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IUPHAR Nomenclature Report |
-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. UpdateDepartment of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, California (R.W.O.); and Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S.)
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I. Introduction: Definition of GABAA Receptors |
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-Aminobutyric acid (GABA1), the major inhibitory neurotransmitter in the brain, exerts its action via ionotropic GABAA and metabotropic GABAB receptors. GABAA receptors (GABAA-Rs) are the major inhibitory receptors in the central nervous system (CNS). They were first identified pharmacologically as being activated by GABA and the selective agonist muscimol, blocked by bicuculline and picrotoxin, and modulated by benzodiazepines, barbiturates, and certain other CNS depressants (Macdonald and Olsen, 1994
; Sieghart, 1995). GABAB receptors are activated by GABA and the selective agonist baclofen but are not sensitive to typical GABAA-R ligands, such as bicuculline and muscimol. GABAB receptors couple to several different effector systems, depending on the associated G protein (e.g., Gi/o), such as activation of inwardly rectifying K+ channels or inhibition of high voltage-activated Ca2+ channels, involving regulation of cyclic AMP or inositol phosphate signaling (Bowery et al., 2002; Bettler et al., 2004).
The objective of this review is to define GABAA-Rs and, in so doing, to summarize classifications and provide guidelines on nomenclature. This should serve to update the nomenclature suggested by Barnard et al. (1998), which remains useful and relevant, for most issues. GABAA-Rs are chloride channels that are gated by GABA and mediate rapid phasic inhibitory synaptic transmission and also tonic inhibition by producing current in extrasynaptic and perisynaptic locations (Mody and Pearce, 2004; Farrant and Nusser, 2005). They are abundant in the nervous system of all organisms with a nervous system, including invertebrates (Buckingham et al., 2005), which also express GABAA-Rs on some muscle cells (Robinson and Olsen, 1988), where they also mediate Cl--dependent inhibition. GABAA-Rs are found, but in a limited capacity, in non-neural tissues [such as the pancreas (Borboni et al., 1994)], where their functional roles are still under study and their pharmacological relevance remains to be established. Consistent with the emphasis of the Nomenclature Committee of the International Union of Pharmacology (IUPHAR), the receptors considered here are limited to mammalian species, with emphasis on humans. Because of their widespread localization throughout the mammalian nervous system, GABAA-Rs play a major role in virtually all brain physiological functions and serve as targets of numerous classes of drugs, used both clinically and important as research tools. The GABAA-Rs are a family of probably many receptor subtypes, but so far only a few dozen subtypes have been identified with reasonable certainty. Because of recent advances in knowledge of their molecular makeup, identification of native subunit compositions, and relevance to pharmacological specificity, an update of the list of GABAA-Rs is in order. This list is necessarily a continually changing work in progress.
A. Ionotropic GABA-Gated Anion Channels
Mammalian GABAA-Rs are all anion-selective channels. Increased chloride permeability generally reduces neuronal excitability (inhibition), because the Cl- equilibrium potential in most mature neurons is near the resting membrane potential and the concentration of chloride within the neuron ([Cl-]i) is much less than that within the extracellular fluid ([Cl-]o) (Martin and Olsen, 2000). However, depending on expression of Cl- transporters, [Cl-]i can increase, leading to a Cl- equilibrium potential that is less negative than the resting membrane potential. Under such conditions, activation of GABAA-Rs can cause membrane depolarization, possibly sufficient to elicit action potential discharge (excitation). The situation occurs in nature and is especially relevant in early development (Ben-Ari, 2002). In addition, on strong activation of GABAA-Rs, the resulting increase in [Cl-]i might shift the membrane potential toward the firing threshold, causing rebound excitation of neurons (Marty and Llano, 2005). GABAA-R channels can conduct other anions with variable permeability ratios relative to Cl-. HCO -3 flux could be physiologically relevant under certain conditions (Kaila et al., 1997). The importance of bicarbonate varies with tissue and is dependent on carbonic anhydrase activity, including the tissue isozyme expression, as well as anion pumps (Rivera et al., 2005).
