Minireview Vol. 267, No. 24, Issue of August 25, pp. 16747-16750,1992 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

y-Aminobutyric Acid* Receptor Structure and Function*

Timothy M. DeLorey and Richard W. OlsenS From the Department of Pharmacology, Mental Retardation Research Center and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024

The major type of receptor for the most important inhibi- tory neurotransmitter y-aminobutyric acid (GABA)' in the mammalian central nervous system is the GABAA receptor. The far reaching functional significance of GABA suggests a role for GABAA receptors in human neurological and psychi- atric disorders (1) and in the mechanism of action of numer- ous clinically important drugs (2). The GABAA receptor is a member of a gene superfamily of ligand-gated ion channel receptors which are typified by a hetero-oligomeric complex that spans the membrane to form an ion channel. Binding of a specific neurotransmitter ligand to the receptor protein controls the opening and closing of the ion channel (3, 4). The GABAA receptor channel is constituted from a family of 15 or so genes encoding for related but different polypeptides. Homologous sequence domains among members of the family and superfamily are believed to have conserved structural and functional features. This article will briefly summarize the latest information available on the GABAA receptor-ion chan- nel complex. The literature cited is illustrative, not exhaus- tive.

GABA Receptor Physiology and Pharmacology The GABAA receptor is a macromolecular complex, the

GABA-benzodiazepine receptor/chloride channel protein (2, 4). The complex consists of five major binding domains: sites for GABA, benzodiazepines, barbiturates, picrotoxin, and the anesthetic steroids. These binding domains serve to modulate the receptor's response to GABA activation. An integral part of this complex is the chloride channel. GABA analogs such as muscimol, an ingredient of the hallucinogenic mushroom Amanita muscaria, act as agonists, binding to the GABA site and opening the C1- channel. The plant-derived convulsant bicuculline is considered the definitive competitive antagonist of GABAA receptors (2).

Electrophysiological investigations of GABAA receptor function including single channel recording indicate that it mediates an increase in membrane chloride permeability, resulting in inhibition of electrical activity of the postsynaptic cell (5, 6). Ion channel activation involves complex kinetics of opening and closing on a millisecond time scale and desen- sitizes in the continuing presence of GABA. 36Cl- flux studies

NS22071, NS28772, AA07680, and HD06576. * This work was supported by National Institutes of Health Grants

$ To whom correspondence should be addressed Dept. of Phar- macology, UCLA School of Medicine, Center for Health Sciences, Los Angeles, CA 90024-1735. Tel.: 310-825-5093; Fax: 310-825-6267.

The abbreviations used are: GABA, y-aminobutyric acid PKA, cyclic AMP-dependent protein kinase; PKC, Ca2+-phospholipid-de- pendent protein kinase C; TBPS, t-butyl bicyclophosphorothionate; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-l-propanesulfo- nate.

with cells, brain slices, or homogenates (7) suggest the pres- ence of GABA binding sites specific for the initiation of desensitization that are distinct from the sites mediating opening of the channel.

Central nervous system depressant drugs of several cate- gories appear to act by enhancing GABAergic transmission (2). Benzodiazepines, used for anxiolytic, muscle relaxant, sedative, and anti-epileptic actions, increase the probability of channel opening in response to GABA (8). Benzodiazepine binding sites measured in vitro are allosterically coupled to GABA, barbiturate, steroid anesthetic, and picrotoxin sites. Drugs acting at this site exhibit a continuum of intrinsic efficacies, ranging from full agonists to partial agonists, to antagonists, to partial inverse agonists, to full inverse ago- nists. For example, several P-carbolines not only antagonize the effects of agonists by occupying the benzodiazepine recep- tor sites but also induce pharmacological effects opposite those of the classical benzodiazepine agonists; thus they are termed inverse agonists. Barbiturates, used for anesthetic, sedative, and anti-epileptic medications, enhance GABAA re- sponses by increasing mean channel open time and also modulate binding to various sites on the GABAA receptor complex. Channel blockers, such as the plant convulsant compound picrotoxin and penicillin G, cause a decrease in mean channel open time (5). The synthetic anesthetic steroid, alphaxalone, as well as naturally occurring metabolites of steroid hormones, ethanol, volatile gas anesthetics, and pro- pofol, have been found to enhance GABAA receptor chloride channel function and to modulate binding in vitro (2, 9).

