1 .
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Feedback Error-corrections, cnhr~nccmentmttd extensions 10-57-01: Plcasc nnt ify spccific crrnrs, omissions, updates and cnmmcnts on this cntry hy contrihutinpl to its e-mail feedhack file ({or &tails, scr: Rcsor~rucJ, Scorch Critrril~ CSN Development). For this entry, send email mcssaEcs To: [email protected], indicating thc appropriate paragraph hy cntcring its six-figure index numhcr (xx-yy-zz or othcr identifier) into the Subject: field nf thc messagc (c.g. Suhiect: 08-50-07). Please fcedhack on only one specified paragraph or figure per message, normally by sending a corrected-replacement accortiiny: t o thc guidclincs in Fccdhnck t?.) CSN A(,r.r,,ss . Enhanccmcnts and extensions can also he s u ~ c s t c dhy this route (ihid.). Nntificd changes will hc indcxcd via 'hotlinks' from the CSN 'Hornc' pagc [http://www.lc.ac.uk/csn/] from mid- 1 996. -- -
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Enrry srlpport ,qroaps and e-mail ncjwslcttcrs 10-57-02: Authors who hnvc cxpcrtisc in one or morc ticlds of this cntry (and arc willing to providc ctlitorial clr othcr support fnr dcvclnping its contents) can join its support group: In this casc, scntS a message To: CSN108le.ac.uk, (entering the wt~rds"support grtnrp" in thc Suhicct: ficl(l]. In the mcssaEc, plcasc indicate principal in tcrcsts (see fir,ldnamr critrria in t hc Int rodrrct inn {nr covcrt~gc) tngcthcr with any rclcvnnt http://www site anti details of any othcr possiblc contributions. links (ustahlishcd or pn)posc~lJ In due coursc, support group mcml>crs will (optionally) receive c-mail ncwsIctters intcndctl to ca-rrrdinatc and devetop thc present (text-hascd] cntry/ficldnamc frameworks lntn a 'library' c ~ f intcrlinkcd rcsourccs c o v c r i n ~ ion channcl signalling. Other (more gencrnl) information of interest to cntry contributors may also hc sent to the ahovc address tor - group clistrihl~tionand fccdhsck.
Woodward, Mol Pharrnoco1 (1992)41: 1 107-1 5 . Lcv~tan,Nrl!nrc. (1988) 335: 76-9. Rurt, FASER 1 [ 1 991 ) 5: 29 I 6-23, 4 Rarhaccia, Ncrlrochcm Ilcv ( I Y Y O J 15: 161-8. Cic~hart,Trcnds Phorrnacol Scr ( 1 992) 13: 446-50. ' Mncdonald, Annrr licv NCurosci (1994) 17: 569-602. ' M ~ c h ~ l s o SnC, I I J R C( 1~99 1 ) 253: 142C3. N~cclll,I ' h y ~ ~ oRcv l (1990)70: 513-65.
' '
entry 10
I
' Rerger,
Neurosci Res (1992)31: 21-7.
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entry 10
"R
Stephenson, FERS lctt (1')891 243: 358-62.
" Sigcl, 1 Riol Chcm (19831258: 6965-71. "" Stephenson, Eur I R i o c h ~ m(19821167: 291-8. Vandenberg, Mol I'hmrmatrol (199,3) 44: 198-20.3. " Angelotti, I Ncuro.~ci(1993)13: 1418-28. "
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entry 10
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Robcllo, Neuroscience (1993)53: 13 1-8. Shirasaki, I Physiol Lond (1992)449: 551-72. Osmanovic, Phhysiol Lond ( 1 990) 421: 151-70. ""Fatimashad, Proc R Soc Lond (Hiol) (1992)250: 99-105. Lcstcr, Annu Rcv Riophys Aiomol S t r u ~(1992)21: 267-92. Ncwlanc!, Physiol Lond (1991 ) 432: 2 0 3 3 3 . Khrestchatisky, Neuron (1989)3: 745-53. Xic, Nature (1991)349: 521-4. ""esthrook, Nature (1987)328: 640-3. ""mart, 1 I'hysiol Lond (1992)447: 587425. "'Draguhn, Ncriron (1990)5: 781-8. Kilic, El11 J Neurosci (1993)5: 65-72. " 9 Chow, Rr 1 Pharmacol(1986)88: 54 1-7. '2"wyman, Riophys 1 (1991)59: 256a. Izquierdo, Trends Pharmacol Sci (1991)12: 260-5. Lange, PfIugers Arch (1987)410: 648-51. ""rerew, Ne~irosciLett (1984)52: 3 17-2 1 . 12* Study, P ~ O Natl C Acad Sci U S A (1981 ) 78: 7180-4. ""Edgar, Mol Pharmacol (1992)41: 1124-9. '" Olsen, Adv Exp Med Riol (1988)236: 1-14. Puia, Neuron ( 1990) 4: 759-65. Prince, Riochem I~harmacol(1992) 44: 1297-1302. '29 Newland, I 1'hy.siol Lond (1992)447: 19 1-213. Concas, Rrain Res (1991)542: 225-32. Hales, Rr I Pharmacol(1991 ) 104: 619-28. '"' Kocni~,Riochem Pharmacol(1992)44: 11-15. .'?' Possani, Hiochim Riophys Acta (19921 1134: 210-16. '.34 H A S , Eur I l'harmacol (1992)210: XZ946. '""oody, Etrr I l'hormacol (1989)164: 153-8. Pitler, 1 Neurosci (1992)12: 4122-,32. Mouginot, I'hysiol Lond (1991)437: 109-.Z2. '." Akaikc, I Physiol Lond (1987)392: 543-62. Reynolds, Brain Res (1991)564: 138-42. 14" Waffnrd, Neuron ( 1 99 1 ) 7: 27.33. 14' Gonzales, Trends Pharmacol Sci ( 1991 ) 12: 1-3. 14' Sudzak, Proc Natl Acad Sci USA (1986)83: 4071-5. 14' Hoffmann, I Neurochem (1989)52: 193740. S i ~ e l FERS , Lett (1993)324: 140-2. Twyman, Physiol Lond (1992)456: 21 5-45. '41 Sieghart, I Neurnchem (1983)41: 47-55. I"' Liiddcns, Nature (1990)346: 648-51. '41 Puia, Proc Natl Acad Sci U S A ( 1 992) 89: 3620-4. '49 Schumacher, Mol Neurohiol(1989) 3: 275304. IS* Knapp, Ncurochem Res (1990)15: 105-1 2. '"I Miihler, Proc Nat1 Acad Sci U S A (1980)77: 1666-70. Hawkinson, Mol lJharrnncnl (1992)42: 1069-76. ""ctkc, Mol P?~ormacol( 1992) 42: 294-301. Sqal, I Netrrophysiol (1984)51: 500-15. 155 Ropcrt, I'hysiol Lond (19901 428: 707-22. 'OR
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'"' Mistry, Pflugers Arch (1990)416: 4 5 4 4 1 . '" Robertson, I Physiol Land (1989)411: 285-300. Peters, Pflugers Arch ( 1989) 415: 95-1 03.
'"' Schmieden, Science ( 1993) 262: 256-8. Rowcry, Trends Pharmacol Sci (1989)10: 401-7. '"' Gage, Trcnd.~Neurosci [I9921 15: 46-5 1. Inn
Rowcry, Annu Rev Pharmacol Toxicol(1993)33: 10947. '""ahner, FASER J ( 1 99 1 j 5: 2466-72. la Qian, Naturc (1993)361: 1 6 2 4 . '""~cruki, I Neurophysinl ( 19921 67: 79 1-7. lM Chvatal, Pfliigcrs Arch (1991)419: 2 6 3 4 . ln7 Kawakami, R i d Pharm Rull(1993) 16: 7 2 6 8 . Narahashi, Trends Pharmacol Sci (1992)13: 2 3 6 4 1 . Shirasaki, Rrain Res (1991)561: 77-83. 17" Mocchctti, Neurnchern Res (1990)15: 125,ZO. 17' Rormann, R c ~ u Pept l (Srrppl) [1985)4: 33-8. 17' Olscn, FASEH 1 ( 1 990) 4: 1469-80. 17.z Bormsnn, Trends Nettrnsci (1988)11: 112-16. '74 Huganir, Nerrron (1990)5: 555-07. f75 Lister, Neuropharmacology (1991 ) 30: 143540. DeLorey, I Riol Chern ( 19921 267: 16747-50. Psychint (1991)148: 162-73. 17' Zorurnski, Am f7R Henlcy, trend.^ Pharmacol Sci (19911 12: 357-9. "' Olsen, Annu REV Phurm~1~01 T o ~ i c o [l1982) 22: 245-77 Nattel, Drugs 11991) 41: 672-701. In'Rauh, Trends Phnrrnncol Sci [I9901 11: 325-9. lR2 Hacfely, Neumchem Res (1990)15: 169-74. 1R.3 Sicghart, Trcnds l'harmacal Sci (1989)10: 407-1 1 . Farrant, Neumchcm Res (1990)15: 175-9 1. ""ldefrawi, FASER I19871 1: 262-71. Narahashi, Adv Exp Mpd R i d ( 1991 1 287: 61-73. IR7 Schofield, T r ~ n d sI'lhormacnl Sci (1989)10: 476-8. In"Costa, Ncuropsychopharmncology ( 1 99 1 ) 4: 225-35. IRP Ruck, Riotechniqucs (1991)11: 636-8. Grayson, Methnd.7 Neuro.~ci(1993)12: 191-208. Kirkness, Trends Pharmacol Sci ( 1989) 10: 6-7. Lamhert, T r e n h Pharmacol Sci (1987)8: 224-7. 1P.3 Stcphcnson, Rinchcm 1 ( 1988) 249: 2 1-32. ' 9 4 Porter, Nerrron (1990)5: 789-96. 16'
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Inhibitory receptor-channels gated by glycine
Edward C. Conley
-
Entry 11
Abstractlgeneral description 11-01-01: The amino acid glycine is an important inhibitory+ transmitter in the spinal cord and brainstem, participating in the regulation of both motor and sensory functions. Upon release by an inhibitoryt neuronc, glycine hinds and opens post-synaptic glycine receptor-channels (GlyR) causing neuronal hyperpolarizationT through their permeability to chloride ions. Generally, glycine inhibits glutamate-evoked depolarizationt and depresses firing of neurones. 11-01-02: Although gamma-aminohutyric acid (GABA) is generally considered to he the major inhibitory neurotransmitter in higher rcgions of the nervous system (see ELG Cl GARAA,entry lo), the wide distrihution of GlyR subtypes (as detected by in situ hybridization, for example) also suggests important roles for glycinergic transmission in higher brain functions. GlyR are expressed in relaying centres for processing pain and sensory information and deficiencies in glycine receptors lcads to spasticityt and loss of motor control in various neuronal disorders. 11-01-03: Post-synaptic inhibition of neuronal firing by GlyRs is selectively antagonized by the competitive antagonist strychnine and receptors specific for glycine were originally identificd by strychnine radioligandt-binding studies1**. Strychnine exerts pro-convulsant effects resulting from excitatory depolarization by Na'-influx. Thus in strychnine poisoning normal glycinergict inhibition.is aholished, giving risc to the muscular convulsions hy 'overexcitation' of the motor system.
11-01-04: Comparative studies suggest that both glycine and GARAAagonistactivated channels (see ELG C1 GARAA,entry 10) act as multi-ion pathways and have similar permcationt characteristics (although sevcral important pharmacological distinctions exist) (see Table 7 under Selectivity, 11-40). The sequences encoding the chloride-selective glycine and GABAA receptor-channels display multiple regions of homologyt. A marked outward rectificationt is ohscrvcd when GlyR channels conduct in the steady statet. This rectificationt is likely to reflect a voltage-dcpcndencct of gatingt, hut not a voltage-dependence of ion perrneationt.
11-01-05: Molecular cloning studies have revealed a heterogeneity of glycine rcccptor subtypes in thc CNS. ~ a t i v c tGlyR are assembled from subunits encoded by five distinct hut related genes ( r l , y2, r,?, r 4 and PI. Intact rcccptor complexes are likely to consist of a pentameric assembly of x and P subunits, and co-expression studies in Xenopus oocytcs havc established that the GlyR TIPhetcro-oligomersj are present in an invariant (3:2)stoichiometry. 11-01-06: Purification of the GlyR to hornogcncityt usingaffinityt ligands such
- as Zaminostrychnine typically resolves thrcc polypcptidcs of 48 kDa
(a
entry 1 1
s ~ ~ h u n i58 t ] ,kDa (P sabunit) and 93 kDa (gephyrin, sec S r ~ h c e l l r ~ l ~ r l o c a ~ 11ions, 1 0 ) following SDS-17AC;Ei analysis. Fnllowing dctcrgcnt-soluhilizationt,thc GlyR sediments as a macromolecular complex of approximately 250 kDa. I
I I
1 1-01-07: Site-dircctcd rnutaRcncsist of GlyR ct~dingscqucnccs have idcntilicd protein suhunit dnmainst involved in ligand discrimination, agonist efficacyi, antagonist binding and ion channel fmrmation: In cnrnmon with other cxtr3cellular ligantl-gated anion channcls [c.g. the GARAAR)aromatic hydroxyl gmups appcar crucial tor ligand discrimination while the prcscncc of net positive charges in ClyR-chnnncl vcstihulcs arc associatcrl with 'attraction' c ~ f anic~nsinto the hydrtlphilici pclrc. Thc a subunits contrihutc thc strychninehinding site of intact pcntamcric GlyRs. T h e GlyR T suhunit M2 domain is likely to 'linc' the pore, which is similar in arrangcmcnt to othcr cxtrnccllular ligand-gated channels (scc Ilonrmin conserv(~tion,1 1-28).
11-OlbR: Extracellular signals rclcasctl from prc-synaptic ncurnncs (such as neurotransmitters and neuropeptides) as well as certain extracel!ular matrix proteins may modulate Gly K function and rcccptor dcnsity in vivo. In commcm with other cxtracclluIar ligand-gatcd rcccptnr-channels, the GlyR contains a large (putativclv) intmccllular loop that contains n numhcr of consensus sitcs for protein phasphorylation (.scr~l'mteinp~~o.sphnryI~~tic~n. 1 1 32). 1 1-01-09: Dcvclopmental variants of GlyR T suhunit gcncs havc hccn discovcrcd r t(~mil,v.I 1 - 0 . 5 ) . via scqucncing ot cllNA cloncs ( s i p ( > 2 , ) r~nrlz?* t ~ n d i ~C;ranr Amino acid suhstiti~tionsin thc T ? ' suhunit can account for a 500-fold lowcr strychnine sensitivity obscrveil in neonatal rats comparcd tr) adults. Thc r ? suhunit cxprcssion is predominant in foetal or n c w l ~ o mtissucs anrl 'dcvelopmental switching' from T ? to 2 , occurs within .1 wccks following birth, ncct~mpanicrlhv :I strony: post-natal incrcasc in high-affinity ['~]-str~chnine hinding. This tlcvrl(~pnicnt;lltmnsiticln from 'neonatal' to 'atlult' C;lvI< ~stlfornisappears t c ~he pcrt~trhcrlin t h r mutant mouse spastic which acquires ;I severe motor disease a l x ~ 1 ~ wrcks ~t after Ilirth. Note: <;lyR protein codirr.~ r r ~ i o n s nppriir i ~ nh:ingctl c in .spuatic mice, sumcsting that an shurrnnt rcgulntion tjf ion ch:inncl xcnc sw ltching underlies t lic zliscasc phcnotypcf.
u 1
11-01-10: GlyR P suhunits s h ( ~ wwiilc distribution in hrain and spinal cord with :I common (non-viiriant) scyucncc. p subunits arc not rcilt~ircd for high-affinity agonist or nntagclnist Iinding and thcy show constitutive expression t h r o u ~ h o u t dcvclopnicnt. Sitc-dircctcd mutagcncsisi stutfics havc prctlictcd the cxistcncc of 'assembly domains\~n thc /I suhunit which may havc cvolvcd t o prcvcnt homo-olignmcrizationt. Thcsc domains may function in a 'sequential assembly' pathway forming functional GlyRs [i.c. 2!1! 4 x / p + r -. 3!1/j?j. 'l'orc-liningJ rcsiducs within thc M2 of t h e /i'subunit arc major tlctcrrninants of picmtoxinin resistance. T h c 0 suhunit is cxprcsscd in areas c ~ f hrain whcrc none c ~ f thc prcscntly characterized s ~ ~ l ~ u n (or i t s thcir mRNAs1 can hc dctcctcrl, whicli sumcsts further ('unclr)ncd'l GlyR suhunit gcncs cxist.
Category (sortcode) 11-02-01: ELC CI CLY, i.c. cxtraccllular li~and-gatcdchloride channels t c d retrieval code [unirluc nctivatcd hv glyc~tic T h e ~ u ~ e ~ clectrnnic
cntry 11
P
embedded identifier or UEI] for 'tagqing' of new articles of relevance to thc contents of this entry is UEI: GLY-NAT (for reports or reviews on nativct channel properties) and UEl: GLY-HET (for reports or rcvicws on channel pmpcrtics applicable t o hctcroloRouslyt expressed recomhinantt suhunits . a dr~crr.csiono f the advantn~csnl UEI.7 cncodcd hy C D N A S ~or R c n e s t ~For and ~nidclineson thelr implemcntatron see the .~ectionon Rc.~ourccJ under Inlroduction nnd layout, cntry 02, d for further rlctnils, sce Rcsourcc J - Search crrteria d CSN devrlopmen~,entry (15.
1
Channel designation
11-03-01: The GlyR; the 1GlyR; ionotropict glycine receptors; GlyR-C (the glycinc rcccptor-channel). T h e neonatal [fmeta!) forms of native GlyR channcl complcxes have been given the d e s ~ g n a t ~ oGIyRN n to d~stinguish thcm from thosc predominantly expressed in the adult (GlyRA1 (see Gene family, I 1-05,and Developmental regulatlon, 11 - 1 I).
0 -
Current designation 11-04-01: Of the form 5,,,,
ex.
hy,
C
~
~
J
Gene family The GlyR gene family 11-05-01: Thc gcncs encoding ~ l y c i n ereceptor-channels form part of the cxtraccllular ~ i ~ ~ n t l - ~ a [ELG) t c d t channcl genc surcrtarn~lyt (d~scrjhcd under ELC: Kcy facts, cntry 04). Cnnvcntional cross-hornologyt screening has [thus far) identified five distinct hut related genes encndlng suhunits which assemhlc into nativc GlyR complexcs (rl,all rl, a4 and 11 - see Trrhlc 1 /or comparison o f features). ~ o n o c l o n a ~antihodics f raised against thc native+ GlyR have hccn shown tcl irnmunoprccipitatc hoth a- and P-chains, and limitcd protcolytict clcavagc of thcsc proteins show similar pattcrns.