B. The Cys-Loop Pentameric Ligand-Gated Ion Channel Superfamily
GABAA-Rs are part of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily, including nicotinic acetylcholine receptors (nAChRs) (Corringer et al., 2000; Lukas and Bencherif, 2006), glycine receptors (GlyRs) (Breitinger and Becker, 2002), ionotropic 5-HT receptors (5HT3Rs) (Davies et al., 1999; Thompson and Lummis, 2006), and a Zn2+-activated ion channel (Davies et al., 2003). They differ in structure from two additional LGIC families: the tetrameric glutamate receptors (P2X) (Chen and Wyllie, 2006) and the trimeric purine receptors (Khakh et al., 2001; Khakh and North, 2006). Whereas the nAChR, 5-HT3R, and the Zn2+-activated channels are cation-selective channels and thus excitatory, the GABAA-R and GlyR families are anion-selective channels and, thus, with the exceptions noted above, mediate inhibition. All of the subunit members of the Cys-loop LGIC superfamily show sequence homology on the order of 30% identity but even greater similarity at the level of secondary and tertiary structure. Such receptors are all organized as pentameric membrane-spanning proteins surrounding a central pore that forms the ion channel through the membrane (Fig. 1). They all use similar sequences and functional domains to establish membrane topology, ion channel structure, agonist binding sites, and even binding sites for diverse allosteric ligands. Each subunit consists of a long N-terminal extracellular hydrophilic region, followed by four transmembrane (M) 
-helices with a large intracellular loop between M3 and M4, and ends with a relatively short extracellular C-terminal domain. M2 forms the lining of the ion channel, with a possible contribution from M1 (Corringer et al., 2000; Sine and Engel, 2006). An -helical domain within the M3–M4 cytoplasmic loop has also been shown to influence ion conduction (Peters et al., 2005). The structure of the Cys-loop LGIC superfamily has been investigated by a large number of biochemical approaches, with current work emphasizing domains for ligand binding and coupling to ion channel gating, as well as subunit-subunit interactions and investigation of the role of intracellular domains. All of these data were confirmed and fell into place when combined with X-ray crystallography data on the snail soluble acetylcholine binding protein (Brejc et al., 2001), recently followed by that of the water-soluble portion of the nAChR 1 subunit (Dellisanti et al., 2007). The complete channel structure has been progressively well resolved (currently 4 Å) for the nAChR of Torpedo marmorata obtained by cryoelectron microscopy and image reconstruction (Miyazawa et al., 2003, Unwin, 2005).
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C. The GABAA Receptor Family of 19 Genes
With the complete sequence of the genome for human and a few other vertebrate species, it is now clear that there are 19 genes for GABAA-Rs (Fig. 2) (Simon et al., 2004). These include 16 subunits (1–6, β1–3, 1–3, 
, 
, 
, and 
) combined as GABAA and 3 
subunits, which contribute to what have sometimes been called GABAC receptors. Birds and probably some other species additionally express β4 and 4, but lack and subunits, so also total 19. The sequence of is closely related to subunits (Fig. 2) and to β, suggesting an evolutionary relationship, perhaps more evident in birds than mammals. The list is modified from Barnard et al. (1998) in that we include the , but not the β4, subunit.
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Subunits and the GABAC Receptor Concept: Not Recommended
"GABAC receptors" were originally described precloning, based on a distinctive pharmacology, to encompass responses to GABA that did not fit either the "A" (blocked by bicuculline) or "B" (activated by baclofen) category. Such "nonA, nonB" GABA responses were found in numerous regions of the CNS. Attempts to find such a nonA, nonB GABA-activated current by expressing poly(A)+ RNA from brain in oocytes were unsuccessful but a cDNA expressing this sort of GABA-evoked current was isolated from retina (Woodward et al.,1992). After cloning of the various GABAA receptor subunit genes, it was demonstrated that the subunits are closely related in sequence, structure, and function to the other GABAA-R subunit families designated with other Greek letters and thus qualify for inclusion within that family. It is now clear that there are three subtype genes of subunits that make homopentameric chloride channels. Such -receptors show some of the pharmacological properties of the GABAC receptors, such as sensitivity to the GABA analog, CACA (cis-aminocrotonic acid). They are relatively insensitive to bicuculline and also to GABAA-R modulators such as benzodiazepines, barbiturates, and general anesthetics, at appropriate concentrations. GABAC receptors are sensitive to picrotoxin, neurosteroids, and some other drugs, but the overall pharmacology differs from that of most traditional GABAA-Rs (Bormann and Feigenspan, 1995; Johnston, 1996). The subunits are not simply equivalent to GABAC receptors, because some regions of the nervous system seem to lack subunits and yet exhibit GABAC (i.e., non-A, non-B) pharmacology. The subunits are all expressed primarily in the retina, but unlike the 1, the 2 and 3 subunits are also found elsewhere (Johnston, 2002). At this time it is not clear whether 1–3 can combine with each other in heteromers. Enz and Cutting (1999) claimed that recombinant 1 and 2 subunits can form hetero-oligomers with distinct physical properties. In native receptors this is difficult to prove because there are no antibodies that distinguish between different subunits. Nor is it established whether 1–3 can combine in nature with members of the other 16 GABAA-R subunits. Some evidence suggests these possibilities but it is not decisive (Sieghart and Ernst, 2005). For example, coassembly of and 2 subunits was reported (Milligan et al., 2004; Pan and Qian 2005), and some cells in hippocampus show GABAA-R-like properties intermediate between and 2-containing GABAA-Rs (Hartmann et al., 2004). There is also some evidence for association of with GlyRs (Pan et al., 2000).
The close structural similarities of subunits to the other GABAA-R subunits, the similarities in anion channel structure and function, the important fact that other subtypes of GABAA-Rs differ in pharmacology, such as benzodiazepine sensitivity, from each other to a similar degree as do the receptors, and the possibility of subunits partnering with other GABAA-R subunits, led to the decision of the Nomenclature Committee of IUPHAR to designate the GABA receptors as part of the GABAA-R family and to recommend against the use of the term GABAC receptor. It is especially recommended that the name GABAC receptor should not be used as the sole name for the receptors in an article including, especially, the title and abstract. The subunits are also discussed in section III.C.1.a.
