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Vol. 50, Issue 2, 291-314, June 1998
-Aminobutyric AcidA Receptors: Classification on
the Basis of Subunit Structure and Receptor Function
Molecular Neurobiology Unit (E.A.B.), Royal Free Hospital School of Medicine, London, England; Neuroscience Discovery (P.S.), Lilly Research Laboratories, Indianapolis, Indiana; Department of Pharmacology (R.W.O.), U.C.L.A. School of Medicine, Los Angeles, California; Institute of Pharmacology (H.M.), ETH and University of Zurich, Zurich, Switzerland; Department of Biochemical Psychiatry (W.S.), University of Vienna, Vienna, Austria; Department of Experimental Biology (G.B.), University of Cagliari, Cagliari, Italy; Pharmaceutical Division (C.B.), Schering A.G., Berlin, Germany; Department of Pharmacology, University of Alberta (A.N.B.), Edmonton, Canada; Synthélabo Recherche (L.E.R.S.) (S.Z.L.), Bagneux, France
I. Introduction
A. Earlier Classifications of-Aminobutyric Acid Receptors
1.-Aminobutyric acidA and
-aminobutyric acidB receptors.
2.-Aminobutyric acidC receptors.
3. Benzodiazepine receptors.
4. Excitatory-aminobutyric acidA receptors.
B. Conclusion on-Aminobutyric Acid Receptor Types
II. Approaches to the Classification of the-Aminobutyric AcidA Receptors
A. Transductional Criteria
B. Operational Criteria
C. Structural Criteria
III. The Structures of the-Aminobutyric AcidA Receptors
A. The Repertoire of Subunit Types
B. The Subunit Number per Receptor Molecule
C. The Subunit Isoforms in One Receptor
D. Possibilities for Subunit Stoichiometry
IV. Principles of the Classification
A. Application of Selectivities at the Binding Site for Benzodiazepines and Their Functional Analogs
1. The choice of a classification system.
2. Benzodiazepine-responsive-aminobutyric acid receptor subtypes.
3. Notation.
B. Advantages and Possible Modification of the Classification
C. Nomenclature for the Site Binding Benzodiazepines and Modulators with Similar Activity
D. Benzodiazepine Insensitivity
V. Chromosomal Localization of-Aminobutyric AcidA Receptor Genes
VI. Other Binding Sites in Relation to the Receptor Classification
A. Other Modulatory Sites
B. The-Aminobutyric Acid Recognition Site
VII. Conclusions
Acknowledgments
References
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I. Introduction |
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This article does not aim to review in detail the properties of
-aminobutyric acidA
(GABAA)b
receptors, because recent accounts of that topic are available. In this
same journal, a review of the binding properties and pharmacology of
these receptors has been published (Sieghart, 1995
). Other reviews have
dealt with their ion channel properties as well as their pharmacology
(MacDonald and Olsen, 1994
; Mohler et al., 1996a
,b
), whereas
others have concentrated on their molecular biology and protein
structure (Wisden and Seeburg, 1992
; Smith and Olsen, 1995
; Stephenson,
1995
; McKernan and Whiting, 1996
). Further, two recent books have
provided many short review articles on the functional, behavioral, and
psychopharmacological aspects of GABA receptors (Tanaka and Bowery,
1996
; Enna and Bowery, 1997
) and an account of these latter aspects
will not be repeated here.
Building on that background, we will consider here how our knowledge of GABAA receptor structure and function could lead to a classification system. Such a system is not immediately obvious from those previous accounts, as it probably would have been with a one-subunit receptor of the G-protein-coupled class. It is surely no accident that all the present series of NC-IUPHAR reports in Pharmacological Reviews on the nomenclature of individual receptor types have so far concerned G protein-coupled receptors.c Certainly the G protein-coupled receptor class covers by far the largest numbers of receptor types; it includes most of the cases in which known clinical drug applications can be related so directly to those types that they have given a great impetus to the receptor analyses. However, beyond those considerations, a major reason for the success in classification of those types must surely be the distinction which can be made therein between the subtypes of a receptor, based on the fact that each will be created by a single polypeptide with a pharmacology which is encoded solely by its own sequence. This is not to say that the classification of any of the receptors previously surveyed in this series has been entirely obvious or without complexities. Nonetheless, the problems involved are generally concerned with borderline cases in which the sequence data or the discriminatory pharmacological tools were historically less satisfactory. How one longs for such a one-to-one correspondence in the case of the GABAA receptors!