V0rinnt.q derived from indjviduol GlyR ,penes 11-05-02 Suhtle variants of GlyR
8 suhunit genes have hecn discovered via scqucncing of cDNA clones. For cxamplc, the 'a2" neonatal' subunit [sp: P22771, see Table I and Dntnhase listings, 11-53) is a novcl cDNA variant identical to thc human ' r 2 suhunit', except for five amino acid substitutions a t positions 18, 24, 37, 194 and 404. Substitution at position 194 (Gly194 to GluJ in the .rl' protein accounts for the 500-fold lower strychnine sensitivity obscrvcd in neonatal rats. Rat na* contains Glu at positron 167, as compared to Gly a t pos~tion167 in rz".
/j srl hunit
genes and prot eins fi subunits show wide distribution in brain and spinal cord
11-05-03: GlyR
with a common [nrjn-vnr~ant) scqucncc. Two sttbunits arc prcscnt in cach five-suhunlt nativc GlyR complex ({or d~tnils oJ the stoichinrnetrrct msscrnhlv patterns o f GlyR, see Predicted protein topography, 11-30, rrnd - Trthle I ) . fi subunits arc nnr rcquircd for high-afflnity agonlst or antagonist
Table 1. Genes encoding subunits present in native GlyR-channel complexes (From 11-05-03) Subunit
Description
Encodinga
Mol. wt (from
GlyR alpha-1 subunit = al
~ GlyR. Alpha-1 Component of adult ' n strychnine-sensitive' polypeptides are the principal ligand-binding subunits of adult GlyR complexes (GlyR*).Binds glycine ligand and the convulsant strychnine. Different variants of a1 have been isolated5.
447-449 aa (human)
48 kDa (human)
GlyR alpha-2 subunit variant = a 2
Variant of the neonatal (or 'foetal'), ' nstrychnine-insensitive' ~ GlyR. Alpha-2 polypeptides are the principal ligand-binding subunit of neonatal GlyR complexes ( G ~ ~ R Contains ~ ) ~ ' ~a . glycine residue at position 167. The a 2 gene generates two apparently isofunctional splice variants affecting residues in the N-terminal domain which are co-expressed throughout development (see Gene organization, 11-20, and Developmental regulation, 11-11).
452 aa (rat] Transcript size -2.8 kb in rat spinal cord poly (A)' ~ R N A '
GlyR alpha-2' subunit variant = az*
~ Variant of the neonatal (or 'foetal'), ' n strychnine-insensitive' GlyR. Principal component in neonatal spinal cord GlyR complexes (GlyRN).Contains a glutamate residue at position 167, which is sufficient to explain lower antagonist-binding properties than ax3.az*binds glycine ligand but has 500-fold lower sensitivity to strychnine when expressed in oocytes3. az* is probably equivalent to the 'strychnine-insensitive' GlyRs in native neonatal rat spinal cord3.
448 aa including signal peptide. 425 aa (mature) (rat)
SDS- PAGE^
48 kDa (human/ rat)
GlyR alpha3 subunit = a3 GlyR alpha-4 subunit variant = a4 GlyR beta subunit variant = p
Peripheral membrane protein
Principal GlyR component in post-natal cerebellum8. Binds glycine ligand and the convulsant strychnine. The novel a4 variant was identified during screening of mouse genomic clones9. The predicted cr4 polypeptide displays very high homology to the a2 subunit. a4 mRNA is expressed at very low levels in brain Presumptive 'structural' subunit without ligand-binding sites. The native GlyR is a pentamer. GlyR alphalbeta heterooligomers have an invariant stoichiometry (3 alpha:2 beta]. The p subunit appears to be expressed in a single form throughout the brain at all developmental stages and is not required for ligand-binding7 Co-purifies with the GlyR on affinity column^'^. Associated with the cytoplasmic domains of the receptor core - possibly a glycine receptor-to-tubulin linker protein (gephyrin)'' (for details, see Subcellular locations, 11-16)
464 aa (rat)
48 kDa (rat) 48 kDa (rat) predicted
Transcript size 58 kDa (rat) -3.4 kb in rat spinal cord poly (A]' ~ R N A ~ 93 kDa (rat) (nonglycosylated)
"Shows the number of amino acid residues in the specified channel subunit as predicted from open reading frarnet lengths of cDNA sequences.
entry 1 1
1n
hind in^ and they show constitutive expression thrnughnut dcvclopmcnt. Note: The p suhunit is cxprcsscd in arcas of brain where none of thu presently charactcrizcrl suhunits (nr their mRNAsl can he dctcctcd, which s u ~ c s t further s ('i~ncloncrl')GlyR suhunit gcncs cxist.
G l y R x e n c s nrc h i , ~ h l yrelntcd to tl~osce n c o d i n g GARAA receptors
11-05-04: On the hasis of sharer1 amino acid homologyr, thc chloridc-selcctivc glycinc and GARAn receptorxhannel gcncs arc morc similar to each othcr than either is to the nAChR or othcr cation-sclcctivc rcccptor-channcls. IJcspitc some kcy pharmacologicnl rliffcrcnccs, many similarities cxist hctwccn glycinc- and GARA,-activated currents - e . scc ~ comparative study of tlicsc channcl types in post-natal cuIturcd hippocampal ncuroncs4. (Sirniloritics in t h r sc/ccbtivit,v r:hrjrnc.tr,ristir:s of rhl: GlyR nnd thc C;ARAAR orc ,yivcn in T o l ) I ~7~t1ndr.r Srlcctivify, 11-401.
Trivial names 11-07-01: Thc inhihitory glycinc receptor; the glycinc rcccptor-channcl; the strychninc-sensitive glycinc rcccptor (hrrt nntc .~trycllninc-in.sensitivity of iort(iIlnronntr11G1vR forms - scc Trlhlc I ] : the ionntmpici nlvcine receptor.
Cell-tPpe expression index T ~ ,yIyc.it~~ c r t ' c ~ p t o r - c h c ~ n n eils a b u n d a n t in the spintrl c o r d mnd 17rt~instrmo f rrertrlhrcltlls 11-08-01: Thc GlyRs arc hifihly cxprcsscd in regions of the CNS associateti with spinal neurotransmission and motor control (.%PC Di~v~1opn~rntml rcyrr/cltior?. 1 I - 1 1, n ~ lNl;~rcclls of the mouse retina", cultured hippocampal ncuroncsJ, tclcnst Mauthncr cells" anti renal proximal cells'".
Channel density C l u ~ t c r i n ~ofy ClyRs h y specific intcrflctions with cytoskeletrrl e1~rncnt.c 11-09-01: Glycinc rrccptors arc known to hc associated with cytoskeletal elements which co-purify using Glylt affinity liKands'to2"-23 . Such ~ntcractionsmay control channcl mohility, suhccll~~lar distribution and local expression density ( s r ~ c . (rlso .q~phyrin,rrndcr IJrotcin intcmctions. 1 1 31/.
cntry 11
Comparable expression density of glycine- and GABA-activated channels in hippocampus 11-09-02: Responses to saturating concentrations of glycine and GARA agonistst in cultured hippocampal neurones suggcsts that thcy activate compnrr~hlenumbers of 'anatomically-distinct' channels with 'vcry similar' permcation (see nlso Selectivity, 11-40).
Developmental regulation Post-natal switching o f GlyR r subunits - effects on antagonist binding and glJ~cinergictIPSC
!
11-11-01: Glycine receptor suhunits exist in 'foetal/ncwhornl (c.g. a? and z z b ]
and 'adult' ( x , , a,?]isoforms (see nlso Table I under Gene family, 11-0.5).In rat spinal cord, 32 subunit exprcssion is predominant in foetal or newhom tissues; 'developmental switching' from a t to cc1 occurs within 3 weeks following hirth", T262" . This 'switching' is accompanied hy a strong postnatal incrcasc in high-affinity 13~]-strychninebindin8'. This process of dcvclopmcntal switching of GlyR r subunits also accelerates kinetics of glycincrgic inhihitory past-synaptic currents (IPSCS~,for examplc, in spinal n c ~ r o n e s ~ ' . ~Note: ~ ) . Functional maturation of the nicotinic acctylcholinc receptor (see ELG CAT nAChR, entry 09) is executed hy its gamma-tocpsilon subunit switchinRt.
GlyR phnrrnacological properties in neonates mediated hy the GIyR n*" isoform 11-11-02: It has been known for ovcr a hundred ycars that neonatal rats arc rclativcly 'immune' (resistant] to strychnine p o i s n n i n c . Incrcascs in scnsitivity of spinal cord ncuroncs to glycinc ant1 strychnine comlatcs with thc disappcarance of a low-strychninc-binding p 2 * suhunit isoform (sp: P22771, see Domain conservtltion, 11-28, and Dntahnse listings, 1 1 - ~ ~ 7 ) ~ . Thc variant shows a number of amino acid substitutions which havc cffccts on intact GIyR function (for stn~cturalvarintions, see Gene f(1mi1,v. 11-05). GlyR r z mRNA accumulates in pre-natal development and sharply dccrcascs after birth, whereas transcripts for strychnine-scnsitivc .*I and 23 suhunits appcar only in post-natal hrain structure^'.^.
Constitutive expression o f C3yX jl subunit mRNA throu~hout development 11-11-03: ~ ~ h r i d i z a t i o nsignals t of P subunit mRNA arc ohserved in early embryonic stages and continuously increase to high levels in adult rats7 (see Tnhle 1 nnd mRNA Distrihntion, 11-13).Note: The npporent1,v 'isofunctional' u2 splice variants (see Gene orx(~nizntion.11-20)have hccn reported to he coexpressed throughout cmhryonic and post-natal development".
Regional nccr~mulation o f GIyR trnnscripts
-
11-11-04: Regions1 accumulation of mRNAs encoding GlyR 11, z ~ x, , during ~ dcvelopmcnt has hecn ohscrvcd [sce r c t 7 for original in situ hyhridization reporting thc timc course of regional cxprcssion in hrain). Tahlc 2 summarizes the relative signal intcnsitics rcflccting mean levels dctcctcd for thc principal GlyR suhunit transcriptst in developing spinal cord'.
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Table 2. Kclr~ti v t ~siipnrrl intcnsit i r . ~ rrpflcctin.y nzrrn Icvcls of GlyR .srrhl~ni! trrrn.~c:riptsi n drvrlopir~sspinr~lc.orrl ( F r t ~ n1 ~1 - 1 1-04) ncvclr,pmcnt:d .;taKct Transcr~pt E I 4
PO
1'5
1'1 5
P2n
rnodcr;~te low very low hi~li
hi~h vrrv low vcry low high
h igh
I{)w
very low
mt)rlcmtc
(17
hi~h
hifih
hifih
(I I
undctcctahlc low
very Tr,w low
very Ir)w
(1
I
i
I
EI0
hi~h
very low vcry low
lii~h
i-'hysinlogicnlcnnxequcnr:es nf '.~;equcntiol nctivntion' o f GlyRchrrnnel Rene exprrsxion
11-11-05: The Jcvclnpmentat transition from the nut~natalto the adult GlyR isnklrm appears to hc pcrturhcrl in the mutant mouse spastic which acquires a severe motor disease about 2 wccks after birth"" (see I)/~cnotypiu expres.sion, 11-14). For further details of thc amino acid determinants for diffcrcntial prt~pcrtics hctwccn GlyR r suhunits switched during dcvclopmcnt, see Ilomoin {unr:t ion.9, 1 1-29. For further details of the clectrnphysinlogical changes ohscrvcd during tht. transition from foctnl-type to adult-type ClyR - currents, sue Sin~lc~-c.hrrnni~I cl(~rrr.11-41.
I
mRNA distribution GIyR o suhunit m R NAs display differentin1 spat iaI expression
11-13-01: As su~nrnnrizcdin Tnhle ;l scvcral diffcrcnccs exist in regional and developmental expression of thc diftcrcnt GlyR .r suhunit mRNAs. Thcsc ' h i ~ h l v - r c ~ i c ~ n a l ipatturns zd' provldc cvidcncc tor nt~vcltlrnctions for GlyR prt~tcins in thc mammalinn hrain and spinal cord. Thc wide ('nnnru~ionalizctl'] distrihuticin c l f GlyR P s~tl,unit transcripts+ inclvdc regions lacking r suhunit hyhrirlization simals (sec Trrhlc ,7) and [nay therefore indlcatc thc c x i s t c n ~ e(if further distinct GlyRs in mammalian hrain (the gcncs for which havu not yet hccn cloncd].
Phenotypic expression Generill phenotypic roles o f ,plycine receptor-channels
11-14-01: Thc wide occurrcncc of nhc GlyR and r isoforms throughout the CNS (set. m R N A di~tributinn, 11-13) also suggests scvcral roles for glycinergic transmissiont in higher hrain functions,','. In medullary dorsal llnm anil in thc spinal riorsal hnrn (relaying centres fnr processing pain and sensory information), glycine inhihits glutamate-cvokt.d dcpolarizationt and depresses firing of neumnes. Dcficicncy in glycinc receptors leads to spasticityt and loss of motor contrcll in various nctrronal discn~cs.~~"'.
Persist~nceof the neono!oI isn/orm is a.ssocio~edwith acquired motor disensc in mice 1 1-14-02: The developmental transition from the ncnnatal to the adult GlyR ~sc~frwrn ( r r ~Dr~rlop1i1entr1lre,q~11ot1011. 1 1 - 1 11 is pcrturhcd in the mutant
-
Table 3. Di.~tri!hutiopatterns o f C;l,vR mRNAs (From 11-13-01) Transcript I
(t2
n3
Ij
I
Gcncral distribution patterns". "
In sit11 hybridization of ~ d u l brain t shows ClyR tr, suhunit mRNA to he ahundant in spinal cord, supcrior and inferior colliculi7. r r l suhunit transcriptst arc highly-cxprcsscd in adults (.FCC Dcvelopmcntnl rexulotion, 1 1 -1 11 Exprcsscd in scvcral forehrain regions inclliding layer V1 of the ccrchral cortex, thalamus and hippocampus'. In rat spinal cord, rrx suhunit expression is prcdominant in foetal or ncwhorn tissues; switching from 0 2 to n l occurs within 3 weeks following birth.?. 7.242" (see r)eve1npmental regulntion. 1 1- 1 1) Expressed a t low ahundancc in ccrchcllum, olfactory bulh and hiPPocamY7 Northern analysis"' and in situ hyhridization7 rcpcirt high amounts of r j suhunit transcripts throughout the spinal cord and hrain7 (including ccrchellum and cortex, where none of the presently characterizcd o polypeptides are expressed at comparahlc levels'
"Sce also original in situ hyhridizationt data and full breakdown of regionalized cxprcssion patterns7. he spatial ctistrihution of channel subunit-specific mRNAs prcsumahly rcflccts a functional specialization in particular cell typcs. Mapping of cxprcssion pnttcms is a complcx task and has to take many vnriahlcs into account, such as in situ localization, dcvclopmental regulation, suhunit stoichiomctry, and factors regulating overlapping or co-cxprcssion. Notes (In thc intcgraticin of computer-based information resources ahlo to crossrcfcrcncc thcsc divcrsc factors influcncing cxprcssion can he found in Fig. 2 undcr Fecdhnck L53 CSN nccess, Pntry 12 nnd Appendix 1 - Serlrch crirerln OJ CSN devcloprnent, entry 65. I, The pcissibility of the r j suhunits participating in other ELC, rcccptorchnnncl typc complexes (c.g. GARAAR, putative taurinc/,-l-alanincrcccptors and NMDAR) has been discusscd3*. mouse spastic (spa)which acquircs a severe motor disease ahout 2 weeks aftcr birth,'". Some key phenotypic features of this disorder arc listed in T a b k 4 (limited to factors capable of disturhing thc normal halance between inhibitory and excitatory ion channel function]. The neurophysiological and neurnpharmacological aspects of thcsc disorders arc further discussed in rcf~""..~'.
Long-term potentiationt o f inhibitory circuits and synapscs in the CNS 11-14-03: ~ ~ ~ c i n e rinhibitiont ~ict in tclcost Mauthncr cells (produced by stimulation of thc contralateral eighth ncrvc) cxhihits long-tcrm ptcntiationt following classical tctanizationt of thc pathway'". ~ o t c n t i a t i o n t (cnhanccment) occurs at the synapscs hctwccn primary afferentst on tci second-order interneurnnest and tho connections hctwocn
cntry 1 1
-
Table 4. Sornc kc!? phctlOl!./Ji~' fcrrtllrr,~(I/ t h r , ric:r]!~irc~rj moror rij.wc~sr' o/),scbrvc~rl in t hr rnutrlrlt rnorrsr spcr.v!ir (From 1 1-14-02)
Phenotypic fcnturc
(~hsrrvatioil/dcscr~ption/infercncc
Time of onset Ah(lut 2 wccks foll
Kcfs
"'
"
"
Sclcctivc deficit Lcvcls c ~ GlyR f {as ~ncasurcdhy [ ' ~ ] - s t r ~ c h n i n c and of the a d i ~ l GIvR t hiniling and clcctrophvsioln~icnlprr~cvrl~irvsf are citnticlns isoform in sprl drasticnlly rvducvd in hclrnozyKelust spr~Jspr, thcrvin 1 1 1 1 ~ ~ rnicc. Thc spr~\tiriii~tt;~tion sclcctivcly intcrfcrvs with the post-natal accnmulation c ~ f rn flNA mcc~rlinl:the atlrilt iso6orln yRA. I'c.rinnt;lli cxprvssioil c ~ the f nctlnatal isofortn CFvRN is I I J ? J J ~ I ~ I ' I I ! ( V11n;lffcctcd. A l t h o i ~ ~ Y ~ nillNA 'Ivvrls' vnce~dinr:EilvK 'adult' r t I suhiinits :ire nt~rin:~l, the sclcctivc cffcct c ~ the f sprr ini~taticlnrli(ry : ~ c at t h t ~ t hthe trnnscriptionalt Icvcl (i.c.;~ffectingrcccptr,rstehilbty, ttirnovcr or intcr;ictions with ophcr pmtuins) sntl/or thc post-transcripticlni~ITlvvcls .79 f Svmptclms o f strychnine poisoning at Mimicking c ~ sprr phcnc~typchy suhconvulsivu tlr~sesin nurmal ~ r ~ i rcscrnl~le cc GlyR antagc~nists the sprrstic. phcnotvpu. Administration of picrotoxinin (ser I
Apparent Ir1c.k of ClvR gcnc striicti~r~l changcs in s p r micc
x4' Changes in functicin or structure of the GlyR prottins appear r!nolCr.ctcdin spnstrc mice, i;ictors which hsvc hccn taken as furthcr cvidcncc for n nyrllr~trryrather th:m n stn~ctllrnl f spastic lnutntion. L~~anrl-hindinp, effect c ~ thv pnjpcrtics, subunit ctimposition an11 synaptic 1oc;llit;ltion of the GlyR ;arc ~ l n o h a n ~ crnt l sprl rnicc. coinparccl to wiltl-typrl
entry 11
Table 4. Contintied Phenotypic feature
Observation/description/inferencc
Refs
GlyR-mediated currents in motoneurones of spn micc
Strychnine-sensitive chloride conductances (which are spontaneously active in normal mice) are rare in spa mice. Current amplitudest are small, resembling GlyR currents inhibited hy strychnine
"
'Compensatory expression' of GARAAR for deficits in GlyR expression in sp(1 micc
A significant increase in GARA,, receptor and density in the lower CNS may serve as a citations compensatory function, partially 'countherein teracting' losses of glycinergict function. Pharmacological fac~litationiof GARAA responses, for example by administration of benzodiazepine agonists or aminooxyacetic acid (which reduces tiegradation of endogenous GARA hy inhibiting GABA transaminasel also allcviatcs symptoms of affected mice (sre ['henotypic expression under ELG Cl GARAA, 10-14).Renzodiazepines (e.g. diazepam, flnni- .?'' trazepam and Roll-6896 (see ELG Cl C;/\l.(AA, entry 101 allcviatc symptoms, and lead to a long-lasting rclaxation o f muscle rigidity
Correlation of S ~ mouse N symptoms with motor disorders in humans
Human hyperexplexiat and some types of spastic paraplegiai show symptoms ri~.semhliri~ thosc of the spalspn mousc
Correlation of spn mousc symptoms with similar disorders in cattle
Inherited myoclonust of cattle is chamctcrizcd " hy hyperaesthesiat and myoclonic jerks of the skeletal musculnturc that occur spontaneously and in response to sensory stimuli. Inherited myoclonus is also associated with a dcf~cicncy of ClyR in brainstem and spinal cord, although altered motor symptoms arc also apparent in prcl-natol calves
'"
thcsc inhihitory cells and the Mauthner neurone. Increases in gain still occur following pharmacological blockade of potcntiationi at the excitatory synapse with glutamate antagonists. Thesc findings imply that in vivo, learning can alter the 'halance' hetween excitation and inhihition within a network hy modifying one or hnth of themIR (scc c~lsoI>cvcIopn~ental rr~ulntion.I 1 - 1 1 , nncl Receptor antc~~onists. 1 1 - 5 1 ) . For (urthrr dc.scriptions o f lon,q-turnpotentiationt processes. S C P Fig. 1 under ELC; CAT CiLIJ N M D A . entry 08.