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II. Structural Basis of Receptor Classification |
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The sequence homology of all 19 GABAA-R gene products to each other and to the Cys-loop pentameric LGIC superfamily lends itself to attempts to classify the receptors collectively. All members of the superfamily are homologous not only in the domains specifying membrane topology but also at the functional domain level, including ligand binding sites (Sigel and Buhr, 1997; Corringer et al., 2000; Olsen and Sawyer, 2004; Sine and Engel, 2006). Thus, homologous M2 residues are involved in channel structure and ion selectivity (Keramides et al., 2004), and the same multiple loops of extracellular sequence domains contribute to agonist/antagonist binding pockets (Galzi and Changeux, 1994; Sigel, 2002). This allows homology structural modeling of the various functional domains comparing GABAA-Rs and nAChRs (Cromer et al., 2002; Ernst et al., 2003; Ernst et al., 2005). Even the binding sites for some allosteric modulators also show considerable homology with agonist binding sites. For example, the major benzodiazepine binding site lies at the / subunit interface and involves residues homologous to the agonist binding loops at the β/ interface, which in turn are homologous in all members of the superfamily (Smith and Olsen, 1995; Sigel and Buhr, 1997; Corringer et al., 2000).
1. Subunit Gene List. The GABAA-R receptor subunit genes form a family of 19 (Table 1).
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2. Subunit Splice Variants.
Splice variants have been reported for only a few GABAA-R subunits, primarily 2 (Whiting et al., 1990; Kofuji et al., 1991). The 2 subunit splice variants differ in only an eight-amino acid stretch of the large intracellular loop that is present in the 2L and missing in the 2S subunit. The sequence includes a consensus protein kinase C phosphorylation substrate serine. No functional consequence of phosphorylation of the unique 2L serine residue has been convincingly demonstrated. The 2 splice variants are both expressed and show differential abundance in different brain regions (Gutiérrez et al., 1994; Meier and Grantyn, 2004). In addition, they show differential aging-related changes in their level of expression (Gutiérrez et al., 1996). Brain membranes from genetically engineered mice expressing only the 2S exhibit increased affinity for benzodiazepine agonists, an effect paralleled by increased sensitivity to such compounds in behavioral responses [e.g., increased "sleep" times (loss of righting reflex)] in null allele mice (Quinlan et al., 2000). The subunit composition of the receptors responsible for such changes is unknown, and, indeed, receptors produced in compensation might include normally non-native oligomers.
Alternative splice products in the intracellular loop in β2 and 3 subunits are found that, like 2L, also include consensus phosphorylation substrate sequences. Alternative start sites create multiple mRNA species for β3, 3, and 5 subunits, and variants lacking one or more exons have been found for 1, 4, β2, and subunits (reviewed in Simon et al., 2004). To date, and this could change, none of these variant polypeptides have been demonstrated to be present within functional receptors, nor do they confer any unique function or pharmacology in recombinant expression systems. There is at least one report of RNA editing in the GABAA-R family (Ohlson et al., 2007), but no evidence so far for functional relevance.
B. Heteropentameric Assembly Produces Complex Subtype Heterogeneity
The assembly of GABAA-R as heteropentamers produces complex subtype heterogeneity in structure, which is the major determinant of their pharmacological profile. These various subtypes differ in abundance in cells throughout the nervous system and thus in functions related to the circuits involved. A major factor in producing heterogeneity is the existence of the six different subunit variants. Some of these physiological receptor subtypes containing specific subunits can be distinguished by ligands that act at the benzodiazepine site (Barnard et al., 1998). In addition, there are now clear examples of β and or or other subunit selectivity for drug action on GABAA-R subtypes (see section III.B.2). Clearly it is the nature, stoichiometry, and arrangement of the subunits that determine details of pharmacological selectivity. Thus, pharmacology can often provide evidence for structural heterogeneity and informs us about the subunit composition of native receptor subtypes.
Evidence is greatly in favor of a pentameric receptor and most GABAA-R subtypes are formed from two copies of a single , two copies of a single β, and one copy of another subunit, such as , , or (Sieghart and Sperk, 2002; Olsen and Sawyer, 2004). At this time the subunit composition, stoichiometry, and wheel alignment are not known for most pharmacological subtypes: tentative definitions of subtypes can be provided as a work in progress. Current knowledge allows elimination of most of the thousands of permutations theoretically possible for combinations of the known 19 subunits into five-part (pentameric) complexes. Barnard et al. (1998) suggested a maximum on the order of 800 combinations and probably far fewer in reality on the basis of current knowledge of identified subunit partnering and apparent rules of assembly in neurons. Very few combinations have been conclusively identified in situ. We suggest some criteria for inclusion in a list of native subtypes and begin to generate such a list. Current evidence suggests that only 11 subtypes can be listed as conclusively identified, and these are reasonably abundant. We have also listed several subtypes for which the evidence is strong but not conclusive (six subtypes). We finish by mentioning subtypes containing one of each of the minor subunits, evidence for whose native existence is tentative (another eight, plus one subtype with two kinds of subunit), for a grand total of 26. An additional similar number of subtypes, which are relatively rare, but are likely to exist, are not listed at this time, but the list will continue to grow as more information becomes available. Each subtype could play a significant role in the cells in which they occur. It should be noted that even these minor GABAA-R subtypes are present in amounts comparable with or greater than receptor subtypes for accepted brain neurotransmitters other than glutamate, that is, the biogenic amines and acetylcholine. The heteromeric LGICs clearly offer much greater heterogeneity than other known receptor subtypes.