The discussion here, therefore, is the first in the classification series to tackle a receptor of their class, i.e., the multisubunit, heteromeric ion channels directly activated by the transmitter. The combinatorial principle of receptor construction (to be discussed below) for these ionotropic receptors, which also is used extensively in glutamate and nicotinic acetylcholine receptors, introduces a higher order of complexity. The functional unit is not the single polypeptide, and further, the functional properties contributed by a given subunit can vary with its interactions with the particular set of subunits in each receptor molecule. This complexity renders the recognition of the structures of receptor subtypes in their natural setting extremely difficult (in fact, at present, usually unattainable). Thus, it is not possible to construct a classification comparable with the comprehensive scheme for native receptor subtypes obtained in the previous articles in this series. Instead, a provisional version is presented which relies on the wealth of sequence and functional data available on the recombinant GABAA receptors.
A. Earlier Classifications of 1. 2.
-Aminobutyric Acid Receptors
-Aminobutyric acidA and
-aminobutyric
acidB receptors.
GABA has been accepted as a
neurotransmitter (in mammals and down to crustacea) for several
decades. It is now evident that GABA mediates most inhibitory
transmission events in the vertebrate brain. It was long clear that the
fast, bicuculline-blocked response to GABA observed was caused by
direct activation of an intrinsic anion channel in an entity
subsequently termed the GABAA receptor. GABAB
receptors were recognized later as bicuculline-insensitive, baclofen-stimulated metabotropic GABA receptors (Hill and Bowery, 1981
)
linked to G proteins. Confirmation by the deoxyribonucleic acid (DNA)
cloning of a GABAB receptor, as a 7-transmembrane domain protein, has been accomplished recently (Kaupmann et
al., 1997
). The complete structural and functional distinction
between GABAA and GABAB receptors has a clear
parallel to that between nicotinic and muscarinic acetylcholine
receptors, between 5-HT3 and metabotropic serotonin
receptors, ionotropic and metabotropic glutamate receptors, or
ionotropic P2X and G protein-coupled P2Y
receptors for nucleotides.
-Aminobutyric acidC receptors.
A third type
of GABA receptor, insensitive to both bicuculline and baclofen, was
designated GABAC (Drew et al., 1984
). The GABAC responses are also of the fast type associated with
the opening of an anion channel; they are, however, unaffected by typical modulators of GABAA receptor channels such as
benzodiazepines and barbiturates (Sivilotti and Nistri, 1991
; Bormann
and Feigenspan, 1995
; Johnston, 1996
). Native responses of the
GABAC type have occurred in retinal bipolar or horizontal
cells across vertebrate species (Feigenspan et al.,
1993
; Quian and Dowling, 1993
; Lukasiewicz, 1996
) and can be expressed
by rat retinal messenger ribonucleic acid (mRNA) injection in the
oocyte system (Polenzani et al., 1991
).
subunits are expressed, and
subunit mRNAs occur
prominently in both human and rat retina (Cutting et
al., 1991
; Enz et al., 1995
; Ogurusu et
al., 1995
, 1997
; Zhang et al., 1995
).
subunits are structurally part of the family of GABAA receptor subunits (Shimada et al., 1992
; Kusama
et al., 1993a
,b
), although their regulatory binding
sites are obviously very distinctive. It would be unsatisfactory to
separate these two branches of the ionotropic GABA receptor family as
GABAA and GABAC receptors, with a metabotropic
family, GABAB, lying between them. Moreover, if the
designation of GABAC were retained, then it would be
difficult to refuse the extension to GABAD, etc., types for
ionotropic receptors which do not match either of the previously
recognized GABAA and GABAC specifications. This
would further decrease the logic of the
GABAA/GABAB classification scheme. Thus, Sato
et al. (1996)
have proposed such a
"GABAD" type, for an embryonic brainstem ionotropic
GABA receptor that is insensitive to both GABAA and GABAB antagonists and is activated by both
GABAA and GABAB agonists. Again, Perkins and
Wong (1996)
have suggested, based on an anomalous current evoked by
GABA in hippocampal pyramidal neurons, that a "GABAD"
channel may occur there with a different ionic selectivity. We
therefore recommend that the term GABAC, as
well as sequential terms for any new classes for ionotropic GABA
receptors, be avoided. The
-containing receptors are best classified
as a specialized set of the GABAA receptors, as will be
shown below.
3. Benzodiazepine receptors.
The interaction with
benzodiazepines (BZ) (fig. 1) has been a
major influence in studies on GABA receptors because of the long
history of therapeutic application of BZs as anxiolytics, anticonvulsants, sedative-hypnotics, and muscle relaxants. Although the
BZs were introduced first into clinical practice in the early 1960s, it
was not until 1975 that these drugs were recognized to act by
potentiating the inhibitory action of GABA in the brain (Costa
et al., 1975
; Haefely et al., 1975
). The
presence of high-affinity, specific binding sites for BZs in the
mammalian brain was then demonstrated (Braestrup and Squires, 1977
;
Mohler and Okada, 1977
). Converging lines of evidence established that
these sites are in the same macromolecule as the GABA sites and the
chloride channel and that all three elements are coupled allosterically
(Chang et al., 1981
; Olsen, 1981
; Paul et
al., 1981
; Sigel and Barnard, 1984
). The term "GABA/BZ
receptor" came into use for this complex (and is still encountered).