1
'Cvtoprotection' by gljrcine and strychnine in renal proximal tubule cells
11-14-04: At certain concentrations both glycine and strychnine (see Receptor onto~nnists.11-51) havc cytoprotective functions in renal proximal tubule cells treated with I [IM antimycin A'" as cstimatcd hy lowered release of lactate dehydrogenaset (a marker of cell dcath/lysis). A critical role for chloride-influx pathways in promoting cell death has been d i s c ~ s s c d ' ~ .
-
Protein distribution
/ ' ~ ] - ~ t r y c h n i nbinding e in CNS sections displays a pronounced rostro-cnudal gradient 11-15-01: Extensive autoradiographic studics employing in situ radiolahellcdt antagonisti hinding have consistently shown a gradient o f binding levels along thc rostro-caudal axisi, with highest binding in the brainstem and spinal cord regions.
Protein and m R N A localizntion techniques report 'overlapping st~hsets'o f total ClyK proteins
-
11-15-02: Binding patterns of strychnine radioligandst and immunocytochemical studies with specific monoclonalt antihodies demonstrate that GlyR subunits are concentrated in spinal cord, brainstem and other areas of the lower neuraxisi. In keeping with the dctcction of GlyR mRNA expression in sornc 11igherbrain regions (sccm R NA distrihutian. I 1 -13) immunocytochcmical prohcs for GlyR prntcins also detect low signals in olfactory hulh, midbrain, cerebellumJ7 and cortex. In general, mRNA distribution stutiits- have dctectcd a considerably wider distrihution pattern of GlyR suhunit cxprcssion than that rcvcaled by immunochcmical/autoradingraphic methods in previous studies. This apparent discrepancy may he explained hy (i)not all GlyRs binding thc antagonisti radioligandsl with high affinity and ( i i ) not all GlyRs posscssing cpitopcst for the antihodies in use.
I
Subcellular locations
The GlyR channel is an immohilizedt post-synaptic peripheral memhranc protein
11-16-01: Thc 93 kDa polypcptidc (gephyrin) which is co-purified using affinityt ligands' of the GlyR1" (see I'rotein mnlccttlar wei,yht (purified), 11-22) h,as hecn localizcrl as a peripheral memhrane protein by immunoelectron' microscopy. The 93 kDa subunit has a cytoplasmic location in post-synaptic meml~rancs'~.Gcphvrin is thought to form a bridge or linker protein hetween cytoplasmic domains of the GlyR and tuhulin, therehy immobilizing thc GlvR receptor-channel complex. The 93 kDa component has bccn cloned and sequcnccdl' (see Dntahase l i ~ t i n ~11-531. x.
Transcript size 11-17-01: Scc Tahlr 1 under Cene family. 1 1 -0.5. Selective mRNA hybridanestt techniques were originally used to define thrcc distinct mRNA species encoding GlyRs in developing rat CNS".
entry 11
Notc: TIIEsymhol II'LITM] denotes an illustrated feature on the channel protein domain topography model (Fig. I ) .
Chromosomal location The gene defective in mouse spastic mutants has not been colocalized to GlyR structural genes 11-18-01: The spastic mutation affecting the regulation of lycinergict inhibitiont (see Phenotypic expression, 11-14) is an autosomal recessive1 traitt carried hy a single en deli ant gene with full penetrancet of the phenotype in h o m o ~ ~ g o t e s t The " ~ . spastic gene locus ~ 1 x has 1 been located on mouse chromosome dm. An allele of the spa gene displaying a very similar (hut more severe] phenotype in mouse has been defined as spaA"' "'. The mouse GlyR r 2 and x 4 genes have heen mapped to the X chromosome9.
P:
Gene organization The genomic organization o f GlyR n. suhunit genes is conserved across subtypes 11-20-01: Analysis of genomict clones covering the coding regions? of the murine GlyR 2 1 , r 2and r4suhunit genes has shown that all genes contain eight intronict regions with precisely conserved houndaries9.
Alternative splicing of a, transcripts 11-20-02: A variant rat zl cDNA has heen identified originating from the selection of an alternative splicet-acceptort site at an cxont encoding the cytoplasmic domain adjacent to transmemhrane domain M3. Splice variant rzi,, has an extra eight amino acid segment containing a novel consensus phosphorylation site (forfurther details, see [PDTMI. Fi,p. 1 and Protein phos-
phorylation, 11-32].
Apparently 'isofunctionnl' N-terminal substitutions in nz introduced b y alternative splicing 11-20-03: In the rat, alternative use of two closely spaced versions of exon 3 in the r2gene can produce two r z subunit isoforms, which differ by only two isofunctionalt substitutions in the N-terminal extracellular region? The rl splice variantst arc co-expressed throughout embryonic and post-natal devclopmcnt'.
1
Protein molecular weight (purified)
Affinity purification of GlyR subunits 11-22-01: Purification of the GlyR to homogeneityt has been achieved using matrices1 derivatizedt with Zaminostrychnine as an affinity ligandt. SDSPAGE? analysis resolvcs thrce polypcptidcs of 48 kDa (a subunit\, 58 kDa (fl subunit] and 93 kDa (gephyrin, .see Suhcellulnr locations, 11-101. Following detergent-soluhilizatid, the GlyR sediments as a rnacromolecular complex of approximately 250 k ~ a ~ " " . (See ~ 1 s ochemical cross-linkina st udie.~under Predicted nrotein tonoaranhv. 1 1-30.)
Molec~~lor wei,yht det ~ r t n jtions n ~ following heterolo,yous expression 11-22-02: Human homomcrici' 7 , GlyR channels cxprcsscd in thc haculovimst expression system can he g;itcdt hy glycinc, hut not in thc prcscncc of strychnine. An immunorcactivc 48 kI)n protein is apparcnt fr,llowing SI>S-PAC;E' of whole-cell lysatcst with maximal expression 3 days post-infcction5'.
Protein molecular weight (calc.) 11-23-01: For prcclictctl (non-glvcos)llntccit) subunit molcculnr masses determined from c n N A ccqucnccc, ccc the respective subunit gcnc name in Tahlc 1 tinr1r.r I:r.nr f(lrnrlv. 1 1-05,
Sequence motifs
T l ~ disulphidc c loop rnotif is conscrtred cross a large number o f ELG chunnels 11-24-01: An invr~rinntfc>oturcof all mcmhcrs of the cxtraccllular l i ~ a n d gated channel-ruccpt(?r supcrfnmily is the prcscncc of sn uxtracell~tlar dist~lphideloop motif1 (c..y. scr ELC; CI C;ARAA. cntry 10. ELC; CAT 5-HT,?. cntry 05. E L I : CAT nAChR, cntrj. 09). As may he cxpcctcd from its occurrence in several ligand-specific receptors, this small d n t n ~ i ndocs not fr~rrnpart of an agonist-hindinp, site as rcccptors with mutations within the loop (C.X.Lysl4.3 Ala14,Z) display few functional ~ h n n ~ (srr c s {~P ~ D T~M I , Ficy, 1 (ind T ( I / J ~h L~, r i d IIort~(~in ~~r f ~ i n c ~ i o 1~1 i-29). ,~,
-
Conscnsu.~sitcs for signnl pr11ti~lrC ~ C ( I V ( I S P ,d i ~ ~ i l p h ihonds d ~ and ~qlj~cine-l~indinx ,sirtrs 11-24-02: In common with other ELT. ch:~nncls,GlyR primaryt amino acitl scqucnccs show n numhcr of conscnsust sitcs for the formation of disulphidc hontis, hr-glycosylation and protcin phosphorylation (src, Trihlr I 1 ~ 1 l l f ( ' El,(; ~ K1.y f(ir.ts, rnt r!, 04, r1rlt1 I'rotcin ~~l~osphorylot ion. I 1-32). h numbrr of consensusi sitcs arc listed in Tahlc 5 (lor (in illl~strc~tinn ol' thc~ t~ppro.urnin!r~ positions o f thr~sc.r?iotjfv or7 ncitirvl pciit(irni.rir (;lvHs. . ~ c r , /IIIITM/,Fi\y, 1).
Tahle 5. Fxnniplr~niotif po~itron\ N O t ( ~ p o r t ~117d C:cnHonkn cntncl; (From 1 1-24-02)
Subunit scquuncc signalt pcptidc (rat1 cleavage motifs GlyR
rr2'
1-27
GlyR rr?
I -<3.Z
GlyR . I
1-22
Disulphidc hond formation motifs
N-glycnsylation motifs
1 72-honcl- 186 2(12-hond-24,1 17 1 -bond-185 2.3 1 -honiI-242. 18.1-bond-197
72, 10.7 71
54, 242
Norc: For sequence motifsf and species not shown, refer to cntrics rctricvnhlc I>y the accession numbers in the n(~trrl~rl.sc listinss, 1 1-57.
Note: The symbol IPDTMI denotes an illu.~tmtedfeature on the channel protein domain topography model (Fig. I ) .
Amino acid composition
U
11-26-01: Amino acid hydropathicityt analyses of all GlyR .x and P suhunits show a pattern of hydrophohict domains (MI-M4) typical of the extmccllular ligand-gated ion channel superfamily (see IPDTMI. Fig. I ) .
Domain arrangement Architecture of the glycine-hind in,^ domain 11-27-01: A multisite model of the GlyR ligand-hinding region (based on analysis of agonist responses in site-directedt mutants) which includes hoth high- and low-affinity agonist suhsites within an extended hinding domain has heen proposed2*. In this model, 'ligand recognition' implies that several side-chains fold together in the assemhlcd GlyIi complex, including those in the second half of the extracellular N-terminal domains of rl suhunits (see IPDTMI. Fig. 1). This pattern appears to he conserved amongst other ligand-gated ion channels such as the nAChR and GARAA receptorchannels (see ELG CAT nAChR, entry 09 and ELG C1 GARA,,. entry 10. resprct ively).
Net positive charges are associated with GlyR vestibules 11-27-02: The existence of channel vestihulest containing net positive charges are consistent with pcrmeahility~ properties of glycinc receptorchannels (i.c. anion-selective) and may function to attract anionsf2 (compare with the 'rings' of negatively charged residues with111 the vestihulest of the cation-selective nicotinic acetylchol~ncreceptor - srr Domain arrangement under ELG CAT nAChR, 09-27).The GlyR z suhunit domain M2 is likely to 'line' the hydrophilict pore, which is similar in arrangement to other extracellular ligand-gatcd channels (Fee Domaln conservation, 11 -28).
Domain conservation Conserved features of the M2 (pore-linin~) domain 11-28-01: In general, there is a high degree of sequence conservation of transmemhranc segments MI-M.7 hetween the GlyR and the GARAAR,indicating their potentlal importance in chloride channel formation55p5n.The M2 domain shows the h~ghestconservation (see Domain functions, 11-29). Ry analogy to other ELG channels, the M2 domain can he covalentlyt lahelled hy crosslinkedt non-competitivct channcl hlockers and determines the s~ngle-channel conductancct properties (0 compar~san o f permear ion+ properties o f these two channrl types appears under Srlectivlty. 11-40).
entry 1 I
-
Conserved residues and motifs hetwecn GlyR and GARAn channels 11-28-02: Roth glycinc and GARA* rcccptor-channels display (i)an invariant prolinc residue at mid-position in the M1 domain; (ii) a common hydroxyrich scqucncc Thr-Thr-Val-Leu-Thr-Mct-Thr (Scrl and a total of eight Scr or Thr in each M2 domain; (iiil a prolinc rcsiduc at the fuurth position preccdud hy a phcnylalanine rcsiduc in the M 4 domain; [ivl rclativcly high positive charge density within eight rcsiducs of the ends of the transmcmhranc domains on the cxtraccllular sides (I7y contrast, cntinn-selective ELG channcls display negatively chsrgcti residucs in irddition to positive (v)potential to form a fi-loopt hetwccn CyslB9 charges hordcring ~2""~); and CyslS.3, with K of 15 positions heing identical or highly conserved in all suhunits"".
Amino acid conservation amoncq CC:IyRsulwnits 11-28-03: The rl ligand-hinding ClyR suhunitx exhihits -82-8,3?h amino acid identity to the previously characterized rat and human r l and r2 suhunit sequences. In Xcnopus oocytcs, r3 homomultimersi form functional glycinc-gated channels which show low glycinc affinity and small responses t o taurincH.
Domain functions (predicted) Propcrtics o f hctcrolo,you.slyt expressed Cl,vK suhunits resemhlc notiveT receptor-channels 11-29-01: Glycinr can elicit large chloride currents following transient+ or stahlei hctcrologous expression of 2 1 , r? or r 3 GlyR suhi~nitcDNA in X~,nnpns oocytes or mammalian cells in cult~rc~~'.''".Gcncrally, clcctrophysiological and pharmacological characteristics of h e t c r o l o g o ~ s l ~ t cxpresscd channels arc similar to those ohscrvcd in cultured spinal ncuronal cells (sr~c, c7.g..re/.'2/.
Dclincntion o f rimino r~ci(lresidues involved in antagonist hind in,^ in ClyR n suhunits 11-29-02: r suhunits contrihutc the strychnine-binding site of intact pcntamcric ClvRs. Studies involving proteolytict ciigcstion of ['HIstrychnine-lahclld glycinc rcccptorsnl.A" have shown that covalent incorporation of the antagonistt occurs hctwccn amino acid positions 170 and 220 of thc r l suhunit, close to thc first transmemhrane region ( s c ~ ~ [I'DTM]. Fig. I ) . Photoaffinity-lahclling has furthcr localized this site to hctween rcsiducs 177 anti 220 (see rcf."") (scc 01.~0 hcloccrl. Hascd on structnml considerations, a strctch of charged residues around two cystcines preceding the first transmcmhranc scgmcnt has hccn implicated in ligand hinding to the GIVR". Flinctionc~l nnalvsis of transiently expressed+, mutated ClyR cDNAs in mammalian (HEK-293) cells has also shown that residues from two scprlratc domains form the r l suhunit strychnine-hind in^ site. The first domain includes thc amino acid residues GlylhO and T y r l h l , and the second domain includes the residues Lys200 2 ~ hclorz,). - and ~ y r 2 0 (scc
entry 11
-
Distinct amino acid residues critical for GlyR agonist and antagonist binding 11-29-03: Independent reports have concluded that hinding of the GlyR antagonistt strychnine requires an interaction with residues Lys200 and Tyr202 of the G l y ~ " ' with an agonist-binding site of the GlyR being located at the residue ~ h r 2 0 4 ~ "These results demonstrate that agonist- and antagonistt-binding sites are composed of distinct amino acid residues (in contrast to other studies of ligand-gated receptor-channels). Chemical modification of lysine and histidine residues of the GlyR abolishes agonist (glycine) Note: Interactions of the GIyR but not antagonist (strychnine) with agonists are likely to he mediated by hydrogen hondingt and not by ionic interactions.
Residues important for taurine activation - distinct agonist srlbsites on rr and o2 GlyRs 11-29-04: Whereas r l receptors are efficiently p t e d t by 1-alanine and taurine, z2 GlyRs show only a low relative response to these agonists, which also display a reduced sensitivity to inhibition by the glycincrgict antagonist1 and convulsantT strychnine. Construction of an z2/rl subunit chimaera1 and site-directed mutagenesist of the extracellular region of the .rl sequence has identified amino acid positions 11 1 and 212 as important determinants of taurine activationzR ( ~ e eIPDTM], Fig. I ) . These results suggest that the ligand-binding pocket is formed from discontinuous domains of their extracellular region and indicate the existence of distinct ~ .diagrammatic representation suhsites for agonists on r l and r2 C I ~ R S(A
o f predicted agonist and antagonist interactions with the G1yR is included as an inset to the [PDTM],Fix. 1.
Critical residues for a~onistefficacyt 11-29-05: Mutational exchanget of the amino acid residues Phe159 and Tyrl6l in the 2 1 (ligand-binding) subunit of the rat GlyR increases efficacyt of amino acid agonists6'. Doubly-mutated (Phc159 to Tyr, Tyrl6l to Phe) r l homomcric1 GlyRs require -0.7 mM /I-alanine for a half-maximal response when expressed in Xenopus ocytes (an affinity - 1 10-fold greater than that of the wild typet). Note: P-Alanine displayed greater potencyt than glycine in these cxperimcntsfl.