C. Criteria for Inclusion on a List of Native Receptor Subtypes
1. Subtypes Based on Structure, Pharmacology, and Function: Nomenclature Guidelines. Criteria are needed to define which receptor subtypes can be accepted as being native to neurons. We suggest basing the criteria on structure, pharmacology, and function. The list of subtypes will necessarily be a work in progress as information on all LGIC families is currently incomplete. We propose five major criteria: two for recombinant studies and three for native studies, each with subclassifications, for inclusion of a subunit combination on the native receptor subtype list (Table 2). None of these criteria by itself is sufficient. Because five criteria are rarely, if ever, met, it is the remit of a committee of experts (e.g., Nomenclature Committee of the IUPHAR subcommittee) to decide which candidates qualify. At this time we choose to be relatively strict regarding inclusion, including subtypes that either must, or are highly likely to, exist, rather than including all subtypes that might exist. We have decided to include more possibilities in the list for GABAA-Rs (Table 3) by dividing it into three categories, depending on the number of criteria that are met: A) "identified," B) "existence with high probability," and C) "tentative."
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2. Discussion of the Criteria.
A brief discussion of the available pertinent evidence and its value in determining the existence of native subtypes is in order. The expression of recombinant receptors, the determination of their subunit composition and arrangement, and their biophysical and pharmacological characterization is obviously an important part in the identification of native receptors. But expression of recombinant receptors is insufficient to prove their existence in vivo, because gross overexpression of subunit proteins can result in hetero-oligomeric combinations that are unlikely to occur in nature. For example, recombinant receptors containing both the 2 and subunits have been generated and characterized (Saxena and Macdonald, 1994; Hevers et al., 2000), whereas the actual existence of such receptors in the brain is highly questionable (for discussion, see Sieghart and Sperk, 2002). In addition, coexpression of subunits does not always result in the exclusive formation of the expected receptors. Thus, several reports have shown that coexpression of , β, and other (, , and ) subunits in oocytes or cell lines can lead to considerable expression of β receptor channels, without , , or . One must either precipitate the receptors via the third component subunit measuring subunit composition by Western blots and pharmacological properties via binding studies or use a pharmacological test to demonstrate the expression of this subunit and its contribution to the channel currents. In any case, it has to be kept in mind that receptor heterogeneity might influence results of the subsequent characterization of recombinant receptors. To increase the formation of receptors containing and β, as well as , , or subunits, higher amounts of mRNA or cDNA for the third component subunit often are used, as well as longer times for expression (Boileau et al., 2002, 2005; Olsen et al., 2007), despite the risk of non-natural or pathological pentamers with too many copies of a given subunit by the use of such extremes.
Thus, in recombinant expression studies, one must demonstrate that the subunits under study are indeed expressed, combine into receptors, form pentamers with defined subunit stoichiometry and arrangement, and have function. If possible, the properties of receptors should be compared with those of receptors with defined subunit composition and arrangement using concatemers. One might add that researchers also need to pay attention to the source of their receptor subunit clones and specify the source and sequences of those they use. A single point mutation in the gene of a subunit isolated from the brain could change the properties of the resulting recombinant receptors (Sigel et al., 1992). Then one can examine the receptor for unique biophysical properties using electrophysiology, assessing agonist and modulator mechanisms, determined, for example, from single channel and macroscopic current kinetics, single channel conductance, and possibly conductance substates. Finally, one can examine the receptor for unique pharmacology, using subtype-selective ligands, including radioligands, and measuring relative affinities, potencies, and efficacies for a series of ligands.
Expression in the brain at the mRNA (in situ hybridization, RT-PCR, single cell RT-PCR) or protein level (immunostaining) is a good starting point to indicate that two given subunits may be coexpressed in a given cell and which types of cells express them. A combination of in situ hybridization and immunohistochemistry is stronger than either technique alone. But colocalization of two subunits, although necessary, is not sufficient to establish whether these subunits are partners in a pentameric receptor subtype. Individual subunits alone most often do not define a receptor and might even have functions different from those of receptors. Microscopic colocalization of subunit proteins is one of the factors used in deciding native subtype composition, but colocalization at the light microscope level is not conclusive evidence for coexpression within a pentamer. Likewise, colocalization at the electron microscope level is useful evidence but does not necessarily define receptor subtypes, because even subunits located side-by-side with immunogold labeling could belong to adjacent receptors. Anatomy compendia are being developed by experts, and these play a role in deciding on inclusion of native subtypes. The questionable specificity of many antibodies, however, suggests that great care must be taken to analyze the published work on subunit localization (Rhodes and Trimmer, 2006; Moser et al., 2007). Because most antibodies used are polyclonal and because the composition of polyclonal antibodies is different in each donating animal and even in each blood sample, such verification of the specificity of the antibodies has to go on until highly selective and well characterized monoclonal or recombinant antibodies are available. All antibodies used for these compendia should thus be used in wild-type and knockout mice to be sure that they unequivocally identify the correct protein only. But even if the data are correct, most data on localization are not in themselves sufficient for defining receptor subtype composition.