Biochemical Properties A typical in vitro cell-free binding assay for the GABA

receptor in mammalian brain (10) shows multiple high affinity sites (Kd < 1 PM) in membrane homogenates, thoroughly washed to eliminate endogenous ligands and assayed in so- dium-free buffer at 0 "C to inactivate the transport system. Binding also can be studied with synaptoneurosomes, homog- enized membrane particles containing membrane-enclosed pre- and postsynaptic elements and having the ability to maintain a membrane potential, as well as to respond func- tionally to GABA agonists measured with 36Cl- flux under similar conditions used to assay binding (11). The GABAA receptor complex also can be assayed with radioligands bind- ing to the picrotoxin/convulsant sites and the benzodiazepine sites. Binding of diazepam to saturable sites in brain shows pharmacological specificity expected of benzodiazepine recep- tors (12, 13). Co-purification with GABAA binding activity, and eventually cloning and expression, have proven that the benzodiazepine receptor is one and the same with the GABAA receptor complex (3, 4). The binding of radiolabeled picro- toxin (14) is inhibited allosterically by barbiturates and com- petitively by cage convulsants, one of which was radiolabeled to provide a higher affinity ligand, [35S]t-b~tyl bicyclophos- phorothionate ([35S]TBPS) (15). TBPS binding is allosteri- cally modulated by GABA, barbiturates, benzodiazepines, and steroids and co-purifies with the GABAA-benzodiazepine receptor complex (2-4).

A variety of mild detergents has been used to solubilize GABAA receptor binding activity from brain membranes. Deoxycholate produces good yields, GABA and benzodiaze-

16747

16748 Minireview: y-Aminobutyric AcidA Receptor Structure and Function

I FIG. 1. Generic GABAI receptor protein subunit sequence

and proposed topological structure. The NH~-terminal half of the sequence is presumed to be extracellular and contains sites of potential N-glycosylation (small white circles) and a cystine bridge conserved between all members of the superfamily (white l i w con- neeting two blue residues at upper right). This region participates in the neurotransmitter binding site. The four block cylinders represent putative a-helical membrane-spanning regions (3). Benzodiazepine binding is believed to occur between the cystine bridge and the first membrane-spanning helii (3,42, 43). Between membrane-spanning regions three and four is a large cytoplasmic loop. This contains potential phosphorylation sites in several subunit subtypes, and 'in vitro phosphorylation of this domain in the 83 subunit by the cyclic AMP-dependent protein kinase has been demonstrated (56). As in- dicated in the box inset, the colors represent the degree of variability among the first eight rat cDNAs described (al, q2, a4, 81, @, 83, y2, 6). Those amitlo acids identical in all clones are shown in blue, those identical in more than two subunit subtypes are green, those identical in all a but not other classes are yellow, and those that vary are in red (modified from Ref. 4, with permission).

regulatory mechanisms such as phosphorylation (3, 4). The subunits form a quasi-symmetric structure around the ion channel, with each subunit contributing to the wall of the channel (Fig. 2, Ref. 4). The model is based heavily on analogy with the nicotinic acetylcholine receptor, another member of the ligand-gated ion channel gene superfamily. About 10-20s homology exists between all GABAA receptor subunit candi- dates and other gene products of the superfamily (3,4).

The existence of these 15 or so subunit candidates leads to the questions oE (a) how many oligomeric combinations exist in nature? ( b ) for what purpose do they exist? and (c) what is the subunit composition of each? Different combinations of subunits are present in different neuronal populations. The nervous system distribution of mRNAs determined by in situ hybridization is very different for different subunit subtypes (30). For example, the 72 and 6 show almost non-overlapping distributions, suggesting that they rarely exist together in the same oligomeric receptor (23); the p subunit is expressed only in retina (24), while various a! subtypes show very different regional distribution (31). Partial co-localization, together with recombinant cDNA expression studies, suggests some tentative conclusions about major oligomeric assemblies (32- 34).