Progress in this field until recently was driven by the synthesis of a
vast range of BZs and BZ-like drugs, all acting at brain
GABAA receptors and possessing clinical anxiolytic or
sedative potencies correlated to their binding affinities there
(Haefely et al., 1985
).
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-carbolines such as
methyl-6,7-dimethoxyl-4-ethyl-
-carboline 3-carboxylate (DMCM) or the
propyl ester of
-carboline 3-carboxylate (
-CCP) (as well as
1-N-trifluoromethyl-benzodiazepines), which can displace
[3H]BZ binding in a biphasic manner and possess a
different affinity for BZ receptors in the cerebellum than those in the
hippocampus or other brain regions, led to the concept of two
BZ-receptor subtypes possessing a differential localization (Lippa
et al., 1981
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4. Excitatory
-aminobutyric acidA receptors.
Yet another apparent distinction between sets of GABA receptors
arises from observations that GABA can be an excitatory transmitter at
certain loci in embryonic and early postnatal life in the mammal (reviewed by Cherubini et al., 1991
; Ben-Ari et
al., 1997
). The excitatory response also may mediate the
observed trophic role for GABA in nervous system development (Ben-Ari
et al., 1997
). Another form of excitatory GABA response
is seen in tonically stimulated adult hippocampal pyramidal neurons
(Staley et al., 1995
; Perkins and Wong, 1996
; Kaila
et al., 1997
). All the evidence on these excitatory
receptors indicates that they are GABA-activated anion channels, in
general similar to the inhibitory GABAA receptors. GABAA receptors therefore should be classified as one
general type, whether their transduction is a depolarization or a
hyperpolarization of the cell membrane. The subunit composition of
these excitatory receptors has not been determined yet. It is possible
that a different subunit composition increases the permeability of
bicarbonate relative to chloride through the receptor channel, or that
the subtypes involved are not necessarily different from those well known in the adult but that the chloride gradient across the cell membrane is inverted at the sites in question. Either of these situations could explain the observed excitatory GABAA
receptor activity. The relative bicarbonate permeability of the channel rarely has been measured for any identified GABAA receptor
subtype, but the possibility that it is increased in a particular case has been supported by Staley et al. (1995)
and Perkins
and Wong (1996)
. It need not be assumed that this would involve a
receptor outside the range of GABAA receptor subtypes.
Indeed, Kaila et al. (1997)
have shown that
activity-induced changes in intracellular chloride and bicarbonate and
extracellular potassium, along with normal GABAA receptor
function, can account for the GABA-excitatory phase in the tonically
stimulated adult hippocampus. Further, the intracellular chloride
activity of developing neurons (of the rat nucleus basalis) has been
measured using gramicidin-perforated patch recording and shows a large
decrease from the immediately postnatal to the mature brain, sufficient
to account for the excitatory and inhibitory responses, respectively
(Akaike et al., 1996
). Likewise, the internal chloride
concentration can be measured locally by confocal imaging based on a
chloride-sensitive fluorescence, and this has shown that dendrites on
some hippocampal or cortical neurons can exhibit a higher value than
somatic locations (Inglefield and Schwartz-Bloom, 1996
), confirming
earlier suggestions of such a gradient. This also must be distinguished
from subtype difference as a potential cause of the excitatory behavior
of GABAA receptors on some dendrites. As further evidence
for this, in the mature mammal the pituitary melanotropic cells are
known to possess a very high internal chloride level, and activation of
GABAA receptors (of normal pharmacology) there also is
depolarizing (Tomiko et al., 1983
). For all these
reasons, it is unnecessary to provide a specific designation for
receptors that mediate excitatory neuronal responses to GABA.
B. Conclusion on
-Aminobutyric Acid Receptor Types
All the available evidence suggests that GABA receptors can be classified simply as two types, i.e., ionotropic (the GABAA receptors) and metabotropic (the GABAB receptors). The criteria for classification into subtypes will be very different for these two receptor families. The combinatorial basis of GABAA receptor structure produces a remarkable diversity of receptor subtypes and requires a new form of classification scheme. The GABAB receptors must be classified separately and will not be considered further here. Likewise, the "peripheral BZ receptors" are unrelated to any GABA receptors and will not be classified here.
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II. Approaches to the Classification of the
-Aminobutyric AcidA Receptors |
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It previously has been accepted in this series of receptor
classifications (see Hoyer et al., 1994
) that the most
fruitful comprehensive system is one in which evidence from three
approaches, operational, structural, and transductional, is applied.