Coimmunoprecipitation of subunits comes close to defining a subtype because it indicates (but not necessarily proves) assembly. Here again, the antibodies used must be demonstrated to have total subunit specificity as any cross-reactivity contaminates the results. The antibodies must be characterized by Western blotting on crude brain tissue or cells and be shown to recognize the band of correct size, and only that, in normal but not knockout mice, when such mice are available. Specific antibody reagents are currently available for such an approach, and the provider of such antibodies should be asked for data documenting the absence of cross-reactivity. Nevertheless, incompletely assembled intermediates could contribute, and subunits or receptors could associate with each other (natural interaction, artifactual aggregation, or association via cytoskeleton proteins) without being in the same receptors, weakening the strength of the evidence based on the coimmunoprecipitation method. One needs to be careful with the choice of detergent and solubilization conditions to minimize nonspecific protein interactions without destroying oligomers. If one could demonstrate that the associated subunits were present in a detergent-solubilized protein of the correct size for a pentamer this would strengthen the argument, but this is rarely, if ever, provided, and even such studies may be ambiguous because of comigration of multiple hetero-oligomeric protein species, so the results in any case must be regarded with caution. In the case of G protein-coupled receptor heteromultimer possibilities, evidence for true subunit-subunit association considers physical techniques such as FRET (Pin et al., 2007), and this approach may also find use for ligand-gated ion channel receptors. However, the method is extremely difficult to apply to native proteins, requiring the use of transgenic mice. If it is performed with recombinant receptors in a heterologous expression system, there is again the possibility of identifying non-native combinations owing to subunit overexpression. But such studies again would not provide a clear-cut answer because subunits demonstrated to be close together by FRET techniques could have assembled in the same receptor or be located in two different receptors associated with each other. The above considerations seem trivial, but they are not, judged by proposals of acknowledged scientists on how to unequivocally solve the problems of establishing receptor subunit composition that do not survive more thorough discussions.
Thus, the conclusion is that a combination of different techniques has to be used to identify a receptor subunit composition of a native receptor, knowing the limits of each technique. Even then, one cannot be absolutely sure, but at least this is at the limit of the techniques available. What is really needed is "in situ" electrophysiology with a channel characterization and a pharmacological fingerprint of the receptors to prove that the subunits known to exist in that cell contribute to the receptor studied and its physiology. This is extremely difficult, and fingerprints for the different receptor subtypes are not always sufficiently selective, may be host cell-specific, or are lacking altogether. One must take care to be as certain as possible that the subunits meant to be expressed in a recombinant system for obtaining a fingerprint are actually present and contributing to the data and that the pentamer has correct stoichiometry (see discussion above). Recombinant expression suffers from uncertainty that perhaps other gene products present in neurons might not be present in the cells used for recombinant expression (e.g., associated proteins, proteins affecting trafficking, or post-translational modifications) or that they do not exactly match that in the native system because of host cell-specific factors (Birnir and Korpi, 2007). Even a biophysical characteristic as fundamental as single-channel conductance can be affected by the expression system (Lewis et al., 1997), and anecdotes abound regarding different results for the "same receptors" expressed in two kinds of cells. Concatenated subunits may be of use in determining specific subunit composition and wheel arrangement (Im et al., 1995; Baumann et al., 2002; Minier and Sigel, 2004; Boileau et al., 2005; Sigel et al., 2006), although this approach also has a number of problems (Ericksen and Boileau, 2007).
To summarize the strategy used: one first notes which subunits are expressed in a given cell type. Then one looks at evidence that a given pair or triplet of subunits on that list are associated, based on coimmunoprecipitation, coregulation in cells, or knockout animals. The coexpression of 1β22 suggests the possibility of coordinate regulation of expression of chromosomal clusters, but the exceptions are too abundant to consider this a factor in partnering. Then one determines by recombinant expression and electrophysiology the channel kinetics and pharmacology of those subunit combinations, keeping in mind the numerous caveats listed above. Because most of these caveats have not been taken into account previously, one clearly cannot believe everything published on the properties of recombinant receptors. Native receptors with the same unique properties are then sought in the neurons recorded from brain slices, dissociated single cells, or at least cells in culture.
Genetically engineered mice (knockout and knockin) provide some evidence indicating that certain oligomeric receptors really do exist because one can correlate the loss of certain receptor responses and behaviors with the receptor subtypes addressed (Jones et al., 1997; Rudolph et al., 1999; McKernan et al., 2000; Sur et al., 2001; Vicini et al., 2001). The function of deleted genes is sometimes obvious, whereas in other cases, subtle changes in behavior need to be evaluated properly. Evidence from global knockout mice is often complicated by possible compensation, causing the observer to miss important functions. For example, deletion of the 1 subunit of the GABAA-R results in loss of nearly 50% of the total GABAA-R population in mouse brain and ablates fast synaptic transmission mediated by the abundant 1 subunit-containing receptors, yet the null mutant mice display a phenotype that is grossly normal (Sur et al., 2001). Diverse transcriptional responses may act to abrogate the effect of the knockout and preserve neuronal excitability and network behavior (Ponomarev et al., 2006). Point mutated knockin mice with altered behavior/pharmacology are more convincing, and several important examples are included in the GABAA-R field. For an overview and discussion, see Olsen and Homanics (2000), Rudolph and Möhler (2004), Sieghart and Ernst (2005), and Atack (2005).