Minireview: y-Aminobutyric AcidA Receptor Structure and Function 16749

FIG. 2. Model of the structure of the GABAA receptor chlo- ride channel complex. The ligand-gated ion channel is proposed to be a hetero-oligomer composed of five subunits of the type shown in Fig. 1, including some of the a, 8, 7, 6, or p polypeptides. The exact subunit composition/stoichiometry is not known at this time. Each subunit has four membrane-spanning domains (cylinders numbered 1-4), one or more of which contribute to the wall of the channel (modified from Ref. 4, with permission).

Expression of various combinations of subunits in oocytes or cultured mammalian cell lines can suggest the likely com- bination responsible for a given pharmacological property, such as the benzodiazepine sensitivity conferred by the pres- ence of 72 (22). Varying the a subunit in combination with constant @ and y suggests differences in benzodiazepine phar- macology, GABA-benzodiazepine interaction, and steroid modulation of GABA responses, but not barbiturate, picro- toxin, and bicuculline sensitivity. Consistent with conclusions from previous photoaffinity labeling (18), the 51-kDa a1 subunit expressed with and y gives a benzodiazepine binding site with “BZ1” specificity, namely high affinity for subtype- selective ligands. On the other hand, a2-6 all show lower affinity for these ligands and may be constituents of what was called “BZ2” receptors, obviously a very heterogeneous group (32-34). Likewise, the a6 subunit in cerebellum appears to be responsible for a novel binding subtype of GABA receptor (27), which binds the partial inverse agonist ligand [3H]Ro15- 4513 but not the classical benzodiazepines like diazepam. This additional layer of complexity may be further complicated by the heterogeneity of the p, y, etc. subunits, the nature of which could affect pharmacological specificity.

Site-directed mutagenesis has confirmed the a sequence specificity for benzodiazepine ligand binding. Comparisons of sequences for al, -2, and -3 revealed differences in the NH,- terminal putative extracellular domain. The synthesis of chi- meric cDNAs with mixed domains of a1 and a3 led to iden- tification of a single residue (glycine 201 in a1) that resulted in high affinity for BZ1-selective ligands or low affinity in a2 or a3 (BZ2), which had glutamate at that position (33). Similarly, the a6 and a1 sequences were compared and a single residue identified (histidine 102 in a1 instead of argi- nine 101 in the a6 subunit) that confers flunitrazepam binding (35). Immunoprecipitation of receptor binding activity with antibodies specific for a1 subtype sequences showed that the a1-containing oligomers were enriched in “BZ1” type binding (36).

Antibodies to synthetic peptides based on subunit subtype- specific clone sequences have been used to identify the subunit on Western blots, to localize in brain sections the presence of subunit subtypes, and to analyze subunit composition by immunoprecipitation and immunoaffinity chromatography. Gene products were identified by Western blotting as a1 (51 kDa), a2 (53 kDa), a3 (59 kDa), a4 (54 kDa in rat, 57 kDa in cow), y2 (43-47 m a ) , 6 (54 kDa), p2 (57 kDa); 83 (54-59 kDa) (Refs. 21 and 36-40). The a6 subunit is believed to represent the 57-kDa polypeptide band photolabeled in cere- bellum by [3H]Ro15-4513 (27). Microsequencing (21) has confirmed identification of some stained bands in the purified receptor preparations. Antibodies specific to a subunit sub- types were shown to immunoprecipitate variable fractions of total receptor, depending on the subunit and brain region, with a small amount of overlap (37,38). Antibodies recogniz- ing all subunits can immunoprecipitate virtually all benzo- diazepine affinity-purified receptors from bovine cortex (38), while antibodies to 83, 72, and 6 each immunoprecipitate about half of the purified receptors (37, 39,41). [3H]Flunitrazepam-photolabeled receptor produced CNBr