How can these be applied here?
A. Transductional Criteria
For an ionotropic receptor the transduction (intrinsic ion channel
opening or closing) is by definition the same for all of its subtypes.
The alternative intracellular pathways used in other receptor classes
have no counterpart here. However, in principle subtle differences
within this single transduction pathway still could occur. Thus, for
two subtypes being activated by the same agonist it might be possible
to measure different kinetic constants in the channel opening or
closing steps, or different desensitization behaviors, or different
distributions of the open and closed channel states. Such cases are
known for subtypes of other transmitter-gated channels, e.g., glutamate
receptors or P2X nucleotide receptors (reviewed
by North and Barnard, 1997
). Differences in the kinetic properties
among GABAA receptor subtypes have been
investigated rarely so far. In one case, Angelotti and Macdonald (1993)
found that some difference in the single-channel properties could be discerned in two recombinant GABAA receptors
expressed in a nonneural cell line. Likewise, expressed recombinant
receptors containing the
6 subunit exhibit, at
least in some cases, distinctive channel properties (Ducic et
al., 1995
). However, it may be difficult in practice to find such
discriminatory differences in channel properties for many of the
subtypes, as well as cumbersome to apply those in classification.
Further, in the native setting it will be difficult, if not impossible,
to determine whether any such difference instead is not caused either
by some intracellular secondary reaction (e.g., a phosphorylation) or
by the availability of a native modulator. Therefore, we will not
consider transductional criteria for classifying these receptors.
B. Operational Criteria
Selective antagonists have been the most powerful operational
tools for discriminating subtypes in other receptor classes (Kenakin
et al., 1992
). However, for the GABAA
receptors, antagonists at the GABA site generally produce convulsions
in vivo. Hence, therapeutic potential is limited and systematic
exploration of antagonists has not been developed. A few compounds
unrelated to GABA, such as certain arylaminopyridazines and cognate
compounds (Heaulme et al., 1987
; Melikian et al.,
1992
), have been developed as potent antagonists at
GABAA receptors generally. Olsen et
al. (1990)
have shown that the binding of such compounds can
discriminate between some subtypes in the brain; their functional study
to identify selective actions on recombinant subtypes could be
rewarding.
GABAA receptors are endowed with a variety of
modulatory sites for which ligands have been found that can
allosterically control the activation by GABA and/or the opening of the
anion channel. With the possible exception of the
N-methyl-D-aspartate subclass of glutamate receptors
(another family of heteromeric ligand-gated ion channels ubiquitous in
the brain), the number of different regulatory sites is greater than
for any other receptor type. Modulatory sites offer the potential for
discriminating among receptor subtypes, namely by the discovery or the
design of agents that can act at these sites but can recognize
differences in a given site as it occurs in different subunit
combinations. Thus far, this possibility has been realized to some
extent with the site at which BZ and molecules with BZ-like activity
bind, as we shall see. Other established modulatory sites, which can
exist on these receptors and which might be used thus include those for
barbiturates, neuro-steroids, propofol, certain other anesthetics, furosemide, zinc, picrotoxin, and some other channel blockers, loreclezole, substituted pyrazinones, and dihydro-imidazoquinoxalines. Those compounds and the evidence of their interaction with
GABAA receptors are reviewed by Sieghart (1995)
,
Im et al. (1993a
,b
), Wafford et al. (1994)
, and
Korpi et al. (1995)
. Only occasional clues to subunit
selectivity have been obtained for any of the latter sites.
C. Structural Criteria
The multisubunit compositions of the GABAA receptors, which create the subtypes, are of primary importance in their classification. In practice, it is not a straightforward task to use the subunit sequences and the subunit assemblies as the primary basis of a classification, a topic which now requires a fuller discussion below.
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III. The Structures of the -Aminobutyric AcidA
Receptors |
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A. The Repertoire of Subunit Types
Cloning from cDNA libraries or genomically so far has generated 19 related GABAA receptor subunits in mammals, which
are each encoded by different genes. These now comprise 6
, 4
,
3
, 1
, 1
, 1
, and 3
mammalian types [for references see
Burt and Kamatchi, 1991
; plus (
) Davies et al., 1997
and
Whiting et al., 1997
; (
) Heblom and Kirkness, 1997
;
(
1-3), see Section I.A.2.; for database
accession numbers see fig. 3]. These
polypeptides are all ~50,000 daltons in size, and each carries four
putative transmembrane hydrophobic segments (TM1-4). Figure 3
illustrates the seven different sequence families into which these fall
structurally and their relationships. A mammalian counterpart of the
avian
4 subunit (Harvey et al.,
1993
) has not yet been isolated by cDNA cloning and so is not included
here. However, the
4 subunit gene, likewise discovered in the chicken (Bateson et al., 1991
), has been
shown more recently in humans (Levin et al., 1996
).