Anion selectivity o f GlyR-chnnnels depends on terminal positive charges in the M2 segment
U
11-29-06: The hydrophilict lining of the ClyR chloride channel includes eight conserved, hydroxylated side chains and a series of positively charged amino acids bordering M2. Synthetic peptides which correspond to segment M2 of the GlyR r l chain of rat (including its N- and C-terminal-adioining arginine residues) form randomly gated channels following the incorporation into lipid hilayersn". Ion selectivity of these 'synthetic channels' can be modified following 'inversion' of the terminal charges of the peptidc, suggesting anion hinding to these residues at the mouth of the channel (compare the 'rings of negatively charged residues' bordering the M2 domain of the nicotinic receptor which determine channel conductivity - sre ELG CAT nAChR, entrv 09, and ref."'l,
Conversion o f cation- to mnion-sr~?cciivcEL<: chcrnncls directed muingenesis
sitc-
17~7
11-29-07: Introduction of three amino acids 'from' the glycinc rcccptor M2 scgmcnt (or the GARAn rcccptor) 'into' thc M 2 scgmcnt of an 2, nicotinic receptor is sufficient t c ~convcrt thc cation-selective nAChR into an aninnselective channel gatctl by acctylchol~nc.T h e critical mutation in thcsc studies was thc insertion of an ~lnc-hmr,yodrcsirtuc ; ~ the t N-terminal end of M2, indicating the irnportnncc of protcin gromrtrical constraints on ion s c l ~ c t i v i ttc r~v b~S(~lrpc*!ivity ~ unricr El,<: CAT nAChR. 00-40).
A uonserverl Axi~148is irnporr(~ntfor C:lyR nsscn~hlyand inflr~ences cln t n,yonis t Ilinding 11-29-08: Site-directed ~ n u t a g c n c s i sof~ the Cl yR x suhunit coding scrlucncct predicts that nn invi1ri;unt rcsiduc (Aspl48)forms part of a receptor assembly/ antagonist-binding sitc (.WE T[il,lr 1,). This structural motif1 may play a rolc in antagonist hintling to other rcl;~tcdr c c c p t o r ~ " ~ .
Tahlc 6 . FfTcr*tco{ cclrr*tcdmlt!mtrnnc on GIIJK functlon (From 11-29-08) Mutation
Functional changes/ nl,scrvatinns
Conclusion/infcrcncc
Lys14,3Ala
Essentially unaltered ClyRs with small tlccrcnscs in strychnine affinity, glycinc displnccmcnt of strychninc hinding, ant1 glycinc activsticln of chloritlc currents"' No binding sites or ion channels were cxprcsscd on the ccll surface5'
Lys 14.3 does not play a major role in cithcr agonist or antagonist binding or agonist activation of the GlvR
Asp148Ala or
AspI48hsn
Asp148CIu (Conservative suhstitution)
Reduced efficiency of expression; a single order of magnitude decrease in strychnine affinity; no change in glycinc displnccmcnt of strychnine binding or ~ l y c i n cactivation of chloridc currcn t ~ " ~
T h c mutation disrupts the cxprcssion and/or asscmhly of the GlyR. Note the conservation of thc Asp1 48 rcsiduc ( s r r ohovr) Asp148 plays an important rolc in GIyR asseml>lyand in antagonist binding, hut n o significant role in agonist binding
Residues in M2 o f t r suhl~nitsdc~cmnincthe main conductunce stritc o I GIyR channels 11-29-09: T h c main conductance statet of GlyR x suhunit homo-oligomcrst exprrsscd in HEK-293 cclls tlcpcnds on rcsiduc 221 which is locatcd within transmcmhrnnc scgmcnt M2. T h e tnutant 2 , C.221 h ~ i v c srise t o a main
entry 1 1
statct of 107 pS, rccordcd at symmetrical C1 concentrations of 145 mM. (cf. 86 pS for wild-typet homo-o~igomcrsi)~' (see Single-channel data, 11-41).
Residues in M 2 o f ij suhunits determine picrotoxinin resistance o f ClyR 11-29-10: Sitc-dircctcd mutagcncsist has identified pore-lining residues within the sccond prcdictcd transmcmhranc segment (M2)of thc /Isuhunit as major dctcrminants of picrotoxinin re~istance''~,' (see Rcccptor antagonists, 11-51). Whcn divcrgcnt rcsiducs of the P M 2 region (which differs markedly from that of the GlyR 1 or GARAh M 2 segment) arc rcplaccd with side-chains from the rl suhunit, heteromcrict a l p channcls gcncratcd with the modified /I suhunit are inhihited hy picrotoxinin. In addition to rcduccd single-channel conductance properties upon coassemhly of the /Isuhunit" (see Single-channel data. 11-41), these results indicate that the M 2 segment is critical for hoth hinding of hlockers and ion flux through GlyR chloride channels.
Predicted protein topography Basic features o f GlyR protein assemblies 11-30-01: Amino acid compositiont analysis of intact, chemically crosslinked GlyR suhunits has shown the GlyR (i) to consist of a pentameric assemhly of mcmhrane-hound suhunits; ( i i ) to possess three copies of thc cy suhunit per complex and two copies of the p suhunit per complex (see /PIITMI, Fig 1, and paragraph 11-30-02). This quatcrnaryt stucturc rcsemhlcs that of the nicotinic acctylcholinc rcccptor (see EL(; CAT nAChR. cntry 09) and other cxtraccllular ligand-gatcrl channcls. Note: An additional 93 kDa non-glycosylatcd peripheral memhrane protein appears to influcncc channel topoRraphyi in sit11 (scBcp S~~hcrllolor lorntions. 11-16, and Protein intcmctions, 11-31).
Functional GlvR rr/il hetero-oligomers have an invariant (3:2)stoichiometry 11-30-02: Cn-expression studies in Xenoprls oocytcs havc cstahlishcd that GlyK -*//I hetero-oligomers have an invariant (3:2) stnichiometry2'. Ry contrast, variable subunit ratios can he distinguished whcn low-affinity mutants of the x2 suhunit are co-expressed with wild-typc rl and r l
Amino ocid sequence motifs governing GlyR suhunit stoichiornetryt and assemhly 11-30-03: Construction of chimaeric receptors hctwccn 2 and /lsuhunits has rcvcalcri that differences in subonit assembly ratios arc dctermincd hy the Nterminal (putatively cxtracellular) regions of tlic s~hunits'.~.Substitution of rcsiducs diverging hetwecn the x and subunits has identified a suhdomain of thc /isuhunit N-terminal region (residues 1-153) that is csscntial for stoichiometric subtinit assembly (see /1)117'hf/. Fig. 1). Compnrativc note: The - trorismcmhranc domains of thc r and p suhunits appcar unimportnnt for
Clsovwd signal peprlde sequence
-"-\
V ,aim) "-(a0
,-(a0 38)
1)
*
Strychnine (antagonist)
Glycine (agonlst)
palenine (agonist)
Taurine (agonist)
(b)
he hydrophllllc
I
Pentamerlc arrangement
-
-
-
.",
(c) Ollgomerlc structure tncludlnn the B3 r 0 cyloplasmicaseoci*tsd protein g*pnyrln
Iunct~ona f # c l d
KEY
Y - Approa1mwis p011tlonof conr*nsua .,@' -
-
Figure I.
for rstarsnces, rat me Protein phosphorylsllon t l s l d )
M-gtycmytmt~onsit* (rctrul poalllon D* 38 - ~ c cSeqwnco motifs). Pmlllon oi con8wouo on0 lor protoin klnau C (aciurl pmltlon DD 3251 Scharnrllc 'ahmms' ot aponint sna nmngonlst rnoleculea rscogniud by the Intact GlyR 1.w Dom*ln tunclions)
,
See ~ I L Oseetlen on 'Invnrlant
1
3 n : 2p stolchiometry' under Prsdkled prolein lopogmphy-
NOTE: All R / u ~ / position# v~ of motlls. domain shapes and sizes are diagrammcrtic and are subbct to re-interpretation.
Chonnml symbol
: j
Monomeric proteln domoin iopogrophv model /PDTM) for the pkcine receptor-chanel /I=lvRIsubunit n 1 . /From 11-30-01)
entry 11
-
correct suhunit stoichiometryf. On the hasis of agonistt dosc-responsechanges with comhinations of GlyR carrying mutations in various 'assembly boxes', it was deduced that the properties of stoichiometrict assembly and homo-oligomert formation were 'mutually exclusive'. These findings may imply that the 'assembly domains' of the /i' suhunit have evolved to prevent homo-oligomerization, and that this may he essential for the 'sequential assembly' of functional ClyR (i.e. m / P + z / p + 2 r.3/r2)75 .
-
,
Arg219 is essential for correct assembly and biogenesis o f n homomeric GlyRs 11-30-04: Site-directed mutagenesist of Arg219 at the cytoplasmic terminus of domain M2 in the GlyR a l suhunit generated homomeric proteins which were only c ~ r e - ~ l ~ c o s ~ l a tThese e d t . mutant regions were retained within intracellular compartments, and aggregated to high molecular weight complexes7? Notc: Arg219 corresponds to an argininc/lysine conserved in other ELG superfamily receptor-channels.
Cross-linked pentameric assemblies o f GlyR 11-30-05: Chemically cross-linked1 glycine receptor subunits have a molecular weight consistent with the assembled receptor-channel complex heing composed of five s ~ h u n i t s The ~ ~ . principal molecular features of the cloned cDNAs for the GI~R"'.~'are illustrated on the channel protein - domain topography model [PIITMI (Fig. I).
-
Protein interactions Receptor-associated proteins can 'c~ptimize'functional properties o f GlyR-channels 11-31-01: Co-expression of the GlyR with the tubulin-linker pmtein gephyrin (see Suhcellulnr locntions. 11-16) changes the agonistt- and antagonist+hinding affinities of GlyRs generated hy r2 suhunit expression in HEK-293 kidney This has hcen interpreted as contributing to an 'optimization' e ~ ~9.3. kDa component of of the post-synaptic neurotransmitter r c ~ ~ o n sThe the native GlyR purified hy affinity chromatography with 2-aminostrychnine prohahly represents the GlyR linked to tuhulin via gcphyrin (see Subcellular locations, 11-16). The GlyR purified without gcphyrin is functional when reconstituted in lipid v e s i c l ~ s ~ ~ ~ .
"
Co-assembly of G1yR n and fl suhunits - dependence on the type of expression system 11-31-02: Although assemhly of GlyR r and /Isubunits is inferred from their co-purification during affinityt chromatography and irnmunoprecipitationt, transient co-expression of a with p suhunits in Xcno us oocytes produces no significant nlterations in agonist-induced gating , hut channels have relatively low agonist affinity3'. Srullle co-expression of r with 11 suhunits in HEK-293 cells results in large whole-cell currents with altered (lowered) picrotoxinin sensitivity when compared to r suhunit homomultimerst [see Domain functions. 11-29. and Receptor anta,yonists. 11-51).
f
entry 1 1
Putmtivc intrrr~c-tionso f ,.Isrll~rlnitswith other ELG chrlnnel suhunits 11-31-03: T h e ubiquitous expression of fl suhunits in hrain (scc rnRNA distrihrrtion. I I-1,7I has s ~ ~ ~ g c s t cthc t i possihlc cxistcncc of native chirnaerici receptors ct~ntnining/I suhunit components (c.g. as part of C;ARAAR, putative taurinclll-alaninc receptors and NMIIARI. Thcsc proposals have hccn tlisci~ssctl~'~~''.
Protein phosphorylation Altcrnotivc vplicin,q o f r phosphorj~lntion site
r ~trrznsc-riptscr~ninc*lutlcor
c~xclude(1 novcl
11-32-01: Sctlucncc analysis of altcrnativc splice+ variants from the ClyR r gene show variation in the prcscncc or ahscncc of a novel consensus phosphorylation site7" (the TI,,,,variant containing an eight amino acid insert in the M.3-M4 intraccllular loop, close to M.3 (srr PDTM]. Fig. I ) . This feature raises the possibility that alternative splicing can contrihute to functional diversity of thr GlyR through regulating susccptihility to protein kinases. Con~l~rlrcitivrnotr: An ccluivalcnt eight amino acid sequence insertion (LLRMFSFK) has hcen identified for the ;.? suhunit of the C.ARAA receptor which has hccn shown t o confer an in vitro suhstratc for protcin kinasc c'" (S(T I'mtrin phosphor!~Icltiot~~ ~ n d Er Lr( : C1 (;ADAA.
f
i
10-32),
'Clusrc?rin,y'o f consrnsrls phosphorvlr~tionsites in t hc putative M.3M 4 loop 11-32-02: Potenti;ll phosphorylation-mod~latorysites exist in the hydrophilic loop separating tmns~nrlnhrancscgmcnts M,? and M4 in GlyR monomers (scr /I'I)TM]. Fix. I ) . No!ra: T h e M.3-M4 loop regions w o t ~ l dhe cxpcctcd to provide inclst c ~ thc f cvtc~pl;~sniic mass of the intact C;lyR complex.
In vitro pho.~pf?orvlation o f GlyR suhunits 11-32-03: Thc GlyR T sllhi~nit can he phosphorylatcd in vitro hy protein kinase C (PKCJ at the Scr.391 residue close to thc fourth transmcmhranc domainH1.T h e same suhunit is a substrate for phosphorylation hy protein kinasc A (PKA) at an undetermined residueR'. Incuhation of intact rat spinal cord ncuroncs wirh specific PKC or PKA activators leads t o increased phosphorylation of the GlyR 2 suhunits, strongly sumcsting a physiological role tor this modification. T h e treatment of oocytcs expressing GlyRs from poolcri rat hrain poly(Alt mRNA with phorhol glycinc-cvokcd currents whereas treatment with dihutyryl cstcrsi dc~crc~nscs CAMP rnhc1nr.r.s glycine-evoked c ~ l n c n t s ' ~ .
I'rot f i n kinase A phnxphorvla t ion increases GlvR currcn ts in mrdullriry riorsal horn nPuronPs 11-32-04: Protein kinase A phosphorylation via a cholera toxin-sensitive C. prc~tcin (G,l tlrnmatically increases glycinc-induced CI currents through the <;lyK of spinal trigcminal ncurones hy increasing the channel I:,,,.,,".
cc~~cm
)rmxmrg~Ek(c~
Current-voltage relation Rectification properties o f whole-cell GlyR channel currents 11-35-01: A pronounced outward rectificationt is observed when ClyR channels conduct in the steady statel. This rectificationt is likely to reflect a voltage-dependencet of g?t,inf, not a voltage-drpencicnce of ion pcrmcationt . The open probah~l~ty(Po,,) depends upon the charge on the cytoplasmic face of the membrane {when it is negatively charged, I',,,,, will he low). Upon depolarization of the neuronal mcmhranc, thc cytoplasmic face will become more positive and P,,,,, of the GlyR will increase ity, (opposing the depolarizing stimulus). (See also Voltage . ~ ~ n . ~ i t i v12-42).
Selectivity GlyR-channels share many ionic selectivity characteristics with GANAAR-channels 11-40-01: Many comparative studies suggest that both glycine and GARAA agonist-activated channels (see EI,G Cl C:ARAA, Pntry 10) act as multi-ion pathways and have similar pcrmcation~ characteristicsR4. A systematic comparison of the selcctivityt mechanisms of GlyR ant1 GARAAR in mouse spinal cord neurones has appeared12 - some of the data applicahlc to GlyR ion permeation properties arc summarized in Tahlc 7.
Estimated open-pore diameters o f GlyR 11-40-02: A pentameric arrangement of a-helices has been predicted to form a central bore of -0.58 nm in diameter, a value which closely matches cstimatetl effective pare diameters using hi-ionic reversal potcntialt measurements [comp;irc 0.52 nm and 56 nm for the open ClyR and GARAhR channels r c ~ ~ e c t i v c l ~The ] ' ~stn~cturc-function . relationshipst for permeation pathways7 in a rangc of ncurtitransmittcr-gated ion channels has hecn rev~ewcd'~.For critical amino acid residues in contrnlling ionic - selectivity, see Domain fi~nctions.11-29,
Single-channel data Characteristic 'bursting' and subconductance hehaviour of native GlyRs 11-41-01: Glycine elicits 'bursts't of single-channcl openings displaying multiple subconductancet states in cultured ~ e l l s ' ~ ~ ' ~,~native * * " ~ spinal " ncuroncs'' and in cells expressing rccomhinantt GIyR {ser Fix. 2). In the falling phase of glycincrgict inhihitory synaptic currents, single-channel currents can he resolved as discrete steps. In cell-attached+ patches, the single-channel slope conductanccs [close to 0 mV memhrane potential) arc 29, 18 and 1 0 p ~ ' In ~ .outside-out1 patches [with equa! extraccllular and intracellular concentrations of 145 mM Cl 1, conductance states of 46, 30, 20 and 12 pS are observed, with the most frctlucntly clccurring suhstatet being 46 PS". (Srr III.FOSelecfivitv. 1 1 -40.1
entry 1 1
Table 7. Similnrity n / .~r!cclcfivitychmracteri.~tric.{or inhihitory GARA* ~ n d ~1yc.incrrccptor-chrrnnc1.r (From I 1-40-01) Ion spccics
Selectivity characteristics
GlyR anrl GARAAR, The E,,, of whole-cell currents shifts 56 mV per tcnfold chloride ion change in internal CI activity, indicating activation of currents CI -sclcctivc channels GlyR and GARAAR, The pcrnmability ratio of K' to CI selectivity over K ' ions
(J)K/J)c'I!
is (0.05
GlyR and GARAAR, SCN > I > Rr > CI > F small anion rclativc permeability scrics GlyR and GARAAR, small anion rclativc single-channel conductance scrics
Single-channel conductanccst measured in equal 140 mM cnncentrations of small anions on both membrane faces rcveal a conductance sequencet of CI > Rr > I =. SCN r F for both anion channcls. Compnrmtivc note: A near-invcrsc permeability sequencet (FCC (l1,oveJindicates the presence of hinding sites for thcsc ions in the channels
GlyR, polyatomic anion pcrmcahility
Phosphate and propinnate ions arc not measurably permcant in GlyK-channels although they support small currents through C.ARAAR-channels. The pcrmcahilitv sequence for largc polyatomic anions through GlyR is formatc > hicarhonate > acetate
Shifts i n single GlyR-chnnnel 17ehnviot1r with incretising rigonist concentration 13-41-02: GlyRs in cultllrcrl mouse spinal cord ncuroncs display increases in total current, single-channel opening frequency and longer opcn times' with increasing an,nistt cnnccntration (0.5-2.0 p ~ ) Thesc . changes are due primarily to shifts in relative occurrence of opening frequencies from the shortest opcn statcst tn two long open states1".
u
Conductance valrles for homo-oligomeric and hetero-oligomeric ClyR-channels in H E K cells 11-41-03: When recorded at symmetrical C1 concentrations of 145 mM in HEK-293 cells, T I , .rz and r l homo-oligomerst display main-statet singlechannel conductanccst of 86, 11 1 and 105 pS, respectively". ~ a i n - s t a t c t conductances of hctcro-r,ligomcrst show reductions to 44 pS (xl/P), 54 pS [ r 2 / / i \and 48 pS (.*,,/PI. Note: Since the lower values arc similar to those found in spinal ncuroncs, this has bucn taken as evidence for nativc GlyRs c x i s t i n ~as r/p hetero-oligomers. Co-expression of native I, with mutant /{ subunits shows that rusi[l~lcs within (and close to1 segment M 2 of the P
cntry 1 1
-
subunit determine the conductance differences between homo- and hcterooligomers7' (see Domain fi~nctions.11 -29).