fragments that could be identified on gel electrophoresis in sodium dodecyl sulfate by radiolabel and immunoblotting with antibodies to the NH, and COOH terminus, tentatively nar- rowing down the possible site of attachment of the photolabel to amino acid residues 59-148 of a1 (42). However, we found that proteolytic fragments (Stuphylococcus uureus V8 digest) of photolabeled preparations could be purified to yield partial amino acid sequences starting with residue 8 (31-kDa frag- ment), another labeled one (30 kDa) commencing at 106, and a 17-kDa fragment starting at 298 which was not labeled, limiting the label to 106-297 (21). Trypsin digestion of pho- tolabeled gel-purified subunits and purification of labeled fragments produced a microsequence starting with residue 223 of bovine a l , with label present at aromatic residues phenylalanine 226 and tyrosine 231, at the beginning of the first membrane-spanning region (43).

Regulation GABAA receptors can be regulated at both the gene tran-

scription level and at the protein level. Different subunit combinations resulting from tissue- and ontogenetic-depend- ent gene expression, as well as susceptibility to dynamic change, also lead to diversity in subcellular locations, affinities for ligands, and ion channel gating properties, and perhaps in regulation by receptor phosphorylation, controlled by other extracellular communication molecules (cross-talk). Distinct receptor subtypes may be responsible for excitatory, depolar- izing responses of GABAA receptors (44) or association with a voltage-dependent anion channel, the function of which could modulate the C1- conductance through the GABA chan- nel and vice uersa (45). Some GABAA receptor subtypes, possibly depolarizing, appear to mediate trophic responses to GABA during development (46), and GABA and GABA- enhancing drugs regulate expression of mRNA for receptor subunits in cell culture and in vivo (47-49).

GABAA receptor function can be controlled by treatments that increase protein phosphorylation, although the receptor itself has not yet been demonstrated to be phosphorylated in uiuo. The cyclic AMP-dependent protein kinase (PKA) re- duces GABAA receptor C1- conductance (50, 51). In oocytes injected with brain or GABA receptor mRNA, phorbol esters turn off GABA receptor function, presumably via activation of Ca2+-phospholipid-dependent protein kinase C (PKC) (52). GABA receptor function also appears to be “maintained” by phosphorylation; the activity “runs down” in some cells when

16750 Minireview: y-Aminobutyric AcidA Receptor Structure and Function

cytoplasmic contents are diluted by patch electrodes and can be maintained by the addition of ATP"? (53).

The GABA receptor clones reveal consensus substrate se- quences in the cytoplasmic loop of several subunits for PKA, PKC, and tyrosine protein kinase. 72 has an mRNA splicing variant 72L containing an 8-amino acid insert with a consen- sus substrate sequence for phosphorylation by PKC (29) that appears necessary for modulation of GABA receptor function by ethanol (54). The GABA receptor protein can be phos- phorylated in uitro by purified protein kinases C and A, but not calmodulin-dependent protein kinase (55). The in uitro phosphorylation by both kinases could be completely pre- vented by preincubation with an antibody prepared against a synthetic peptide corresponding to the consensus substrate for PKA in the p subunits. Further, tryptic digestion and microsequencing revealed that the PKA-phosphorylated res- idue was in a fragment containing the cytoplasmic loop of the p3 subunit (56).

In conclusion, the GABAA receptors in the nervous system consist of a family of hetero-oligomers produced from 15 or so related but distinct genes. Most, if not all, of these isoforms with different subunit compositions are ligand-gated chloride channels involved in inhibitory synaptic transmission. Recep- tor subtype differences in structure, function, and regulatory mechanisms, at the gene and protein level, are under heavy investigation. The existence of multiple GABAA receptor sub- types suggests specific functional and pathological roles, and the potential for dynamic changes in subunit composition relevant to normal central nervous system function, plasticity, and disease processes, as well as pharmaceutical intervention.

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