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This heterogeneity is increased by alternative exon splicing of the
pre-mRNA, which generates two forms of the
2
subunit from one gene (Whiting et al., 1990
; Kofuji et
al., 1991
), which can be distributed differently in the brain
(Glencorse et al., 1992
). Two such forms are also known for
the
2 and
4 subunits (Bateson et al., 1991
; Harvey et al., 1994
). In
each case, the longer and shorter products were designated "L" and
"S," and differ by some form or other of a short peptide in the
long intracellular loop between TM3 and TM4. Splicing also occurs to
express two alternative forms of exon-1 of the
3 subunit (Kirkness and Fraser, 1993
). Three
potential forms of the
5 subunit mRNA also exist (Kim
et al., 1997
) but with unchanged protein sequence. Another product of alternative splicing deletes a short sequence at the N-terminus of the
6 subunit (Korpi et
al., 1994
), although this abolishes the functional receptor
activity in all the combinations tested so far. Therefore, in assessing
possible combinations of subunit types (other than
) to form a
GABAA receptor, we must consider in a given
mammalian species, including the splice variants, at least 7
forms,
7
forms, 4
forms, 1
, 1
, and 1
form. The recently
discovered
and
subunits in each case can combine with
and
subunits to form a functional, BZ-insensitive receptor (Davies
et al., 1997
; Heblom and Kirkness, 1997
; Whiting et
al., 1997
). The
subunit has been detected clearly so far only
in certain peripheral tissues (Heblom and Kirkness, 1997
), and its range of combinations has not been defined yet. The
GABAA receptors in the central nervous system
(CNS) are formed, on present knowledge, by combinations of both
and
subunits with one or more of the
,
or
subunit types (or
possibly, exceptionally, of
and
types alone). In addition there
are three known
subunits that occur in the retina:
1 (Cutting et al., 1991
);
2 (Cutting et al., 1992
; Kusama
et al., 1993b
);
3 (Ogurusu and
Shingai, 1996
; Shingai et al., 1996
). In co-expressions,
evidence was not obtained to show that a
subunit can participate in
combinations with the aforementioned
,
, or
types (Shimada
et al., 1992
; Kusama et al., 1993a
), although
more recently a
1
2
heteromer forming in heterologous expression was suggested (Pan
et al., 1997
). In the rat retina, however, a recent study by
immunofluorescence microscopy showed punctate localizations of non-
GABAA receptors and of
-containing receptors,
which occur at different synapses and do not overlap (Koulen et
al., 1998
). Hence, a pool of at least 20 subunit types may be used
in forming combinatorially the CNS GABAA
receptors, plus at least 3
subunit types which assemble in a
restricted manner.
B. The Subunit Number per Receptor Molecule
To understand the construction of GABAA
receptor subtypes from this repertoire of subunits, it is necessary
first to establish the total number of subunits in each receptor
molecule, then to know whether this number is constant for all the
native compositions, and finally to know the stoichiometry of the
subunit types within that number. Regarding the number of subunits per
receptor, the suggestion often has been made that this will be the same
(five subunits) as for another transmitter-gated ion channel where the composition has been established unequivocally. Thus, the
GABAA receptor subunits share a low but definite
(~25%) amino acid sequence homology with the subunits of the
nicotinic acetylcholine receptors, both being in the same superfamily
of the transmitter-gated ion channels (Schofield et al.,
1987
; Barnard, 1996b
). The muscle type of that receptor occurs in
Torpedo electric organ at such a high density in large
postsynaptic membrane sheets that it is possible to prepare membranes
containing a surface lattice of the receptors, from which a
low-resolution three-dimensional structure of the molecule could be
obtained by electron optical diffraction techniques (Toyoshima and
Unwin, 1988
; Unwin, 1993
). Those studies clearly showed that the muscle
type nicotinic receptor is pentameric, with the ion channel located in
the center of a rosette formed by five homologous subunits (with the
stoichiometry
2 

).
For the GABAA receptors, the situation is
necessarily more complex, because the unique situation in the
Torpedo postsynaptic membranes does not recur in the
mammalian CNS and because there are many types of subunits involved, in
varying combinations, in the receptor population. It is preferable,
therefore, to use the natural GABAA receptor
population from the brain rather than a selected recombinant
composition expressed in a nonneural cell, which may or may not be
representative of the native population; further, when direct analyses
are made on the latter, these will not be limited by an assumption of
the subunit classes to be taken as co-assembling. Using purified
GABAA receptors from pig brain cortex and image
analysis in the electron microscope, dispersed single receptor
molecules can be visualized and analyzed (fig. 4). This method yields a power spectrum
for each particle with a peak at its dominant symmetry. Figure 4
illustrates that this symmetry is five-fold, over the population of
particles analyzed (Nayeem et al., 1994
). Further, the
negatively stained images obtained for all the receptor particles
indicated a central pore in the pentameric rosette. These data
correspond to the images observed with negatively stained
Torpedo receptor particles, because of a central channel in
the membrane enclosed within the pentameric receptor in the latter case
(Toyoshima and Unwin, 1988
). The particles isolated from brain will
comprise a variety of GABAA receptor subtypes.