Developmental changes in open-time kinetics of single-channel G1yR currents 11-41-04: In rat spinal cord, a2 subunit expression is predominant in foctal or ncwhom tissues, and developmental switching from rrrz to z, occurs within d weeks following 13irth.l.7*24-2(1 (see Devciopmcntnl regrrlatian. I I I I). In direct comparisons, mcan channcl opcn timcst of GlyR r l and mature nativct glycinc rcccptors (rat spinal cord) arc cqually short, whereas both the recombinant 22 and [native] foetal rcccptors show significantly longer opcn timest''. GlyRs in native spinal cord ncuroncs show a striking c o n t i n t ~ o ~fall ~ s in mcan opcn timet following birth (typically from 40-50 m s at birth to 2-10 m s at agc 20 days)*'. Consistcnt with thcsc results, thc dccay timc of thc glycincrgict inhihitory postsynaptic cuncntst (IPSCs) in spinal ncuroncs hccomes shortcr during post-natal dcvelopmcnt.
Comparison of single-channel ct~nductancelevels for typic01pre- and post-natal GIyR components
-
11-41-05: In outside-outf patches from Xenopus nocytes expressing GlyR at or a? subunit C R N A S ~ ,application of glycine induces single-channel currents exhibiting multiple amplitudes2' (see Fix. 2a nnd c). Ity plotting single-channel amplitude histogramst for currcnts supported by homomcric'i al rccuptors and homomerict a r . ~ receptors, distinct singlechannel (peak)conductancesf can hc rusolvcd a t 75, 59, 4.1 and 25 pS for a l rcccptors (Fig. 21)) and 88, 72, 42 and 24 pS for a2 rcccptors (Fig. 2d), With thc exception of the 88 pS lcvel (ohscrvcd only for thc 'foetal' 9,. rcccptor) thc condl~ctnncelcvcls for channcls supportcd hy thcsc n l or z2 homomultimcrst arc broadly similar. Analysis c ~ glycinc-gated f channcl currents of nativet GlyRs in rat spinal ncuroncs at [octal stage (E20) and post-natal stage (P16) also show broadly similar conductance lcvcls except for a 94 pS level which is obscrvcd only in the foetal neumncs2'. Mcthodolo~icalnotr: Ity fitting for Gaussian curvcst using the Icast-squares rncthodt, singlcchannel conductancest can he estimated from the peak amplitudct of the Gaussian curvct.
Voltage sensitivity Gating processes o f GlyR-channels are voltage-sensitive 11-42-01: Current-voltage (I-V) relationst of transmitter-activated currents obtained from whole-cell mcasurcmcnts in mousc spinal cord ncurones, show outward rectification [with 145 r n M Cl intracellularly and extmcellularlyl1". In voltage-iumpt cxpcrirnents, thc instantancoust I-V relations are linear, and thc steady-statct I-V rclations arc outwardly rcctifyingt, indicating that the gating of GlyR channels is voltage-sensitive (sr~crllso Current-voltage relrrtiorl, 1 1 -.351.
I c r , -subunit cRNA
in oocytes
nr-subunit cRNA in oocytes t)
,
*I"-<,
-
I
- - --
-
I
1 it,-subunit
cRNA in oocytes
1
u,-subunit cRNA in oocytes
-T
Figure 2. Cornprrrisorl of \rr~,ylr~-c.hrnnnt.l c.on(fl~c.t(inc.c> 1cvc.lc {or t1pic.01 p r r r*on?ln)ncnt\ lNi1prodoccrl 1z1tJ1 pcrrnr\\ion from rirlrl post-nr~l(il <:hi-? Trrlrrillr151111.1 (11 /I9021 Ncuron 9 1155-61 I. (From 11-41-05)
Blockers I'icrotoxinin scnsitivitv i s g r c ~ ~ t rcduc-cd ly in hctcro-oli,qomcric Glylis 11-43-01: Inhihition of I homo-oligomcrict C.lyRs by picrotoxinin (a noncompetitive hlockcr of ion flow, and sornctirncs dcscrihcd ss 'sclcctivc' for C.hRAA chloride channcls) is rcduccd 50- t o 2 0 0 - h ~ l d for rill hctcrno ~ i ~ o r n c r ireceptors c~ gcncratcrl hy co-tmnsfcction7* . Notc: For molecular dctcrminnnts of picrotoxinin resistance on /I suhunits, scc llornliin fi1nc.r ion\. 1 1-29,
Channel modulation lnlpartanc*r ol pho~phomodultrtion for C;IyR-chcrnncl f~rnctron 11-44-01: In conitnon w ~ t h othcr cxtr:~ccllulnr Iignnil-gntcrl rcccptor-
-
channels, the GlyR contains a large (putatively) intraccllular loop that contains a number of consensus recognition sites for protcin kinascs (see Protein phosphorylatron, 11-.12). Extrace!lular signals rcleascd from the prcsynaptic neumne, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein ( ~ c cnotc helow], arc known to r e p l a t e several functtonal characteristrcs of ligand-gated ion channels hy phosphorylation (see r e v i e d 7 nnd E L G Key iocts, entry 04). Note: Coexprcssiont of the GlyR/tuhulin-lrnker protein gephyrin wl th the GlyR influences agonist- and antagonrst-binding affinities (.rep I'rotern ~nteractions, 11-31).
Penicillin G-induced potentiation of Ic[, 11-44-02: Penicillin G (PenGJ has bccn ohservcd to iatet GlyRmediated chlondc current in rat vcntro-medtal hypothalamic neuroncsRR. Whcn PcnG is applied simultaneously with ~ l y c i n e(Gly), PcnG dcprcsses IGl, like a C 1 channel hlocker (from -30 units/lO ml to a maxlmum hlockadc a t 600 unitslml). When test solutions containing PcnG plus Gly are raptdly substituted with one containing glycine alone, a new 'rcboundllkc transient current' (IT) is ohserved which passes through the Cl channcl. The peak amplitudet of IT induced hy PcnG (>lo0 un~ts/lOm!) is grcater than that induced by glycine alone {i.e. PenG-induccd potentiation of lc;rylRR.
I
Equilibrium dissociation constant
Antagonist affinities 11-45-01: The 'high-affinity' hindiny: of [3~1-strychnineantagonistst to membranes of rat spinal cord has a typical Kd in the range of 2-12 n ~ . ' * ~ + ' ~ Thts hindtng is displaced hy thc aRonl.;tst glycine, p-alanine, taudne and Paminoisahutyric acid (see Domitin funct~onq,11-29].
Differential ngonist nffinities of cloned subunits
11-45-02: Diflercnt GlyR a subunit isnforms (e.g. al, ax, zz', a,%)form rcccptors with distinct agonist affinities, allowlng their discriminat~on by doscrcsponsc crltcria 'm''H*2h211.
Hill coefficient Co-operativity in agonist-ind~lcedgating
11-46-01: nl subunit R N A or poly(A)+RNA from hrain or spinal cord injected into oocy tcs supports the expression of GlyR which display Hill cocfficicntsi of n = 2.5-3.3 ( c . ~ .ref.2R), suggest in^ that a mrnirnurn of thrcc glyclnc molcculcs are required to open thc channel. High co-operativity may indicate a mechanism tor gattngt withln a narrow cxtraccllular aRnnistt concentration range.
Dependence o f Hill coefficient on receptor suhlrnit stoichiornerry 11-46-02: Dose-response curvcst of whole-cel~tcurrents in MEK-2'13 cells cxprcsslng rccomhlnant nl suhunits drsplay an avcraRc Hill cocfficicntt of
entry I I
4.2. Co-expression of GlyR .xl and P suhunit cDNAs in HEK-293 cells markedly increases glycine-gatcd wholc-cell currents7' and alters the mean Hill coefficient to 2.5 {see Single-channel data, 11-41).
Ligands 11-47-01: The principal radioligandt used for GlyR binding assays is I 3 ~ J strychnine, which is antagonizetl hy (in order of relative potency) glycine > p-alanine > taurine =1, L-alanine, L-serine > proline. Cross-linking' of [' H Istrychnine to nativc and hctcrologouslyT expressed GlvRs have determined the ligand-binding site to he located on r suhunits hut not P suhunits (for dctnilr, scc I>omnin Cr~nctions,11-29).
Mechanism o f ligand discriminntion 11-47-02: Gamma-aminohutyric acid (GABA) and mserine, amino acids that do not activate wild-typc r l homomeric glycine receptors, efficiently p t e t a mutantt glycinc receptor-channel (a FlSYY, Yl(i1F amino acid exchange of the GlyR r , suhunit demonstrating that aromatic hydroxyl groups are c n ~ c i a lfor ligand discrimination at inhibitory amino acid receptor^"^ (see also Domnin irinctic>z~s. 11-20).
Receptor agonists (selective) The GlyK is activated hv n range o f n and , I amino acig.9 11-50-01: In general, potcncyi of GlyR activation hy ajionistsi decreases in the order glycine > p-alanine > taurine L-alanine, L-serine > proline"''. In rat brain slice suhstantia nigra ncurones, taurine acts at the same recognition sitc as glycinc on the ~ ! ~ ~ - c h a n n c l ' ~ .
Comparntivc note channels
- glycine
is olso a co-agonist for NMDA receptor-
11-50-02: Glycinc or a glvcinc-like m o l c c ~ l eis an important modulator for thc NMDA rcccptor-channel (.we ELG CAT GLU NMDA. entry 08). ~cscnsitizationt of NMDA receptor-mediated currents elicited hy glutamate in newly exciscdt outside-out patches is reduced in the presence of sc~turt~tin,y concrntrations of glycine, prohahly due to the allosteric coupling of the glutamate- and glycine-hinding sitesP3 (see Receptor ci,ynnict.s under ELC; CAT GLU NMDA, 08-50).
Functional note
- ~ I y c i n eis o structurallv simple molecrzle 11-50-03: The agonists for inhibitory receptor-channels [glycine and GABAI arc of relatively simple structure (see [PIITMI. FIR. 1) and in consequence,
-
the numhcr ot spccitic interactions they can have with their respective receptors is likcly to he limited"". This factor is of significance in determination of structure-function relationshipst of the GlyR, as removal of any one d these specific interactions can he expected to result in dramatic reductions in agonist affinityh5.
entry 11
Receptor antagonists (selective) The plant alkaloid strychnine is n well-characterized, high-nffinity antagonist for adult GlyRs 11-51-01: The GlyR can he identified hy the convulsant strychnine', which hinds at a site distinct from glycine65 (see Domain functions, 1 1 -29). In strychnine poisoning, normal glycinergict inhibition is abolished, giving risc to muscular convulsions by 'overexcitation' of the motor system. Strychnine can he used to distinwish the GARAA receptor from glycine receptor C 1 channels [comparc pAz values against muscimol and ~ l y c i n e of 5.3 and 6.0 {neonatal)or 8.0 (adult] respcctivcly). Note: Some antagonistic action of strychnine has heen reported a t the GABAA receptorv'.
Database listings/primary sequence discussion 11-53-01: The relevant database 1s indrcated b y the lower case prefix [e.g.gb:), which should not be typed (see lntroduct~ond layout o f entries, entry 02). Database locus names and accessron numbers immediateIy follow the colon. Note thnt a comprehensive listing of all available accessron numbers is superfluous for Iocat~ono f relevant sequences m GenRank" resources, whrch are now available with powerful in-built neighbonr~ngt analysis routines (for description, see the Database list~ngsfield in the introduction el layout o f entries, entry 02). For example, sequences o f cross-species variants or related gene familyt members con he readily accessed by one or t w o rounds o f neighbouringt analysi~(which are based on pre-computed alignments performed using the R L A S T ~algorithm by the N C R ~ ~Thi.7 ). feature rs mo7t useful for r ~ t r ~ e v aolf wqnence entries depo.sited in databases Inter than those listed helow. Thus, representntive m e m hers o f known sequence homology groupings are listed to permit initial direct retrieval^ h y accession nrrmher. nuthorlre ference or nomenclnture. Following djrect accession, however, nergh houring analysis is t strongly rccontmendcd to idcntr{y newly-reported and related sequences.
Nomenclature
Species, cDNA Original source isolate
Human 421 aa Glycine receptor alpha-1 (principal adult 148 kDa chain precursor GlyR in spinal protein] cord); Glycine ligand-binding subunit
Accession
Sequence/ discussion
gh: X52009 ch: YO0276 gh: Mh.3915 pir: A27141 pir: JN0014 prosite: PS0023h sp: PO7727
Grcnningloh, Nntrrre (1987) 328: 2 15-20. Sonthcimcr, Neuron (1989) 2: 1491-7.
Nomenclature
Spccics, cDNA O r i ~ i n a l s~)urcc istilatc
Accession
Scqucnccl discussion Ruiz-GOmcz, tlrocht.mi.stry ( t 990) 29: 70.7,140. Malnsio, J
R~olChem (19911266: 2048-5.3.
Lnngosch,
Fur I Hiochrm (1990)194: 18. Glyc~nc Human receptor alpha-1 chain prccursc~r
44'1 aa
gh: X5200X sp: P23415 pir: S12,782 mim: 138491 pro~itc: PS002.36
Grcnningloh, EMIT0 (199019 : 771h.
Glycinc Rat rcccptor alpha-I cham precursor
449 aa
gh:n008.73
Akagi, Ncum.sur Kcs (1991)11: 2X-
40.
Glvcinc Rnt rcccptor alpha-1 chain prccur.ior
Glyc~nc receptor alpha-2 chain precursor
457 a;]
gh: 5524h
H u ~ n n n'foctrti. 452 nn gh. X52008 nu ~ t r r ~ u h n ~ nGrly - at an 167 sp: F2.3416 rrncitlvc GlvR' (cf. Glu 167 In MIM: 305990 0 1- 1 prtislte: I'S00231,
GI ycinc Rnt rcccptor alpha2A
452 aa
gh:Xh I I SY
Kuhsc, Malos~o, Ilctz, Unpuhttshcd (1990).
Grenningloh, FMRO 1 (f990)9: 7716.
Kuhsc, FERS Let; (1991) 283: 73-7.
cntry 1 1
-
Nomenclature Species, cDNA Original source isolate
Accession
Scqucnce/ d~scussion
Glycine rcccptor alpha2' chain precursor
gh: X57281 em: X57281 PIR: S14816 sp: P22771 prosite: PS00236
Akagi, FEES Lett (1991) 281: 1 6 0 4 . Kuhsc,
Rat, Wistar, 10 day old (principal neonatal GlyR in spinal cord); Glycine ligandhrnding suhun~t;'foetal, nM strychnineinscnsitivc GlyR'
452 aa (48 kDa protein) Glu at aa I67 (cf. Gly I67 in nl)
Glycine Rat [principal 464 aa receptor alpha3 adult cerebellar (48 kDa chain precursor isaformj protcin)
gh: M55250 gh: M38385 pir: A23682 sp: P24.524
Glycinc Mouse receptor alpha4 gcnomic gcnc
not found
not found
Net~ron ( 1990)5: 86773. Kuhsc, FERS Lett (1991) 283: 73-7.
Kuhsc, (1990)
1 Riol Chem 265: 223 1720 Matzenhach,
1 Rio1 Chem ( 1994) 269:
2607-1 2. Glycine Rat receptor beta chain precursor
496 aa (58 kDa protein)
Glycine receptor peripheral membrane component
193 kDa not found protein, nonglycosylated, cytoplasmic locatton)
Co-purifies with the GlyR on affinity columns'"; gephyrln
pir: JHOl65 PROSITE: PS00L36 sp: P20781
Grcnningloh, (1990) Neuron 4: 963-70 Prior, Neuron ( I 992) 8: 1161-70.
A
n Related sources & reviews
U
11-56-01: Major quoted sources - GlyR structure-functi~n~~~~~-~'; general reviews5; ncurotransmittcr action and opening of extracellular ligand-gated ion channcls"; phosphorylation of ELG channcls, including the G ~ ~ R ~ ' ; permeation pathways of ncurotransmitter-gated ion channclsR'.
Feedback Error-corrections, enhancement and extensions 11-57-01: Plcasc notify spcci~icerrors, omissions, updates and comments on this cntry hy contrihuting to its e-mail feedback file (for detoils. see Resource 1, Search Criteria d CSN Dcvelopm~nt).For this entry, send c-
-
mail messages To: [email protected], indicating the appropriate paragraph by entering its six-figure index number (xx-yy-zz or other identifier) into the Subject: field of the message 1c.g. Suhiect: 08-50-07). Please feedhack on only one specified paragraph or figure per message, normally hy sending a c o r-r.e c.t e d ~ a c e m e n taccording to thc guidclincs in Feedhack el CSN Accrss . Enhanccmcnts and extensions can also he suggested by this route (ihid.]. Notifiud changes will hc indexed via 'hotlinks' from thc CSN 'Home' page (http://www.le.ac.uk/csn/l from mid- 1996.
Entry support groups nnd e-mail newsletters 11-57-02: Authors wh(1 have cxpertisc in one or morc ficlds of this cntry (and are willing to provide editorial or othcr support for developing its contents) can join its support group: ln this casc, send a message To: CSN1 [email protected], (entering the words "support group" in the Suhiect: field]. In the message, please indicate principal interests (see fieldname criteria in the lnrroduct ion tor coverage) together with any relevant http://www site links (cstahlishcd or prnpnsed) and details of any other possihle contrihutions. In duc course, support group memhcrs will (optionally) receive e-mail newsletters intended to co-ordinate and develop thc prcscnt (tcxt-hascd) entry/ficlJnamc frameworks into a 'library' of interlinked resourccs covering ion channel si~nalling. Other (more general) information of interest to entry contributors may also he sent to the ahove address for eroun distribution and fcctIback.