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III. Working List of Native GABAA Receptor Subtypes |
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It was found early on in recombinant GABAA-R studies that robust GABA-activated channel formation occurred with combinations of and β subunits and also with , β, and subunits. The latter turned out to be the prevalent native combination (Barnard et al., 1998). The vast majority of GABAA-Rs in the CNS contain the 2 subunit, and this is the most abundant subunit in rat brain and in most regions based on in situ hybridization of mRNA (Wisden et al., 1992; Laurie et al., 1992; Persohn et al., 1991, 1992) and immunostaining (Fritschy and Möhler, 1995; Pirker et al., 2000). The 1 and 3 subunits are rarer but have some role in discrete regions, probably with well prescribed subunit partners. Thus, approximately 75 to 80% of GABAA-Rs contain the 2 subunit (Whiting et al., 2000; Sieghart and Ernst, 2005).
The 1 is the most abundant of the subunits and is often colocalized with its chromosome partners, the likewise highly expressed β2 and 2 subunits (Sieghart and Sperk, 2002). As noted above, knockout of the 1 subunit causes total GABAA-R content in mouse brain to decrease by 50% (Sur et al., 2001). The 2 and 3 subunits are moderately abundant and 5 is relatively rare except in the hippocampus, as indicated by regional distribution and immunoprecipitation studies (Pirker et al., 2000; Sieghart and Sperk, 2002). The 4 and 6 subunits are reasonably highly expressed in forebrain and cerebellum, respectively. Among the β subunits, β1 is least common, β2 is most abundant and most widespread (knockout results in a 50% reduction in GABAA-Rs in mouse brain) (Sur et al., 2001), and β3 is reasonably highly expressed, but more discrete. Furthermore, it is more dense perinatally than in adult brain (Zhang et al., 1991; Laurie et al., 1992). The identity of the β subunit often cannot be determined, because on precipitation of GABAA-Rs with , , or subunit-specific antibodies in most cases all three β subunits are coprecipitated. Only in some rare areas where there are cells with only one type of β subunit contributing to functional GABAA-Rs (Persohn et al., 1991, 1992; Wisden et al., 1992; Laurie et al., 1992; Pirker et al., 2000) can the type of β subunit be predicted. In these areas, however, no subsequent immunoprecipitation or electrophysiological studies have been performed to investigate possible assembly partners, and, thus, actual assembly of defined receptors and their composition cannot be derived with certainty. For this reason, it has been concluded that all of the β subunits exist in functional receptors, usually with only one type per pentamer (Whiting et al., 2000).
Based on the frequent colocalization in the brain (Fritschy and Möhler, 1995; Pirker et al., 2000) and in neurons (Klausberger et al., 2002), on results from coimmunoprecipitation experiments (Jechlinger et al., 1998; Pöltl et al., 2003), and on the pharmacology described in section III.B, the 1β22 subunit combination is considered to exist in the brain, probably in large amounts (Benke et al., 1991; Somogyi et al., 1996; Whiting et al., 2000). Recombinant 1β22 receptors have been shown to have a 2-2β-12 stoichiometry [denoted (1)2(β2)22], and this stoichiometry is supported by coimmunoprecipitation results from rat or mouse brain (Sieghart and Sperk, 2002). It is generally assumed, without conclusive proof, that other and β subunits also combine with 2 in combinations of 2-2β-12 (Sieghart and Ernst, 2005). Other subunits are also colocalized with and have been shown to coprecipitate with all β and the 2 subunits (Sieghart and Sperk, 2002). Receptors containing these subunits also have a specific pharmacology (see section III.B) and thus also seem to exist in the brain.
The other "minor" subunits, notably , are thought to be able to replace in the pentamer, as in the 2-2β- combination (Barrera et al., 2008). However, 4 and 6 subunits are just as often combined with the subunit as with 2 (Sieghart and Sperk, 2002). The subunit is obligatorily partnered with the 6 subunit in cerebellar granule cells and is primarily associated with the 4 subunit in forebrain areas including dentate gyrus, neostriatum, some layers of cortex, and a few other areas. The subunit seems to have a perisynaptic/extrasynaptic localization (Nusser et al., 1998; Peng et al., 2002; Wei et al., 2003). The subunit is rare but can substitute for or in some areas, such as hypothalamus, and the and subunits are only sketchily characterized (Korpi et al., 2002; Sieghart and Sperk, 2002).
The 2 subunit is required for synaptic localization of GABAA-Rs, usually associated with the 1, 2, or 3 subunits. Nevertheless, substantial numbers, possibly a majority, of 2 subunit-containing GABAA-Rs are extrasynaptic, because of the much greater area of extrasynaptic membranes. This includes some of the 1/2/3 and the great majority of 4/5/6 combinations. In contrast, combinations in which other subunits, such as and , replace 2 are considered to be exclusively nonsynaptic. The physiological and pharmacological importance of perisynaptically and extrasynaptically localized GABAA-Rs has recently become increasingly appreciated (Mody and Pearce, 2004; Semyanov et al., 2004; Farrant and Nusser, 2005).