These data show that at least the majority of those receptors are
pentameric; a deviating small minority with an atypical subunit number
would not be distinguished from the experimental noise. Independent
evidence to support the pentameric structure has been obtained in
several ways. Hydrodynamic estimates of the size of
GABAA receptors, either native (Mamalaki et
al., 1989
) or
1
3
2
recombinants (Tretter et al., 1997
), in solution are consistent with the pentameric molecular weight. Further, the integral
ratios of the subunits combined in several forms of functional recombinant receptors, as determined by diverse methods, fit best in
each case with a subunit total of five (Im et al., 1995
;
Chang et al., 1996
; Tretter et al., 1997
). For
parallel evidence, a method similar to that of Nayeem et al.
(1994)
has been used for the native 5HT3
receptors by Boess et al. (1995)
and there is supporting
evidence by other methods for the neuronal nicotinic receptors and the
glycine receptor (reviewed by Barnard, 1996b
), all of these being in
the same superfamily and all being deduced to be pentameric. In
view of this concurrence with other receptors in the same superfamily,
it is presumed that the pentameric structure that has been observed,
within experimental error, for GABAA receptors holds for at least the great majority of that receptor type.
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It can be concluded that the repertoire of (at least) 24 mammalian
subunit isoforms described above is drawn on to a total of 5 for each
receptor molecule. Evidence discussed below will show that in most (but
not all) GABAA receptors
,
, and
subunits co-exist in one molecule, and that yet other combinations
exist accommodating in special ways the other subunit types,
,
,
, and (separately)
. It is assumed that the
-containing
receptors are also pentameric, but this question has not been studied
as yet.
C. The Subunit Isoforms in One Receptor
The majority of GABAA receptors contain, as
noted,
,
, and
subunits, whereas the total number of subunits
per receptor is five (fig. 4). Hence the receptors in this set can have
at least one of three general compositions:
2
.2
.
; 2
.
.2
;
.2
.2
. Here, a notation is introduced in
which the numeral represents the number of molecules of a given subunit
class (
,
, etc.) present in one receptor molecule and not the
isoform identity within that class; separating points are then used,
and are absent when the stoichiometry is not being indicated. Such
additional cases as 3
.
.
and
.
.3
are theoretically
possible, but measurements of an electrophysiological property
determined quantitatively by the number of tagged recombinant subunits
of each type forming the channel (Backus et al., 1993
; Chang
et al., 1996
), in the cases of co-expression of the
3
2
2 or
1
2
2
subunits, have excluded (at least in those cases) the presence of three
of any of those types in one receptor molecule. The next logical step
in enumerating the potential combinations of subunits, therefore, is to
ask whether two isoforms of
or of
or of
can occur in one receptor molecule, e.g., to produce compositions
of the type
(
1
2).2
.
.
In the
subunits, there is a variety of evidence for such a
co-occurrence of isoforms in a minority of GABAA
receptors. This evidence has come first from co-precipitation of a
second
isoform when a brain-derived population of
GABAA receptors is treated with an antibody
specific for a first
isoform. Receptors containing at least the
pairs
1
2,
1
3,
1
5,
2
3, and
3
5 have been detected
thus (each in a minority, with the majority of receptors in the
population containing a single
isoform) (Duggan et al., 1991
; Luddens et al., 1991
; Zezula and Sieghart, 1991
; Endo
and Olsen, 1993
; Mertens et al., 1993
; Pollard et
al., 1993
; Khan et al., 1996
; McKernan and Whiting,
1996
). Further, for the
6 subunit that occurs
(in the mature brain) only in the cerebellar granule cells (Laurie
et al., 1992
; Thompson et al., 1992
) and in the
similar granule cells of the cochlear nucleus (Varecka et
al., 1994
), antibody reactivities show
1
and
6 co-occurring in one cerebellar receptor
(Pollard et al., 1995
; Khan et al., 1996
),
although not for all the
6 subunits there. For
the
subunits, the similar use of isoform-specific antibodies has,
on brain extracts or purified receptor preparations, shown evidence for
the co-occurrence of
2 with
3 and also of
2L with
2S (Khan et al., 1994a
,b
; Quirk
et al., 1994a
).