Young, Proc Natl Actid Sci IJSA (1973) 70: 2832-6.
' Young, Mol Pharmacol(1974) 10: 790-809. 3
Kuhsc, Neuron (1990) 5: 867-73. Fatimashad, Proc R Snc Lond /Rioll[ 1992) 250: 99-105. "angosch, Eur J Riochem (1990) 194: 1-8. ' Kuhsc, FERS Lett (1991) 283: 73-7. Malosio, EMRO {(I9913 10: 2401-9. Kuhse, I Riol C h ~ m( 1 990) 265: 22.31 7-20. ' Matzcnhach, J Riol Chem ( 1994) 269: 2607-12. '" Schmitt, Riocherni~trv(19871 26: H05-11. l1 Prior, Neuror~( 1992) 8: 1 l h 1-70, l2 Rormann, 1 Plzysiol Lond (1987) 385: 24.3-86. Hamill, Nr~ture( 19833 305: 805-X. 14 Takahashi, Neumn (1991) 7: 965-9. 15 Twyman, 1 I'hysiol Lond (1991)435: 303-31. lh Cohcn, J Physiol Lond [ 1YKY) 418: 53-82, " Suzuki, 1 Phpsiol Lond (1990)421: 6 4 5 4 2 . l R Korn, Proc Nr~tlAL'IIII' Sci IlSA ( 1992) 89: 440-3. l V Miller, 1,ilc Sci (1993)53: 121 1-15. Kirsch, J, Him1 Chcm (1991) 266: 22242-5. '' Pfeiffcr, 1 Riol Chcm (1982) 257: 9389-93.
'
'"
entry 1 1
" Langosch, Proc Natl Acad Sci U S A (1988)85: 7394-8. Garcia-Calvo, Riochemistry ( 1 989) 28: 6405-9. Recker, EMRO I(1988)7: 3717-26. 2s Akagi, Science (1988)242: 270-3. Akagi, FERS Lett (1991)281: 160-6. " Takahashi, Neuron ( 1 992) 9: 115541. " Schmicden, EMRO 1 (1992) 11: 2 0 2 5 3 2 . 29 Falck, Arch Ges Physiol (1884)34: 375-81. Reckcr, Neuron (1992)8: 283-9. Crcnningloh, Neuron (1990)4: 963-70. .3," Malosio, E M R O I ( l Y 9 1 ) 10: 2401-9. k t = , Trends Neurosci (1991)14: 4 5 8 4 1 . White, Nature (1982)298: 655-7. Hayashi, Ann Neurol(1981) 9: 2 9 2 4 . Hall, Neurology (1979)29: 262-7. Gnndlach, FASER 1 ( 1 990) 4: 2761-6. Reckcr, FASER 1 (1990)4: 2767-74. Heller, Rrain Res (19821 234 299-308. " White, Nature (1985)298: 655-657. " Riscne, 1 Physiol (1986)379: 275-292. Riscnc, Rr Phormacol(1982)75: L3-5. 43 R~SCOC,1 Physiol (1980)379: 275-92. Sacnz-Lopc, Ann Neurol(1984) 15: 36-41, '"cllcr, Rrain Res (1982)234: 299408. 46 Harding, J Neurol Neurosurg Psych 11981) 44: 871-83. Pfeiffer, Proc Natl Acad Sci U S A (1984)81: 7224-7. 4' Trillcr, 1 Cell Riol (1985)101: 683-8. 49 Akagi, Proc Natl Acnd Sci U S A (1989)86: 810.1-7. Eichcr, 1 Hered (1980)71: 315-18. Whitc, J Neurogenet ( 1 987) 4: 253-8. " Graham, Riochemistry (1985)24: 990-4. " ~ a s c i o1, Riol C h e m (1993)268: 2213542. "4 Vandcnhcrg, Mo1 Pharmacol(1993) 44: 198-203. k t z , Neuron (1990)5: 383-92. Grcnningloh, Nnture (1987)330: 25-6. Numa, Harvey Lect (1989)83: 12145. Nnda, Nature (1983)302: 528-32. Rarnard, Trends Riochcm Sci (1992)17: 368-74. * Snntheirner, Neuron (1989)2: 1491-7. Graham, Eur 1 Riochem (1983)131: 519-25. Crcnningloh, Nnture (1987)328: 2 15-20. 63 Ruiz-G6mcz, Biochemistry ( 1 9901 29: 7033-40. Vandcnherg, Proc Nntl Acad Sci U S A (1992)89: 1765-9. " Vandcnhcrg, Neuron (1992)9: 491-6. Ruiz-COmcz, Riochem Hiophys Rcs C o m m u n (1989) 160: 374-81. "'Schmicdcn, Science (199.3)262: 256-8. "'Langosch, Riochim Riophys Acta (1991)1063: 36-44. "'Imato, Nature (1988)335: 645-8. " Galzi, Nature ( 1 992) 359: 500-5. " 24
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Prihilla, EMHO / (19941 13: 14c1,Z. Kuhsc, Ncrlron (I99.1l 11: 104C)-56. 7,5 Rercndes, S C ~ F ~( 1T093) C 262: 427-.30. Langosch, FEHS Lctt (199.3)336: 540-4. 77 Grcnningloh, N(ltr1rr ( 1987) 328: 2 15-20. 7R Tnkagi, FERS I.rtf ( 1992) 303: 178-80. " Malosir), / Iliol Chpm ( I991 1 266: 2048-5.7. "" Whiting, I'mc N(ltl Actrd Sci IlSA ( 1990) 87: 9966-70. " Ruiz-GOmcz, 1 R i d Chr'nl (l')')l) 200: 559-66. Vnull(~,/ Hiol Chcni ( 1 994) 269: 2002-H. S m g , N(,turc (1WOl 348: 242-5. Fatimashad, I'roc R Soc Lond /lliol/ (199t31253: 69-75, RS Lcstcr, Annrr IIcv fliophys Iliomol Struc (1992) 21: 267-92. R" Smith, 1 Mcrt~hrHiol IIYX9) 108: 45-52. R7 Swopc, FASEIZ /(1901,)6: 2514-2%3. Tokutomi, Hr I'horr71ncol (19921 106: 73-8. " Rcckcr, Ntutrmchrm Irlf (1988)13: 1.37-46. * Shirnsnki, Ifrcrtn Kc.; (11)l)l) 561: 774.1. 'I Hcnzi, Mill IJh~lrrni~c.r)l (19921 41: 79.3-X01. H~LISSCT, Rr~rinRcs (1992) 571: 10.3-8. 9.7 Lcstcr, / Nr~rlrosl-i(199%1) 13: 1088-96. " Hctz, (1 Rev I
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FEEDBACK & CSN ACCESS
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Entry 12
Consolidation of contents 12-01-01: This crlition c ~ Thr f Ion Chnnnr1.s F(~ct,sRookhas hecn compilcd hy 'scanning' thc litcmturc up to thc end-of-year 199,1 (for vol. I), to near the cnci-of-year 1994 (for v t ~ l2 ) and to mid-1'$95 for vols 3 and 4. Indexed information supplcmcntary to the cntrics (i.c. appcying aftcr the publication dcadlincs) will I,c acccssihlc ovcr the Interneti using a World Wirlc ~ c h tbrowser from mid-1996 (rcc hclow). This arrangcmcnt facilitates a mechanism for hoth error correction and consolidation of thc information sct. To hcgin with, incrcnicntal changes to cntrics will he loggcct on a host computcr within the C M H T [Lciccstcr, UK - see c:ornprIter ncidre.ss h e h w ) hut other sites will contrihutc to the proicct in due course. Each entry or rcsnurcc is supported hy a unique c-mail fccdhack filc. The fccdhack format is intended to providc an 'open' and informal mechanism for entry valid~tionhy specialists on each subtopic. ~ h neth c hod also prr~vidcsan cfficicnt means to communicate changes and extensions to cntrics, and ciffcr a forum for dchatc. Most of all, 'communities' linked hy the core interest in the cntrics can contrihutc to thc evolution of the datasct as a comprehensive 'informatic tool' hy structurcrl dcvclopmcnt of thc ficld contents ( X C P N ~ r o t ~ t /r '-~S~arc:hcsJ crit rvrirr PJ C S N rlovc~lol?rn(~nt. (lntrv 05). ~-
-
n The need for feedhack 12-01-02: An ohicctivc of Thr, Firt.rsllook is that the sccipc ;ind arr;ingcmcnt of the information should hc r.on.~!mntlyr ~ f i n r dt o contclln whmt is n70,vt u ~ ~ f u l clnr? nrlthr~ritrrtivc- fcudhack Irom individual users is an csscntial part of this process. Incvitahly, with so much divcrsc and detailed information, specialists will discover crrors, ~nisintcrprctationsand significant omissions from thc cntrics as prcscntctl. Howcvcr, if cornmunicatcd to thc appropriate c-mail fccrlhack filc (srr, hrlow), these points will he attcnrlcrl to directly.
To feedhrrck an crror correction or notify significant omission: For each individual comment or point, send a separate e-mail message to thc fccdhack file supporting the entry in question as indicated in Feedback ({icld 57) (c.g.To: CSN-19ble.ac.uk). lndicatc the appropriate paragraph under discussion hy cntering its six-fiwre index number (xxyy-zz) into the Suhiect: ficld of the mcssagc (c.g. Subject: 19-29-02).For optimal correction, updating o r tor simalling a significant omission, include a piccc of 'corrected' tcxt or send a re-drawn figure designed to the existing tcxt or figure without loss of information.
entry 12
1 If a field, or 'sipnifcant foct' is not covered for a particular channel, yet the relevant information is available from a published source, please advise on the best accessible reference (hut see note 2). 2 'Updates' for the purposes of 'feedback' are published 'facts' which actually invalidate a statement as presented witllin the entrim. New papers in the published literature following the FactsRook deadlines do not require notification as they will normally be incorporated as part of the literature 'scanning' and updating procedure. 3 If a 'fact' is incorrectly referenced please indicate accordingly. Full referencing for each separate 'fact' has proven impractical for a printed format but further specific citations could be incorporated if considered useful o r necessary. 4 The 'running order' of entries within The FactsRook is not absolute, and subsequent descriptions of novel channel types (or the cloning of the genes that encode them) are likely to extend and modify the present 'working arrangement'. There will be a shift towards classification by gene structure whcn more channel genes are cloned and a true consensus on classification and nomenclatures is reached - see the IUPHAR entries under the CSN (below) for developments in this area. S Electronic mail (e-mail) is strongly prcferrcd for receiving feedback, however formatted text on disk and graphical materials illustrating or enhancing the entry text can he sent by surface mail to: Dr Edward C. Conlcy, c/o Ion Channel/Gene Expression, University of Lciccstcr/ Medical Research Council, Centre for Mechanisms of Human Toxicity, PO Rox 138, Hodgkin Building, Lancastcr Road, Leicester LEI 9HN, UK. 12-01-03: Consolidation of the existing database in this way is vital to achieve the original aim of systematic coverage for ion channel molecular properties. Following the establishment of this information framework, an increasing proportion of 'incremental' entries (containing new data) will be maintained via a n international network of specialist authors on each type of channel below). or ccll-signalling molecule
n Posting of updates on the Cell-Signalling Network (CSN)
12-01-04: Follnwing receipt of update suggestions by electronic mail, new or modified entry paragraphs will be posted in computer files as part of the CMHT's Cell-Signalling Network (CSN). Anyone with access to ~ n t e r n c t t 'hrowsingt' software such as ~ c t s c a p e can ' ~ access, search and down-load these corrected/upgraded paragraphs over the WWW (World Wide ~ e h t ) network. ' ~ o t - l i n k s t 'between different parts of the database (activated hy 'double-clicking' objects or words highlighted in boldface underlined type)
entry 12
can he configured hetween files compiled in multiple ccntrcs around the world - expansion hy 'double-clicking' will automatically trasversc the 'Weh' to the appropri;~tc scrvcri upd:ltcd by spccialist contributors or ccntrcs. 'Navigaticln' through the Wch is strai~htforwarrl- 'douhlc-clicking' items of intcrcst will open new 'linked windows' as yo11 go.
Accessing the Cell-Signalling Network (from-mid-1996) 12-01-05: In adtlitinn to updates, the CSN 'homc (indcxl pagc' (URL: http:// www.le.ac.uk/csn/index.html~(Fi,y. I ) , proviilcs access to several appendices and reference indexes supporting thc contents of Thr FartsRook (fclr a
listin$ o f tllcsr. on-1inc support rr;lpcndiccs, rcpfrrrnc~indrxrs nnd ~ l o s s a r y itrms, srr thr drssc-riptic~nsrzrldcr Fi,ys 2 rind 3). In timc, the CSN will aim to provide a convenient index for a11 information rcsourccs that arc of potential rclcvancc to cell signalling and that arc presently available from ~ n t c r n c t tsites. In common with othcr WWW resources, the CSN can be accessed from any typc of computer. Users unfamiliar with Internet hrowsingt should ask their local nctwork administrator about WWW/ Nctscape". Oncc launched using the above URL' (the uniform resource locatorT, an 'address' or 'pointer" to another document on the nctwork), thc 'Wclcomc to the CSN' filc on the 'homc page' will provide all of the othcr information needed to use the service (rcc focsimilc. Fig. 11.
Exploring the CSN by hypertexti links 12-01-06: An important criterion for the CSN is that many different groups of specialists, expert in each typc of cell-signalling molcculc or topic will contrihutc to the development of an intc,yrrlt~ddatasct (scc thc scctions i ~ t t i t l r d'Tli(,C S N (1s ( 1 co-opcrotivc proicct ' rlnd 'How to cr~ntrihurcto thr C S N ' . I~clr1r4.).Thus, c.;~chcontrihutor or Rroup of contrihutclrs will aim to compile an authoritative 'repository' of information on a molecule or topic of intcrcst 'hypcrlinkcdt' to othcr information already on the nctwork. Compilation o f such a comprehensive 'consensus archive' will incvitahly take some timc to implement properly, hut the proccss has hcgun. The availahilitv of free software tools designed to assist high-quality document preparation and exchange over the Internet will make the whole process rclativclv stmightforwarti. For instance, the graphical user interfacei (CUI) provided by the Mosaic or Nctscapc browsert programs, integrates the proccss of finding, cross-referencine and down-loading information of intcrcst. Other 'browsers', 'gophersT1 and document-exchange software (170th commcrcial and public domain) will hccomc availablc in due course. Following publication of Tlir Fnctsliooks, the latest information on the development of thc CSN using these and othcr tools will he made available 11. directly from the its 'homc p a ~ c (Fi,y. '
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The CSN as a co-operative project 12-01-07: Consolid:ltion of knc~wledgcahout all of the different classes of molcculcs affecting s i ~ n a ltransduct~onin cells remains a significant organi-
entry 12
zational challenge. Rcscarchcrs arc constantly faced with the prohlcm of integrating new information into what is already known (perhaps ahout a molcculc or phenomenon which is not within their immediate ficld of cxpcrtisc). However, genuine 'hrcakthroughs' in untlerstanding oftcn come from relating uncxpcctcd dcvclopments in parallcl fields. Furthermore, in many arcas it has become difficult (even for experts) to 'keep pace' with emerging information, which can leave significant compamtive data undiscovered. Thc relational linking inhercnt in the CSN at least offers scope for penetration of untamiliar ficltls, together with facilities for integration anrl communication of the latest dcvelopmcnts in familiar ones. Lcicv\tcr / h.lrdical Rv\carrh Cotrrinl C'c,ntrr for Mt*rhanicms:of Human I-oxicity
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A common, rational framework for dissemination of research results in cell signalIing 12-01-08: As already dcscril.lcti, The Cell-Signalling Nctwork (using the hypertcxt facilities of the World Wide ~ c h tcotrld ) offer part of thc solution to the ahovc prohlcms. Howcvcr the utility of the collective resource held acrnss different sites will depend on some fr,m ol common 'architccturc' to prevent the data accumulating in a haphazard way. T o provide a framework for dcvclopcrs, somc of thc key applications of thc CSN and their requirements are outlined hclow. For rictailcd discussion o f these ~.F.FUP.F. scc Rcsorrrce - Srarch criteria d CSN rlrrvclopmcnt, entry (15. Conceivably, cach of these resources should hc addrcssahlc from within any document alrcatly on the nctwnrk hy means of a U R L ~embedded within the sourcc document. The versatility of ernl,edded 'parinterst' to other documents is illustrated in Fig. 4, which dcscrihcs the integration of applications relating different aspects of cell-signalling ~nolcculc gene expression. In this example, complex ciatasets such as thosc reporting patterns of in s i r u hyhridization, immunnlocalization and devclopmcntal lincagc can he rclatcrl to hasic information a l ~ o u tgcnelprotein rnn!ecular typc and physiologicnl functic~n. Thc same hierarchy allows crossreferencing of the molcculnr typr t o sr~hstnntial existing information resources covcrinp, thc ~ c n c t i cdctcrlninants of ce!lular pathology [ c . ~the . on-line vcrsinn of McKusickJs Mc~nrklirrn Inhrritut1c:r in MrlnJ and/or to databases supportrd hy thc HMCW (Human Gene Mapping Workshop1 pmicct. 12-01-09: The cholcc of cell-s~gnallinr: molcuulc5 as the clrganlzlng prrnclpk 31qo ;111ows d ~ r c c tintcgmtlon of inf(lrmat~onrcsourccs relating sspccts of protcln structure and function, as tllustratctl In Fig 5. In these examples, po~ntcrqi to rlocumcnts containing primary qcqucncc and crystalFograph~c co-c~rtl~natcs [ava~lahlcfrom cstahlirhcd datahascq such as thc Rrnakhaven Protein Datahank, PDRJ can hc incorporated Into qource documents. In lt~ fmm clrdcr t o keep pacc wrth thc large numhcr of r c ~ i ~ emergent ~nutat~onal/functu,naIanslys~s of specified proteins, somc form of ~nteractive protein dnmaln topology modelling and paoteln ctructurul function mapping could he developed as shown (I.IK. .51.