B. Pharmacological Evidence for Subtypes
1. Benzodiazepine Site Ligands Distinguish between Subtypes Based on and Subunits.
The typical GABAA-R is positively modulated by diazepam-like benzodiazepines and binds radioligands for the GABA site (e.g., [3H]muscimol), the benzodiazepine site (e.g., [3H]flunitrazepam, [3H]flumazenil, or [3H]Ro15-4513, and the picrotoxin/convulsant/channel sites (e.g., [35S]t-butyl bicyclophosphorothionate) (reviewed by Macdonald and Olsen, 1994; Sieghart, 1995; Barnard et al., 1998; Korpi et al., 2002; Johnston, 2005). Barnard et al. (1998) listed approximately 20 chemical classes of ligand that bind to the benzodiazepine (BZ) site on GABAA-R, including the structures of approximately 40 compounds. Additional chemical classes of ligands are listed in Gardner et al. (1993), Olsen and Gordey (2000), Sieghart and Ernst (2005), Atack (2005), and Whiting (2006), which should be consulted for chemical names and structures. In each of these chemical classes compounds are available that enhance (positive allosteric modulators or BZ site agonists) or reduce (negative allosteric modulators or inverse BZ site agonists) GABA-induced chloride ion flux via the same BZ binding site. In addition, in each of these chemical classes there are compounds that do not modulate GABA-induced chloride flux, although they interact with the BZ binding site (neutral BZ site ligands or BZ site antagonists). The latter compounds, however, are able to inhibit the action of BZ site agonists or inverse agonists. In between the full BZ site agonists or inverse agonists, there are compounds that elicit less drastic allosteric enhancement or reduction of GABA-induced chloride currents; these compounds are called partial agonists or partial inverse agonists at the BZ site, respectively.
The BZ binding site is located at the interface of an and a subunit. Its pharmacology is thus influenced by both of these subunits, whereas β subunits, although needed to construct a channel, do not greatly affect the sensitivity of the GABAA-R to BZ site ligands (Hadingham et al., 1993). The traditional BZ site agonists (GABA-enhancing CNS depressants such as diazepam) are active on the GABAA-Rs containing a 2 subunit (Pritchett et al., 1989), a β subunit, and one of the subunits, 1, 2, 3, or 5. Receptors containing the 2 subunit exhibit a higher BZ sensitivity than those containing the 1 subunit (Sieghart, 1995; Khom et al., 2006); the 3-containing GABAA-Rs are modulated by some BZ ligands but with altered selectivity from those incorporating the 2 subunit (Sieghart, 1995; Hevers and Lüddens, 1998). The BZ-sensitive GABAA-Rs can be further subdivided, in that receptors containing the 1 subunit have a higher sensitivity to a subpopulation of BZ site ligands, the benzodiazepines quazepam and cinolazepam (Sieghart, 1989) or nonbenzodiazepines such as zolpidem (an imidazopyridine) and a few others, including CL218-872 (triazolopyridazine), zaleplon, and indiplon, and abecarnil (β-carboline), (Olsen and Gordey, 2000; Korpi et al., 2002; Sieghart and Ernst, 2005). Furthermore, receptors containing the 2 or 3 subunit have an intermediate affinity for zolpidem, whereas those containing 5 have very low affinity for this drug. The differential zolpidem affinity demonstrated by recombinant GABAA-Rs containing different subunits can also be found in the brain (Itier et al., 1996; Whiting et al., 2000; Sieghart and Sperk, 2002) and individual cells can be shown to exhibit more than one GABAA-R with varying affinity for zolpidem, depending on subunit subtype expression (Criswell et al., 1997). Again, consult the cited reviews for chemical structures and analogs.
Receptors containing the 4 or 6 subunits, together with β and 2, do not bind the traditional BZ agonists, including zolpidem, but demonstrate high affinity for some ligands, notably the imidazobenzodiazepines such as flumazenil and Ro15-4513, or bretazenil (Korpi et al., 2002). Both the potency and efficacy for BZ ligands depend on the nature of the subunit.
The benzodiazepine site ligands so far available do not distinguish well between the 2 and 3 or between the 4 and 6 subunits. All four, however, can produce functional channels in vitro when coexpressed with other subunits, and their differential distributions in the brain suggest they will modulate different behavioral circuitry. Behavioral effects of drugs predicted from subtype selectivity in vitro has been successful in a few cases, such as the 2/3-selective triazolopyridazine anxiolytics based on CL218-872: L838,417, TPA003, or TPA023 (Atack, 2005; Morris et al., 2006; Atack et al., 2006).