A second method for investigation of possible co-occurrence of
particular isoforms is the application of isoform-specific antibodies
in situ, i.e., in light or electron microscopic studies (table
1). Thus, by confocal laser microscopy
with double or triple immunofluorescent staining, Fritschy et
al. (1992)
and Mohler et al. (1996a)
have found that
certain
pairs were co-localized on the membranes of various neurons
(table 2). Co-localization of
1 and
6 subunits in
single synapses of rat cerebellar granule cells also has been
demonstrated by double-antibody labeling in postembedding electron
microscopy (Nusser et al., 1996
), although when used in a
freeze-fracture method on such cells in culture a co-localization of
1 and
6 was not seen
(Caruncho and Costa, 1994
). Third, some electrophysiological properties
of a recombinant 

assembly containing two isoforms of
can
be distinct from those with either isoform separately, demonstrated
with
1
3 or
1
5 pairings (Ebert
et al., 1994
; Verdoorn, 1994
).
|
|
Further, the
subunit often has been found to replace
subunits:
Quirk et al. (1995)
found that
and
are completely
separable by antibodies [although Mertens et al. (1993)
found some co-existence].
subunits were present in only 11% of
all the receptors in rat brain but in 27% of those in rat cerebellum,
from which both
6
n
and
6
n
2
combinations can be isolated (Quirk et al., 1995
) and where
Caruncho and Costa (1994)
found in situ that the receptors contain
either a
or a
subunit, but not both, by a
label-fracture method. The subunit
(which has some similarities to
) also may replace
in some cells of the hypothalamus and
hippocampus (Whiting et al., 1997
).
Receptor gene knock-out can provide additional evidence. In favorable
cases it can show the co-occurrence of certain pairs of subunits. Thus,
the homozygous mice lacking the
6 gene also lack the
subunit protein in the cerebellar granule cells and the
results obtained support other evidence that
6
and
are paired in receptors there and not
1 and
without
6
(Jones et al., 1997
). The specific pharmacology of
6
-containing receptors was confirmed in
vivo in this system (Mäkelä et al., 1997
).
D. Possibilities for Subunit Stoichiometry
On the basis of the extensive evidence reviewed above, that two
isoforms of the
subunit can sometimes occur in one receptor, the
receptors are considered as having two
places in the pentamer. Pollard et al. (1995)
supported this by quantitation in the
1
6-containing cerebellar receptor. Likewise, Khan et al. (1994a
,b
) and
Quirk et al. (1994a
,b
) found that two different
isoforms can co-occur, although Mossier et al. (1994)
and Im et al. (1995)
did not find this; if the former
statement holds two
places can also be in the pentamer. However, it
is not known whether any conclusion of this type would apply to the
entire native population of GABAA receptors.
Because
has been observed at some sites (see above) to occur with
and
subunits only, a plausible model is that either
or
(and perhaps
) subunits can occupy the
places (in different
receptors). We will give an illustration here, only of the basis on
which the theoretical maximum number of receptor compositions may be
assessed.
Some of the native GABAA receptors may have the
stoichiometry 2
.
.2
(with the
subunits in some cases being replaceable by
or by
). Several
lines of evidence support this. Thus, for the
2-containing receptors, there is evidence from immunoprecipitation analyses (as noted above) that the
2
3 pairing within one
receptor molecule can occur in some cases (Khan et al.,
1994b
; Quirk et al., 1994a
) and also that a subset of
receptors in the cerebellum has the composition
1
6.
.
2S
2L (Khan et al., 1994b
, 1996
).
Further, Backus et al. (1993)
have deduced, by incorporating
mutant subunits with altered electrophysiological effects in the
recombinant
3
2
2
receptor [expressed in human embryonic kidney (HEK) 293 cells], that
the 2
.
.2
composition best fitted the
properties found.
On the other hand, Chang et al. (1996)
, using a similar
principle (in oocytes and using
1, not
3 subunits), found that there the evidence
apparently favors the 2
.2
.
composition. The same stoichiometry also was derived for
1
3
2
receptors, when expressed in HEK 293 cells, from the staining ratios of
those subunits when separated in Western blots (Tretter et
al., 1997
). Moreover, the co-occurrence of
1 with
3, and of
2 with
3 (but not
1 with
2), isoforms
has been indicated in some of the receptors from rat cortex by
immunopurification (Li and De Blas, 1997
) and likewise in rat
cerebellum (Jechlinger et al., 1998
). Benke et al. (1994)
compared the fractions from rat whole brain containing
1,
2, or
3 subunits by immunoprecipitation and also
excluded the
1
2
combination; however, in contrast to the findings just noted, they
found that the
1
3 or
2
3 pairings also were
absent. Overall, it is desirable to allow for possible
2
.2
.
forms in the nomenclature. In view
of this situation and of the evidence for
2
.