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A single reference source Enr official nomenclatures of receptors, ion channels and other cell-signalling molecules 12-01-10: An important application of thc CSN is in standarrlization nnd cli.;scrnin;ition of official norncnclaturcs for cell-signalling rnolcculcs and in classificatians of dn~gsthat act at thcrn ((I.\ rrxcl~~pliCird in Fig. 01. Thc grcat tlivcrsity of cell-signalling mc~lcculcs nntl thu 'av;ilanchcl of new
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information forthcoming from refined methods for their identification and characterization has led to significant prohlems for those concerned with proposing current and accurate nomenclaturcs [see, for example, Vanhoutte et (11.( 1994) Pharmc~colRev 46: 1 11-1 6). In particular, thc rapid cloning of genes encoding multiple isoformst of receptors and channels has outpaced the ability of the research community to characterize thcir function in nativct tissues. Furthermore, since the proposal of an official nomcnclaturc . functional and undcrcsscntially involves multiple critcria ( e . ~structural, standing of signal transduction mechanisms) it necessitates the input of expertise from a wide range of disciplincs. Finally, once a nomenclature committee has produced a consensus, there still remains the problem of efficient disscmination of the proposals, so that researchers around the world can follow them. 12-01-11: The relational linking structure of tho CSN can help alleviate many of these problems, i f only hy providing a convenient and automatic means to distribute 'high-quality' update dncumentation electronically to interested parties. In common with other proposed applications of the CSN (Figs 1-s), relational links to on-line glossaries (e.g. pharmacological tcrms and pharmacopeia) could provide important s u p p l e m c n t a ~ information for the
entry 12
application of official nomenclatures. Overall, the implementation of the CSN approach would help expert suhcommittccs (c.g. those of the IUPHAR) involvcd in proposing drug, receptor ant! channel nomenclatures to create, cxchangc and then ti~sscminatcofficial rccommcndations directly to rcscarchcrs, as well as providing an important f o n ~ mfor feedback and debate on current proposals.
Integration with on-line bibliographic resources and conventional 'hard-copy' publishing 12-01-12:Ry analogy to the use of accession numbers for rctricval of primary sequence data from tlat:ihascs, thc growth of on-line bibliographic resources in recent years makcs it likcly that rctricval of I>~hllographicdata hy acccssion numhcr will hccomc commonplace (minimally as citation data and abstracts, hut in a few instances as 'clcctronic ic~ztmals',and 'printahlc' facsimiles of articlcsl. For thc time-hcing, retrospcctivc, scarchahlc compilations of the litcrnturc offcr opportunities for creating relational links with cell-signalling molcculc data. The intc~ration of sequence dntahasc cntrics (in ~ c n ~ a n for k ' instance) ~ 'pointing to' related papcrs in ~ c d l i n & ' ~ ' via eight-figi~rc acccssion nulnhcrs has already hccn implcmcntcd in thc Enfrtlz proicct pr~ducctt hy tho NCRI, and hoth scqucnccs ant1 articles can he usctl for 'ncighhn~lringsnalysis' (for further [lcscription, scr Introdllc-tion 07 L(IJ'(III~ ~ c c t i o nI I I T ~ C TFI'cIA nurnl~cr53: Dr~trtl~r~ livtings/prininr!~ s~ sPqllrncc d i . ~ c n . ~ . ~ i cIn ~ n ) .principle, such relational indexing could he incorpnratcd in compilations c ~ fd(~cumcnts within the CSN, thus providing ;in unamhiguous link from thc molccular datahasc to electronic rctricval of ahstractcd or full papers. Alternatively, supporting documents that arc too unwiclrlly for inclusion in convcntional pnpcrs (such ns gene-family a l i ~ n m c n t s ,large-scalc in .situ lncalizations or molccul:ir structi~rc/functionmodcls) c t ~ ~ r lho d rcfurcnccrl ant1 rctricvcrl hy mc:ins of :I URLi. Portable document software which makcs the frce cxchangc of high-quillity rlcctronic documents stmightforwartl irrespective of computer platfornm (c.g. A(lohc Acrtlhat'", Microsoft Intcrnct A s s i s t a n t T " ' / ~ o r d viewer-Iu and scvcral othcrs in dcvclc~pmcnt) arc therefore likcly to he of great iitility in the CSN proicct.
How to contribute to the CSN 12-10-01: Some suggestions on how individuals or groups can contrihutc new information resources as part of the nctwork ot availahlc information are outlined in Fig. 7. As well as individual contributions, collaborative (multiauthor) documents can hc created on personal computers or networks (contributor networks) for editing and cc)nsnlidation. Following achievement of 'consensus', one can pithlish thc document as an option acccssihlc from your own 'home' or 'intlcx' page on a computer connected t o thc WWW/lntcrnet (computcr nctwork ad~ninistratr~rs will tcll you how to do this). Contrihutions on cell-signalling molecules not already cc~vcrcd arc particularly valuable, hut somc effort should he made to refine the
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Step 1: Set up a PC or Macintoshm linked t o ma Internet. running a WWW browsor (e.g. Mos8rc) and Web-compatlble authoring software (e.g. M~crosotfWordm versron 6 wfth lnlernel sss~starrl) lsee note 7 )
Step 2: Prepare documonta by typ)ng Into Mank Yreldname templates' [with embedded raphrcs 11necessary]. t o r guidelines on blank floldname template deslgn. see note 2
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The daummf(s) are now a c c c l n i b b by a simp+e http: commmd and can ba acc.rssd worldwid. by a n y o m wtth the appropriate 'browsing' h a m . *lch is generally avallable frecof-charge (see note 1).
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informntic~n through inter-laboratory links ;~nil hy co-opcrntion with inrlivitl~~:ils wr~rkingIn the salnc ficltl (st1 that ;I truly comprchcnsivc and authoritative 'conscnsi~s'c ~ prr~pcrtics f is rcachcrll. Authors sh~)uldtake care not to duplicate unncccssarily infc~rnintson;~lrcndyavnilahlc (covcragc to date will 1 ) ~ ' posted on thc C S N 'home pagc'). Errors ant! misintcrprctati~)ns sl~ouldhc cotnmunicatctl hack to contril>utnrs hy c-mail, as this minimizes papcrwtlrk. T h c CSN mny also hc usctE t c ~raise awareness of particular topics within thc hroatl ficltl c ~ cell f si~nnllinr:nnd, via thcir own hnmc p ; l ~ c c;m , pul~licizc the ;ictivitics o f rcscnrch ccntrcs, I:It?oratorics .or othcr 17('14J r~,.w)iIrt.i,,< ol it~jorttiotii)~~ l ) i ~ ( ~( t~nv i~~ ; / r ior/ ) li f~ ~ n s t i t i ~ t i o nW17r,r3 s, , S V I I ( / i / ~ t i ~ r o/ i , s .v(~)pi,, ril~tl~or.~ i,xi,stin,y rra.vol]rciJ,$ (Irc I I O ! inrlr,xt,(f r~lri~orl~,. t i!ut I O I T ' , ~ f JI-!l2/h[tp: (~rhir(,,s,s z i l ( I ! ) (,-tt~(iiJ i?~(~.v.~(i~qc to t / w r~ridt!ir / t i ) , ~ ir7,vt [email protected] i:r,dhoc.k lilr, orlrl ~ h r , will , ~ Ilr, ~rlr~rlc r~r~cc~.sil~Ir~ virr nn rlpl~roprirrtr,resource indpx on tllr I:S N 'ho171i'p ~ , y c ' .Many h r t hcr iIctails on thc scnpc and dcvulc~pmcnt thc CSN ciln hc foi111din R ~ s o l ~ r uI rScmrt:h c:ri!ericl i+) C S N r f w rloprncnt , rnlrjr 65.
F i g ~ ~ r7.c How to ( * O I I / rihtrtr, to 11lr' C S N on thrp Worlrl Widr Wch. Nolcs: I . Si,vc,rrrl Iritr,rnr,t/WWM7 'rrrrtltorrr~s' softrfi~rr,pirc:ltrr.yr~s rirr, hr,cornincq t ~ ~ ~ r ~ i J (irlt ~ h~! iOb ,I I tlw ~ Tt ~! (~> \ t( ~ > t ~ \ , i ~(>pi r ~I OrI ~I , ~iwill ~ t !x,t !~o,sc! t htlt ~ ( 1 1 7(111t o~iir~tir~irliy t.or~v(,rt~vor(!-l~ro(.i~s,+i~i! ( ~ U Y II ? I ~ ~(1vit I ? ~11s,yrr~phics) dircctlv t o f h : HTMI. {orrn(~r{(I,V ir~(-rl to ,stbnr!J O ~ I ~ I ? >o ~w #r It I ~ h .W S W W ) . At t !ic t irt~r*o{ go in.^ t c l prclss. Mic.rost>Tt(lnr~ouncwl(in oy)tion for M7r)rd 6 (I?o!h Mrrc: (2nd lJC vr*rsrons) tvhic h r:nn simpli/y trhr prr~p(irrition rind plil,/ict: t ion of piz'qex
ifcvtrnvr! {or t h i ~ W W W . Thr Micn~soit Intr!rnrt Assisrrinr proaqrmm is rir~oil(ll~li, irom !hi, Microstl/t W W W 'Ilclmr p r ~ c ' ,os is I I I C WordVirpwrr r . ~ 1i M S pr({yrt1171. whic.h r~rtt~l?lt*s c i o ~ ~ ~ t ~tot -hrb t ~tpr~d t . ~ hv olhr'r ~ ~ s c(cvivi Worif 1,s ZIO! iri,xtrilli~!l.2. LSibl(u(.tii~ri (J/ '~iiblil~~(~ ,shoiilri n ~ c ~ .iollow v' ( I scat o i r.ri!rrritl for ~~lrir.r~inr~nt o f mrirt~iol((iscbxr,nil)lilir-rl fhr. Fr~r~r,sln!,~ ~,s l(>llotvitigi11rrhr7r ' r i r c t ~ / r ~ ! i o ~ ~ ' rrrrrrinrl t h i ~r.onrrihrrtclr sul)-nrptwork rnrIv I>(, prrhli.~Iur.l(1s 'incrr,m(,nttrl(il(>s' ,x i r r ~(,i~rlj(,r tvur,siotl).4. F(>r/7in/p{);It lw strllct i~ringo{ or (1,s ' ~ I ~ I ( / ( I '/ ~(rt,pli~t.i~~,q IITML lilrs diri~rtorii~s rrnd thr crcrrtion o( 'ho!-links' fir r)thr,r rr!sorirrcs, sec vot~rzlrr t c t 7 0 t I i irrfr7linjslriz!or. Spi!r.ic~lic/slzppclrt {or itlir iot ing or mr~irltrlinin,p rrv.r~ivi~rl (c-tnrrrl to t hi, crppropriatr ion r l ~ n n ~rsntrics ~ r ~ l wor~lrfI ) i u .yrt~tr/t~llj~ 5 7 ol r~1c.hrpntrv). sr~pjwrtsr(111j1., s ~ ifii*Ji/ ,
This Page Intentionally Left Blank
E n t r y 13 Entry (channel type) numbers form the first two numbers xx of the six-figure index number (xx-yy-zz) according to the rubric below, yy is the field i.d. number (see the accompanying field number rubric) and zz is the datatype i.d. number within a field (in the current entries zz simply indicates 'paragraph running order' but this number will eventually define and index specific types of data 'falling under' each fieldname). 'Coverage' under each page header/sortcode as listed below is indicated under C u m u l a t i v e contents, e n t r y 02. Entry
page header / sortcode partying paGa.EADaRSRUBRIC
Seeal. . . . . . .
Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry Entry
01-yy-zz 02-yy-zz 03-yy-zz 04-yy-zz 05-yy-zz 06-yy-zz 07-yy-zz 08-yy-zz 09-yy-zz 10-yy-zz 11-yy-zz 12-yy-zz 13-yy-zz 14-yy-zz 15-yy-zz 16-yy-zz 17-yy-zz 18-yy-zz 19-yy-zz 20-yy-zz 21-yy-zz 22-yy-zz 23-yy-zz 24-yy-zz 25-yy-zz 26-yy-zz 27-yy-zz 28-yy-zz 29-yy-zz 30-yy-zz 31-yy-zz 32-yy-zz 33-yy-zz 34-yy-zz 35-yy-zz 36-yy-zz
Cumulative tables of contents Introduction & layout of entries Abbreviations ELG [key facts] ELG CAT 5-HTs ELG CAT ATP ELG CAT GLU AMPA/KAIN ELG CAT GLU NMDA ELG CAT nAChR ELG CI GABAA ELG Cl GLY Feedback & CNS access- See also Entry 65. Rubrics ILG [key facts] ILG Ca AA-LT[C]4 [native] ILG Ca Ca InsP4S [native] ILG Ca Ca RyR-Caf ILG Ca CSRC [native] ILG Ca InsPs ILG CAT Ca [native] ILG CAT cAMP ILG CAT cGMP ILG CI ABC-CF ILG CI ABC-MDR/PG ILG Cl Ca [native] ILG K AA [native] ILG K Ca ILG K Na [native] INR K [key facts] INR K ATP-i [native] INR K G/ACh [native] INR K [native] INR K [subunits] INR K/Na Imq [native] JUN [connexins] MEC [mechanosensitive]
Entry e-mail feedback file [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN- [email protected] CSN- [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN- [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN-31 @le.ac.uk [email protected] [email protected] [email protected] [email protected] [email protected]
~1~
ELG Key facts
Entry 37-yy-zz Entry 38-yy-zz Entry 39-yy-zz Entry 40-yy-zz Entry 41-yy-zz Entry 42-yy-zz Entry 43-yy-zz Entry 44-yy-zz Entry 45-yy-zz Entry 46-yy-zz Entry 47-yy-zz Entry 48-yy-zz Entry 49-yy-zz Entry 50-yy-zz Entry 51-yy-zz Entry 52-yy-zz Entry 53-yy-zz Entry 54-yy-zz Entry 55-yy-zz Entry 56-yy-zz Entry 57-yy-zz Entry 58-yy-zz Entry 59-yy-zz Entry 60-yy-zz Entry 61-yy-zz Entry 62-yy-zz Entry 63-yy-zz Entry 64-yy-zz Entry 65-yy-zz Entry 66-yy-zz Entry 67-yy-zz
entry 13]
MIT [mitochondrial, native] NUC [nuclear, native] OSM [aquaporins] SYN [vesicular] VLG key facts VLG Ca VLG CI VLG K A-T VLG K DR VLG K eag VLG K Kv-beta VLG K Kvl-Shak VLG K Kv2-Shab VLG K Kv3-Shaw VLG K Kv4-Shal VLG K Kvx (Kv5.1/Kv6.1) VLG K M-i [native] VLG K minK VLG Na Appendix A - G Protein-Linked Receptors Appendix B - Electrical Effectors Appendix C - Compounds & Proteins Appendix D - Diagnostic Tests Appendix E - Book References Appendix F - Subject Reviews Appendix G - Consensus Sites & Motifs Appendix H - Cell-Types Appendix I - Cell-Signalling Molecular Types Appendix J - Search Criteria & CSN Development Multidisciplinary Glossary Framework Cumulative Subject Index
[email protected] [email protected] [email protected] [email protected] CSN-41 @le.ac.uk [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN-5 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] CSN-61 @le.ac.uk [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]
Note: Entry 'running order' is only of significance in book-form publications; computer-compiled updates will use the xx-yy-zz numbers as hyper-relational pointers.