Subtype selectivity for drugs in vivo has been further confirmed in subunit-specific genetically engineered mice (Rudolph and Möhler, 2004; Atack, 2005; Whiting, 2006). Thus, based on the evidence that most of the actions of diazepam are mediated via receptors composed of 1β2, 2β2, 3β2, and 5β2 subunits, a point mutation involving a histidine to arginine substitution was introduced into the genes of the individual subunits, rendering the respective receptors insensitive to allosteric modulation by diazepam. A comparison of drug-induced behavioral responses in the mutated and wild-type mice then allowed the identification of diazepam effects that were missing, or reduced, in the mutant mice. With this approach, it was demonstrated that 1β2 receptors mediate the sedative, anterograde amnestic and in part the anticonvulsant actions of diazepam (Rudolph et al., 1999; McKernan et al., 2000). These two independent studies showed exactly the same behavioral responses, provided the mice were tested under the same conditions (Crestani et al., 2000). The anxiolytic activity of diazepam is mediated by GABAA-Rs composed of 2β2 subunits (Low et al., 2000), and, under conditions of high receptor occupancy, also by 3 GABAA-Rs (Dias et al., 2005; Yee et al., 2005; discussed by Möhler, 2007). The 3-selective drug TP003 also implicates a role for 3β2 receptors in anxiolytic (Dias et al., 2005) and anticonvulsant action (Fradley et al., 2007). The 2β2 and 3β2 receptors are also implicated in some of the muscle relaxant activities of diazepam (Low et al., 2000) and the 3β2 in the antiabsence effects of clonazepam (Sohal et al., 2003). The 3 global knockout mice displayed a hyperdopaminergic phenotype relevant for GABAergic control of psychotic-like symptoms (Yee et al., 2005). The 5β2 receptors seem to influence learning and memory, shown by improved spatial memory in mice with knockout of 5 subunits (Collinson et al., 2000), and trace fear conditioning was facilitated in the point-mutated 5 knockin which unexplainedly showed major 5 subunit knockdown in the CA1 region (Crestani et al., 2002; Rudolph and Möhler, 2004). Furthermore, the 5-selective inverse agonists such as 5IA can improve cognitive function (Atack, 2005; Dawson et al., 2006). Mice lacking the 5 subunit were shown to exhibit reduced amnestic response to the intravenous general anesthetic etomidate but not to the immobilizing activity of the drug (Cheng et al., 2006). Knockout mice have also implicated several GABAA-R subtypes in the reinforcing effects of ethanol, including the 1 (Boehm et al., 2004), the 5 (Boehm et al., 2004; Stephens et al., 2006), the (Mihalek et al.,2001), the 4 (Liang et al., 2008), and the 6 subunit (Hanchar et al., 2005). Years of study on subunit-dependent subtypes have led to drug candidates for modifying selective behaviors and clinical indications (Atack, 2005; Sieghart and Ernst, 2005; Whiting, 2006), and the genetically modified mice have supported this subtype selectivity. Possibly even more impressive than the drug candidates has been the clues generated regarding behavior involving specific receptor subtypes and thus specific brain circuitries (for review, see Rudolph and Möhler, 2004). Although this attribution of the varied behavioral actions of diazepam site ligands and ethanol to different receptor subtypes might only be a first approximation, and the evaluation may be somewhat tentative, it clearly indicates that receptors containing the respective subunits are present in the brain and exhibit distinct functions in different neuronal circuits, verified by consistent results with subtype-selective drugs.
In contrast to 1β2, 2β2, 3β2, and 5β2 receptors, 4β2 and 6β2 receptors are diazepam-insensitive but are still able to bind the imidazobenzodiazepines Ro15-4513, and flumazenil, which act as BZ site inverse agonist and antagonist, respectively. Thus, these receptors can be identified by ligand-binding studies and autoradiography, using [3H]Ro15–4513 in the absence or presence of high concentrations of diazepam or flumazenil, to mask receptors containing the 1, 2, 3, or 5 subunits. Such studies have clearly identified diazepam-insensitive, but flumazenil-sensitive, binding sites in cerebellum and forebrain. These sites disappear in 6 or 4 null mutant mice, indicating that they represent 6β2, or 4β2 receptors, respectively (Korpi et al., 2002). In addition, there are GABAAR-mediated inhibitory currents recorded in hippocampus that are enhanced by Ro15–4513, an effect specific for 4β2 (Liang et al., 2006). So, even with limited pharmacology, some in vivo and in vitro information about selectivity, and the help of genetically modified mice, we can make receptor assignments; e.g., each subunit defines at least one unique subtype, partnered with a β and a 2 subunit. Thus, GABAA-Rs exist with binding properties consistent with high, intermediate, and low affinity for certain BZ site ligands, such as zolpidem, and GABAA-R channels with this pharmacological specificity are localized in certain brain regions where the different subunits are expressed. In addition, all subunits can be coprecipitated with β and 2 subunits in these regions. Therefore, we conclude that the six different subunits occur in the brain, combined with β and 2, and can start our receptor list with all six subunits, each combined with β2, for a total of six subtypes (Macdonald and Olsen, 1994; Sieghart, 1995; Barnard et al., 1998; Whiting et al., 2000; Korpi et al., 2002). As discussed in section III.A, only for the 1β2 receptor subtype does sufficient evidence identify the type of associated β subunit (1β22).
The subunit, generally partnered with the 4 and 6 subunits, produces the seventh and eighth subtypes. This is supported by evidence of colocalization and coimmunoprecipitation (Jechlinger et al., 1998; Whiting et al., 2000; Pöltl et al., 2003; Sieghart and Ernst, 2005), accompanied by electrophysiological evidence for 6β GABAA-Rs in cerebellar granule cells and