.2
combinations, Li and De Blas (1997)
suggested that the ratios of the
and
subunits in the molecule
(within the total of 5) may vary with the isoforms selected.
In the expression of recombinant receptors in either cultured cells or
oocytes, any ternary combination of the
i
j
k type tested so
far can yield a functional receptor in the membrane (e.g., Kirsch
et al., 1995
). The limit to the number of ternary subtypes
in vivo apparently is not set by barriers to the co-assembly in
certain cases but by the program for gene expression of different isoforms in a given cell. However, in a case where this was tested (Angelotti and Macdonald, 1993
), when such an
+
+
set is
expressed the ternary combination assembles (as far as the subunits are available) and is maintained at the cell membrane to the exclusion of
binary combinations. Within the ternary assemblies, there are no
obligate combinations or exclusions of 
pairings known from the
co-distribution data at the present resolution limits. However, some
exclusions are known (see above) at the
/
position. Moreover, the
subunit has a more restricted expression in the brain than
subunits (Wisden and Seeburg, 1992
) and has fewer co-occurrences with
other subunits; the same is true for
(Whiting et al.,
1997
), whereas
is clearly detectable in certain peripheral tissues only (Heblom and Kirkness, 1997
). Hence those subunits cannot be
included on the same basis as the others in permutations of the
possible compositions.
An enumeration is obtained on the basis that, for a given subunit set
which will form one receptor, there will only be one arrangement and
stoichiometry in the molecule. This is found to be so with all other
heteromeric proteins containing tightly-bound subunits; for example,
there is only one cyclic order of the subunits,
.,
.,
.,
.,
, present in the population of
Torpedo acetylcholine receptors (Karlin, 1991
). Moreover,
with those subunits one does not find that the same receptor type, in a
variety of skeletal muscles, can contain another stoichiometry. That
constancy and the circular order of subunits around the rosette are
fixed by the interactions between the interfaces of different subunits. In the case of a GABA receptor (the recombinant
1
1
2S),
supporting evidence for a single configuration in the population, from
the homogeneity of the channel properties, has been reported (Angelotti and Macdonald, 1993
).
Therefore we do not count all possible permutations (n = 36) of 2
isoforms present (out of the 6), but only those for a fixed order (n = 21); likewise, for the others. Splice
variants could increase these numbers on the same basis, except that
two alternatives from one subunit cannot be assumed to be able
necessarily to co-occur. From what is known so far of excluded
compositions and the restricted co-occurrence of subunits in certain
cases, Barnard (1996a)
has suggested that a maximum of the order of 800 combinations, of the types observed so far, would then be calculated. The true number is likely to be far smaller than this, but still much
larger than for other known receptor subtypes.
The
subunits apparently assemble separately from the others
(discussed below). They do not affect the enumeration above, but can
add a few separate subtypes in the total noted above.
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IV. Principles of the Classification |
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|
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A. Application of Selectivities at the Binding Site for
Benzodiazepines and Their Functional Analogs
1. The choice of a classification system.
As noted above, this
modulatory site presently offers by far the richest pharmacology for
distinguishing subtypes of the GABAA receptors. BZs have no
intrinsic activity on mammalian GABAA receptors, unlike
some of the other modulators such as anesthetics (although such a
direct effect of some BZs has been found at invertebrate GABAA receptors; Zaman et al., 1992
). Most
BZs act to enhance the action of GABA by increasing the frequency of
channel openings and their bursts (Rogers et al., 1994
).
This can be explained partly by the ability of BZs to increase the
affinity of GABA at its binding site. However, in tonic activation of
hippocampal neurons by low GABA concentrations they also can increase
the channel conductance (Eghbali et al., 1997
).
; Braestrup et al., 1982
),
for which the late Willy Haefely introduced the term "inverse
agonist," i.e., having negative efficacy at this site (fig. 1). One
ligand class, exemplified by flumazenil, Ro14-7437, ZK 93426, or RP
60503, has such low efficacy (at most subtypes) that they effectively
act as antagonists at this site (fig. 1). The wide range of BZs and
other ligands active at the same site (table
3) that was examined has led to compounds
which discriminate among some of the subtypes. When tested in
recombinant subunits expressed (in either Xenopus
oocytes or transfected mammalian cells) in various combinations, a
variety of such effects can be found, as will be detailed below. For
those cases where subtypes are established, the nomenclature for them ideally would be based only on molecular biology and would express the
subunit composition, e.g., the
"
1
2
2" subtype (which
then could be abbreviated as GABAA122, etc.). Even this
scheme would be cumbersome to use, e.g., needing expansion to cover all
the subunit forms, etc., as discussed in Section IV.B. below. It is acceptable for stating the composition of an experimentally expressed mixture of recombinant