118
E n t r y 13 Field numbers form the third and fourth numbers yy of the six-figure index number (xx-yy-zz)according to the rubric below, zz is the datatype i.d. number within a field (in the current entries zz simply indicates 'paragraph running order' but this number will eventually define and index specific types of data 'falling under' each fieldname). Omission of a field number within the main entries implies information was 'not applicable' or was 'not found' during compilation.. NOMENCLATURES SECTION xx-01-zz Abstract - general description xx-02-zz Category (sortcode) xx-03-zz Channel designation xx-04-zz Current designation xx-05-zz Gene family xx-06-zz Subtype classifications xx-07-zz Trivial names EXPRESSION SECTION xx-08-zz Cell-type expression index xx-09-zz Channel density xx-10-zz Cloning resource xx-11-zz Developmental regulation xx-12-zz Isolation probe xx-13-zz mRNA distribution xx-14-zz Phenotypic expression xx-15-zz Protein distribution xx-16-zz Subcellular locations xx-17-zz Transcript size SEQUENCE ANALYSES SECTION xx-18-zz Chromosomal location xx-19-zz Encoding xx-20-zz Gene organization xx-21-zz Homologous isoforms xx-22-zz Protein MW (purified) xx-23-zz Protein MW (calc) xx-24-zz Sequence motifs xx-25-zz Southerns STRUCTURE & FUNCTIONS SECTION xx-26-zz Amino acid composition xx-27-zz Domain arrangement xx-28-zz Domain conservation xx-29-zz Domain functions (predicted) xx-30-zz Predicted protein topography xx-31-zz Protein interactions xx-32-zz Protein phosphorylation
ELECTROPHYSIOLOGY SECTION xx-33-zz Activation xx-34-zz Current type xx-35-zz Current-voltage relation xx-36-zz Dose-response xx-37-zz Inactivation xx-38-zz Kinetic model xx-39-zz Rundown xx-40-zz Selectivity xx-41-zz Single-channel data xx-42-zz Voltage sensitivity PHARMACOLOGY SECTION xx-43-zz Blockers xx-44-zz Channel modulation xx-45-zz Equilib. dissoc, constant xx-46-zz Hill coefficient (n) xx-47-zz Ligands xx-48-zz Openers xx-49-zz Receptor/transducer ints xx-50-zz Receptor agonists xx-51-zz Receptor antagonists xx-52-zz Receptor inverse agonists INFORMATION RETRIEVAL SECTION xx-53-zz Database listing xx-54-zz Gene map. locus desig. xx-55-zz Miscellaneous information xx-56-zz Related sources & reviews xx-57-zz Feedback In-press updates REFERENCES SECTION
Index The following is a conventional index, note however that a cross referenced subject index for topics appearing within fields covering all entries, appears under the section cumulative page index, as a cumulative topic index. For rapid location of information see entry and field number rubrics, under entry 13. Also note italic page numbers represent entries in figures and tables. A
B
A23187, 241 Abecarnil, 345 Acetylcholine, 3, 4, 6, 28, 35, 42, 53, 279 ACPC, 216 ACPD, 167 Actin depolymerization, 196 Adamantane, 201 Adenosine receptors, 212 Adenylyl cyclases, 184 Aggregates, of nAChR, 269 Agrin, 270 AIDS, 155 Alcohols, 276-7 Alkaline phosphatase, 324 Alphaxalone, 329, 346 Alternative splice variants GABAAR, 320 GluR, 79-80, 87, 89, 92, 100, 101,
B6B21 antibody, 166 Baclofen, 96, 212, 329 Barbiturates, 294, 334, 335, 336 see also individual compounds Basic fibroblast growth factor (bFGF), 269 Benzamil, 156 Benzodiazepines, 294, 314, 317, 319, 334, 335, 343, 345, 376 see also individual compounds Bicuculline, 307, 324, 329, 331, 334, 342, 348 Blockers field, 55 ELG CAT 5-HT3, 27-8 ELG C1 GABAA, 329, 330, 331-32 ELG CAT GLU AMPA/KAIN, 122 ELG C1 GLY, 391-3 ELG CAT nAChR, 274-7 ELG CAT GLU NMDA, 156, 198202 ELG CAT ATP, 63-4 BOA_A, 125 Brain-derived neurotrophic factor (BDNF), 91 a-Bungarotoxin (:~-Bgt), 238, 242, 272, 276, 278, 279, 280, 281 Bursting, 340, 388 GABAAR, 321 nAChR, 274
109, 117-8
NMDAR, 141, 177 Alzheimer's disease, 155 AMPA receptors, 73-139, 188 Androsterone, 341 Angelman syndrome, 302, 306, 309 Aniracetam, 122 Anti-secretory factor (ASF), 331 Antimycin A, 377 AP3, 167 AP4, 167 AP5, 214 Apoptosis, 155 role of P2xR, 36, 40 2-APV, 212, 214 D-APV, 218 Arachidonic acid, 208 Arcaine, 560, 216 Argiotoxin, 122, 127, 217 L-Aspartate, 125 mixed agonism, L-glutamate, 211 ATP-gated channels, 6, 35-73 Atropine, 279, 283 Avermectin, 334, 337 12(
C c-fos, 149 c-jun, 149 CACA, 329 Calcitonin gene-related peptide (CGRP), 283 Calcium/calmodulin-dependent protein kinase (CaM kinase), 9, 147, 149, 155, 181, 184, 268 Calmidazolium, 148, 203 Calmodulin, 203
Index
Calpain II, 155 cAMP-dependent protein kinase, 9, 268 fLCarbolines, 336 Cd2+ ions, 331 CGP-37849, 214, 218 CGS-19755, 214, 218 Chlordiazepoxide (CDPX), 322, 323, 338 7-Chlorokynurenic acid, 166 Chlorpromazine, 274-5, 275 Cholecystokinin, 28 Chromosomal location field ELG C1 GABAA, 309 ELG CAT GLU AMPA/KAIN, 98, 99 ELG C1 Gly, 378 ELG CAT nAChR, 248-9 ELG CAT GLU NMDA, 170 7-C1-Kyn, 216, 218 Clomethiazole, 334, 338 CNQX, 126, 127, 212, 218, 560, 216 CNS1102, 215 CNS1505, 215 Co2+ ions, 158, 322, 331 Co-agonism, glutamate and glycine, 191, 192, 210 Co-transmission, 35, 41, 45 a-Cobratoxin, 2 78 Colchicine, 196 Conantokin-G, 169, 217 Conantokin-T, 217 Concanavalin A, 80, 82, 91, 116 a-Conotoxins, 276 Convulsants, 303, 348 see also Strychnine Convulsions, 304 CPP, 214, 218 Curare, 2 79 Cyclodiene insecticides, 314 D-Cycloserine, 211 Cyclothiazide, 80, 82, 86, 91, 116, 117 L-Cysteine sulphinate, 3 Cytisine, 2 79 Cytochalasins, 196 Cytoskeletal elements, 185, 196, 238, 371
D
DA10, 219 DAA, 214 Dantrolene, 165, 241
DBI, 351 DDHB, 128, 214 Decamethonium, 278 Desipramine, 214, 282 DET, 219 Developmental regulation field ELG CAT 5-HT3, 15 ELG C1 GABAA, 301-302 ERLG CAT GLU AMPA/KAIN, 89-91 ELG C1 Gly, 372-3 ELG CAT nAChR, 24043 ELG CAT GLU NMDA, 147-54 ELG CAT ATP, 40-1 Dextromethorphan, 202, 214, 218 Dextrorphan, 200, 201,214 DGG, 12 7 DHt~E, 261 DHEAS, 348 Diazepam, 308, 325, 334, 342 Dithiothreitol, 203 Dizocilpine (MK-801), 156, 200, 212, 214, 218, 219 DMCM, 349, 350 DMPP, 279, 281,282 DNQX, 127, 156 Dopamine, 28 Doseresponse field ELG CAT 5-HT3, 24 ELG C1 GABAA, 324 ELG CAT GLU AMPA/KAIN, 114 ELG CAT GLU NMDA, 194 ELG CAT ATP, 56-7 Duchenne muscular dystrophy, 267-9 Dystrophin, 267-9
E
Electrophysiology section ELG CAT 5-HT3, 23-7 ELG CAT ATP, 53-63 ELG CAT GLU AMPA/KAIN, 113-21 ELG CAT GLU NMDA, 191-98 ELG CAT nAChR, 269-74 ELG C1 GABAA, 322-30 ELG C1 GLY, 388-91 Eliprodil, 216 Endonucleases, 186 Endothelium-derived relaxing factor (EDRF), 209 12]
Index
Enflurane, 127, 201,305, 336 Epilepsy, 95, 126, 155, 156, 190, 218, 304 Ethanol, 205, 305, 334, 337, 340, 342 Evans blue, 128 Expression section ELG CAT 5-HT3, 14-18 ELG CAT ATP, 39-45 ELG CAT GLU AMPA/KAIN, 86-98 ELG CAT GLU NMDA, 146-70 ELG CAT nAChR, 238-48 ELG channels, 5-6 ELG C1 GABAA, 299-309 ELG C1 GLY, 371-8
F
Felbamate, 212 Flip/flop splice variants of GluR, 80-1, 87, 89, 92, 100, 101, 109, 117-18 Flumazenil (Ro151788), 348 Flunitrazepam, 316, 330, 334, 346 Forskolin, 284 Fos proto-oncogene, 148-9 FPL12495, 215 Furosemide, 219
G G protein-coupled receptors, 3, 12, 38, 65, 142, 186, 294 GluR, 78 G stretch-binding protein, 251 GABA, 3, 4, 6, 10, 28, 366, 393 role of NMDAR, 158 GABA receptors (GABAR), 187, 212, 219 modulation of NMDAR, 167 GABAA receptors (GABAAR), 293-66, 3 76, 388 Gamma amino butyric acid see GABA GAMS, 128, 218 GAP43, 149 GDEE, 127 GenBank{S}R{s}, 379 Gene families field, 142, 144-5, 235, 236-7 ELG CAT 5-HT3, 13 ELG CAT GABAA, 295, 296-7 ELG CAT GLU AMPA/KAIN, 78, 80-85 122
ELG C1 Gly, 368, 369-71 ELG CAT nAChR, 235, 236-7 ELG CAT GLU NMDA, 142-3, 144-5 ELG CAT ATP, 37, 38 Gene organization field ELG CAT 5-HT3, 18 ELG CAT GABAA, 309 ELG CAT GLU AMPA/KAIN, 99-103 ELG C1 Gly, 378 ELG CAT nAChR, 250-53 ELG CAT GLU NMDA, 170-1 ELG CAT ATP, 47 Gephyrin, 378, 379, 387, 393 GluR, non-NMDA, 74-139 L-Glutamate, 3, 4, 6, 125, 307 as co-agonist, 191, 192, 210 mixed agonism, L-aspartate, 211 Glutamate receptors GluR, 5, 9, 74-139 NMDAR, 5, 9, 140-233 Glutamate toxicity, 10 neuronal death, 209 NMDAR, 155, 180 Glutamic acid decarboxylase, 149 Glutathione, 216 Glycine, 3, 4, 6, 10 as co-agonist, 140, 191, 193, 210 Glycine receptors (GlyR), 295, 366-99 Guam disease, 212 Guanylate cyclase, 187 GYKI, 127 H H-7, 283 Ha-966, 216 Halothane, 277, 305, 334, 336 HEK293 cells, 66 Helical diffraction, Torpedo nAChR channel, 264 'Helical wheel' plots, 50, 51 Heptanol, 277 Hexamethonium, 277 Hexanol, 277 Hirsutine, 64 Histamine, 3, 4 Histrionicotoxin, 274 L-Homocysteic acid, 210 5-HTgR, 12-34
5-HT, 12, 53 Huntingdon's disease, 155 Hypoglycaemia, 155 I
I2CA, 215 IBMX, 283 Ifenprodil, 218, 560, 216 Imipramine, 282 Information retrieval section ELG CAT 5-HT3, 31-2 ELG CAT ATP, 70-71 ELG CAT GLU AMPA/KAIN, 129-35 ELG CAT GLU NMDA, 219, 221-5, 225 ELG CAT nAChR, 284-8 ELG C1 GABAA, 351-63 ELG C1 GLY, 394-7 Inositol 1,4,5-trisphosphate (InsP3), 56 receptors, 160, 185 Ionotropic receptors, 5 Ischaemia, 95, 127, 155, 218 Isoflurane, 277, 305, 334, 336 Isoprenaline, 53
J Joro spider toxin, 103, 122, 124, 127, 218 Jun proto-oncogene, 148-9
K
Kainate, 340 Kainate receptors, 74-139, 186 Kainate-binding proteins, 84 Kappa-flavotoxin, 281 KATP, 207 Ketamine, 200, 201,214 Key facts, (ELG), 3-11 Kindling, 156, 190
L L687414, 216 L-689,560, 216 Lanthanum chloride, 158 Ligands field ELG CAT 5-HT3, 28 ELG channels, 8
ELG CAT GABAA, 343, 344 ELG CAT GLU AMPA/KAIN, 123-4 ELG C1 Gly, 393 ELG CAT nAChR, 280-83 ELG CAT GLU NMDA, 208 ELG CAT ATP, 65-6 Long-term synaptic potentiation (LTP), NMDAR, 159, 160-63, 165-8, 190, 203, 204 Lophotoxin (LTX), 277, 281,282 LY-235959, 214
M
MBTA, 255 MDL100453, 214 Mecamylamine, 276 Memantine, 199, 215 Metabotropic receptors, 5 Methoxyindole carboxylic acid, 215 ~,f~-Methylene-ATP, 54, 66, 67, 70 Methyllycaconitine, 276, 278, 283 2-MethylthioATP, 66, 67 Mg 2§ ions, 140, 148, 198, 200, 215 Microtubule assembly, 196 Mixed agonism, L-glutamate, L-aspartate, 211 Mn 2+ ions, 215, 331 MNQX, 216 Modulators Channel Field ELG CAT 5-HT3, 28 ELG CAT GABAA, 323, 324-8, 339, 340, 340-42 ELG CAT GLU AMPA/KAIN, 122 ELG C1 Gly, 391-93 ELG CAT nAChR, 277-8 ELG CAT GLU NMDA, 147, 149, 202-8 ELG CAT ATP, 64-5 Morphinians, 202 mRNA distribution ELG CAT 5-HT3, 15-16 ELG CAT GABAA, 302, 303-4 ELG CAT GLU AMPA/KAIN, 92-4 ELG C1 Gly, 3 73, 3 74 ELG CAT nAChR, 243-4 ELG CAT GLU NMDA, 152-4 ELG CAT ATP, 41 Muscarine, 284
12~
Index
Muscimol, 316, 346 Myasthenia gravis, 246 MyoD 1,252 N
Na§ § ATPases, 200 NANC excitatory transmission, 66 NBQX, 126, 212 Neosurugatoxin (NSTX), 276, 280, 281 Nerve growth factor, 41, 91 Neurokinin A, 209 Neuronal cell lines, expression of 5-HT3, 14 Neuronal-bungarotoxin (n-Bgt), 238, 281 Neurosteroid pregnenolone sulphate, 207 Neurotoxicity, NMDAR, 220 Ni 2+ ions, 331 Nicotine, 234, 278, 279, 283 Nicotinic acetylcholine receptor, 233-92 Nifedipine, 122, 156 Nitrendipine, 205 Nitric oxide, 203, 209 Nitric oxide synthase, 155, 188 Nitroglycerin, 560, 216 Nitroprusside, 560, 216 NMDAR, 140-234 Nocodazole, 158 Nomenclatures section ELG CAT 5-HT3, 12-14, 32 ELG CAT ATP, 35-9, 71 ELG CAT GLU AMPA/KAIN, 130-34 ELG CAT GLU NMDA, 140-46, 222-5 ELG CAT nAChR, 234-8, 286-8 ELG C1 GABAA, 293-301,351-52 ELG C1 GLY, 366-71,395-6 Nonanol, 277 Noradrenaline, 28, 42, 66
O Octanol, 277 Oestradiol, NMDAR modulation, 147, 149 2-OH-Saclofen, 167 ORF 399 aa, 36-73 ORF 472 aa, 36-73 Oxiracetam, 122 Oxotremorine M, 283 124
P
P~.xpurinoreceptor-channels (Pax), 35-73 Parkinson's disease, 127, 212 Penicillin, 331 Penicillin G, 392 Pentobarbital, 329, 342 Pentylenetetrazole (PTZ), 149 Phalloidin, 196 Pharmacology section ELG CAT 5-HT3, 27-31 ELG CAT ATP, 62-72 ELG CAT GLU AMPA/KAIN, 122-9 ELG CAT GLU NMDA, 198-219 ELG CAT nAChR, 275-85 ELG channels, 9-10 ELG C1 GABAA, 329-50 ELG C1 GLY, 391-5 Phencyclidine (PCP), 200, 201, 214, 275-6 Phenotypic expression field ELG CAT 5-HT3, 16-17 ELG C1 GABAA, 302, 304-8 ELG CAT GLU AMPA/KAIN, 94-7 ELG C1 GLY, 373, 375-6 ELG CAT nAChR, 244, 245-6 ELG CAT GLU NMDA, 154-68 ELG CAT ATP, 41-2 Philanthotoxin, 122, 129 Phosphatases, 188 Phosphoinositide, hydrolysis, 35 Phospholipase A2, 155, 189, 337 Phospholipase C, 28, 155, 189 Phosphorylation motifs, 5-HT3, 23 Picrotoxinin (PTX), 313, 329, 331, 334, 336, 347, 367, 391 Piracetam, 122 Pitrazepin, 347 Platelet-activating factor (PAF), 204 Polyamines, 205, 206, 219 PQQ, 216 Prader-Willi syndrome, 303, 307, 310 Pregnanolone, 342 Progesterone, 276-7 L-Proline, 125, 211 Propofol, 334 Protein distribution field ELG CAT 5HT3, 17-18 ELG CAT GLU AMPA/KAIN, 97-8 ELG C1,Gly, 377
ELG CAT nAChR, 246-8 ELG CAT GLU NMDA, 168-70 ELG CAT ATP, 42 Protein domain topography model (PDTM) ELG CAT 5HT3, 19, 20 ELG CAT GABAA, 312 ELG CAT GLU AMPA/KAIN, 107 ELG C1 Gly, 385 ELG CAT GLU NMDA, 174 ELG CAT ATP, 47 Protein kinase A, 320, 387 Protein kinase C, 9, 155, 181, 189, 190, 202, 208, 242, 268, 319, 387 Protein kinase inhibitors, 203 Protein sequence superfamily, 6-7 Protein topography, nAChR, 263, 265, 266
Protein tyrosine kinase, 9 Protopine-hydrochloride, 347 PTMA, 258 Pyrazole, 215
q Quinolate, 3 QUIS receptors see AMPA receptors QX-222, 260
R
Reactive blue 2, 55, 58, 66, 68, 69 References section ELG CAT 5-HT3, 33-4 ELG CAT ATP, 72-4 ELG CAT GLU AMPA/KAIN, 135-9 ELG CAT GLU NMDA, 225-33 ELG CAT nAChR, 289-93 ELG channels, 10-11 ELG C1 GABAA, 361-5 ELG C1 GLY, 396-8 Remacemide, 215 Riluzole, 127 RNA editing, GluR, 75, 102, 110 Rol 51788, 347 Ro154513, 334, 339, 341,349 Ro194603, 349 Ro54864, 331,334 RP-2 cDNA sequence, 40
RU135, 347 Ryanodine receptors, 185 neuronal, 160
S
Scopolamine, 219 SDZEAA494, 214 Selectivity field ELG CAT 5-HT3, 25-6 ELG CAT GABAA, 325-6 ELG CAT GLU AMPA/KAIN, 119-20 ELG C1 GLY, 388, 389 ELG CAT nAChR, 273-4 ELG CAT GLU NMDA, 196-7 ELG CAT ATP, 57, 59-60 Sequence analyses section ELG CAT 5-HT3, 18-19 ELG CAT ATP, 47-9 ELG CAT GLU AMPA/KAIN, 98-105 ELG CAT GLU NMDA, 170-2 ELG CAT nAChR, 248-55 ELG CAT GABAA, 309-10 ELG C1 GLY, 378-9 Sequence motifs section ELG CAT 5-HT3, 19 ELG CAT GABAA, 310 ELG CAT GLU AMPA/KAIN, 105 ELG C1 GLY, 379 ELG CAT GLU NMDA, 170-71 ELG CAT ATP, 46, 47 Serotonin see 5-HT SH-SY5Y neuroblastoma cell line, 238 Single channel data field ELG CAT 5-HT3, 26-7 ELG CAT GABAA, 326-7 ELG CAT GLU AMPA/KAIN, 120, 121 ELG C1 GLY, 389-90 ELG CAT nAChR, 241,274-5 ELG CAT GLU NMDA, 197-8, 199 ELG CAT ATP, 60-61, 62 SKF10047, 214 SKF 89976A, 158, 322 Sp l-like factor, 251 Sphingosine, 203 SR95531, 342, 347 Staurosporine, 209, 242 Steroids, 334, 343 Stilbene isothiocyanate analogues, 69
12E
Index
Stretch-activated channel, 54 Structure and functions section ELG CAT 5-HT3, 19-23 ELG CAT ATP, 49-53 ELG CAT GLU AMPA/KAIN, 105-12 ELG CAT GLU NMDA, 172-90 ELG CAT nAChR, 256-70 ELG channels, 6-8 ELG C1 GABAA, 310-22 ELG C1 GLY, 380-88 Strychnine, 277, 279, 283, 348, 367, 368, 369-71,372, 381,382, 392, 393,394 Substance P, 35, 156, 209 Suramin, 54, 55, 64, 66, 68, 69
T T5M2d, 264-7 Tachykinins, 209 Taxol, 196 TEA, 202 TETS, 348 Thapsigargin, 165 Theophylline, 96, 212 Thiocyanate, 328 THIP, 346 Thymopoietin, 282 TID, 259 Tiletamine, 214 TMA, 258, 279
Tolbutamide, 207 TPA, 181,242, 269 TPBS, 334 TTX, 241 (+)-Tubocurarine, 27, 55, 58, 250, 261, 272, 276, 278, 283 Tubulin, 3 77 Tyrosine kinase, 1'90, 269
U U-78875, 344 V Valinomycin, 61 Verapamil, 158, 239, 322
W
W7 calmodulin inhibitor, 324 Walker type ATP binding site, 47, 48 WAY100359, 348 Willardines, 125
Z
Zn ~§ ions, 64-5, 156, 200, 204, 215, 303, 329, 330, 331-4, 334 Zolpidem, 345
