REVIEW ARTICLE the GABAA Receptors F

Biochem. J. (1995) 310,1-9 (Printed in Great Britain) 1 REVIEW ARTICLE The GABAA receptors F. Anne STEPHENSON School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1 N 1AX, U.K. INTRODUCTION although the classical benzodiazepines, valium and librium, bind y-Aminobutyric acid (GABA) is the major inhibitory neuro- to a single high-affinity site in the brain, other related compounds, transmitter in the mammalian brain. It mediates its effects via which are again thought to act at the benzodiazepine or an the specific interaction with the integral membrane proteins, the adjacent overlapping site, show shallow displacement curves GABA receptors. GABA receptors can be classified according to in radioligand-binding studies. It is now known that these their respective transduction mechanisms following activation, compounds, for example the fi-carbolines, zolpidem and as the GABA and the GABAB receptor respectively. The focus oxoquazepam, discriminate between GABAA receptor subtypes of this review is the GABAA receptor. GABAA receptors are fast- (see below). acting ligand-gated chloride ion channels. Thus receptor ac- Other allosteric modulators of GABAA receptors include the tivation in the brain is followed within milliseconds by the gating barbiturate drugs, non-competitive chloride channel blocking or opening of an integral chloride ion channel which results, in agents [including picrotoxin and the cage convulsant compound, general, in the hyperpolarization of the recipient neuronal cell. t-butylbicyclophosphorothionate (TBPS)], certain neurosteroids, The GABAA receptors are of importance because of both the the anthelminthic agents, the avermectins, Zn2+, ethanol and the pivotal role that they play in the regulation of brain excitability anti-convulsant drug, loreclezole. Each of these compounds and the fact that their function is allosterically regulated by modulates GABAA receptor function by binding to a distinct site several distinct classes of therapeutic compounds. These include within the receptor complex. Figure 1 is a diagrammatic rep- anxiolytic benzodiazepines such as valium, barbiturates, neuro- resentation of the complex pharmacology of the mammalian steroids and some volatile anaesthetics. The first GABAA receptor GABAA receptors. gene sequences were reported in 1987 by Schofield et al. [1]. Since that time, a plethora of homologous genes encoding GABAA MOLECULAR BIOLOGY OF GABAA RECEPTORS receptor polypeptides have been identified. These polypeptides At the current time, 15 mammalian GABAA receptor genes have are thought to co-assemble, predominantly in the brain, to form been identified (for review see [6]). These are classified with heteromeric receptor proteins with distinct biophysical. and respect to the conservation in amino acid sequence homology of pharmacological properties. This review will focus on the their gene products. Thus there are five GABAA receptor subunit developments in this field since the GABAA receptor genes were types a, fi, y, a and p. Within four of these, isoforms exist with first cloned, addressing in particular, the structural and functional the now accepted nomenclature of al-a6, fll-,83, yl-y3 and significances of mammalian GABAA receptor heterogeneity. pl-p2. Isoforms of a single subunit type, for example comparing all the a subunits, have at least 70 % amino acid sequence GABAA RECEPTOR PHARMACOLOGY identity, whereas if the conservation in primary structure is The rich pharmacology ofthe GABAA receptors has had a major compared across subunit classes, for example a versus , subunits, impact in the elucidation of the biochemical properties of the the percentage identity falls to within the range 30-40 %. Some GABAA receptors. Before describing the current developments in of the GABAA receptor genes undergo alternative splicing. The the understanding of their structures, it is necessary to have an most prevalent of these is the y2 subunit which exists in two appreciation of their pharmacological properties. Perhaps the forms, y2Short (y2s) and y2lOng (y2L) [7,8]. But also, alternative most important of these was the realization that the anxiolytic splicing of the human ,B3- [9] and the rat a6-subunit gene have benzodiazepine drugs (e.g. valium and librium) exerted their been described [10]. In the chick, the GABAA receptor subunit action by a facilitation ofGABA neurotransmission (for detailed genes fl2 and f4 have been found to undergo splicing to yield reviews, see [2,3]). Thus it was found that benzodiazepines bound ,f2' and f84' [11,12]. (Note that for the chick, a fl4 and a y4 with high affinity to an allosteric modulatory site of the GABAA GABAA receptor subunit gene have been reported but it is receptor. In the presence of GABA, they potentiated the in- probable that these are the avian homologues of the mammalian hibitory response by an increase in the frequency of chloride ion ,f1- and y3-subunit genes respectively, [13].) Two invertebrate channel opening. The benzodiazepine mode of action permitted GABAA receptor genes have been cloned. They are the Drosophila the purification of the GABAA receptors by benzodiazepine Rdl gene [14], and a GABAA receptor gene from the pond snail, affinity chromatography (e.g. [4,5]). Benzodiazepines such as Lymnaea stagnalis [15]. Interestingly, in amino acid sequence valium, are anxiolytic. Although they are unable to activate comparisons with vertebrate GABAA receptor genes, these are GABAA receptors by themselves, by convention they are termed both most closely related to the , subunits, with identities of the agonists. Other benzodiazepines and related compounds thought order of 28 %. This low percentage ofconservation is in contrast to act at similar sites within the GABAA receptors have been to comparisons between the subunits of various vertebrate identified which are anxiogenic and, in the presence of GABA, species, where values of greater than 90 % are found. Figure 2 decrease the frequency of chloride channel opening. These shows the evolutionary tree of the GABAA receptor and the compounds are termed, by convention again, 'inverse agonists'. related glycine receptor subunit genes reported to date. The The benzodiazepine Ro 15-1788 has no or very low intrinsic lineage is consistent with the evolution from a single ancestral efficacy and is recognized as an antagonist. Furthermore, gene [16]. Abbreviations: GABA, y-aminobutyric acid; y2L, y2Long; y2s, y2Short; TBPS, t-butylbicyclophosphorothionate. 2 F. A. Stephenson (a) 1 226 429 NH2 -A 0 Cys-Cys Ml M2 M3 M4 I 139 153 Clomethiazol ? 313 319 Splice variant y2L insert in a6 Avermectin ? Splice variant in ,3 (b) 'hols Channel-openers o (barbiturates, steroids, [Ca2+1; alcohols) Frusemide (anion-channel blockers) Figure 1 Schematic presentation of the various binding sites associated with the GABAA receptors of mammalian brain Figure 3 Schematic representation of the pertinent features of GABAA receptors Abbreviations: PKC, protein kinase C; PTZ, pentylenetetrazol; [Ca2+]i, intracellular free Ca2+ concentration; misc., miscellaneous. Reprinted from [3] with the kind permission of (a) This shows the numbered amino acid sequence of the bovine al subunit highlighting F. Hoffmann-La Roche Ltd, Basel, Switzerland. domains conserved between all GABAA receptor polypeptides. V, consensus sequences for N-glycosylation; Ml-M4, transmembrane domains; Cys-Cys, conserved extracellular motif common to all members of the ligand-gated ion channel superfamily. (b) shows a schematic view of the receptor oligomer as viewed perpendicular to the plane of the membrane. It represents each of the five subunits of the receptor as a segment within the annular structure where the hole in the middle represents the chloride ion channel. The four membrane-spanning regions within each polypeptide are shown as filled circles with the predicted a helix of M2 lining the wall of the channel. The subunit complements and their ordering around the rosette are not definitively established, but see the text for discussion. The mature GABAA receptor subunits have similar molecular masses as deduced from their respective cDNA sequences. These range from 48000 (y subunits) to a maximum of 64000 (a4 subunit). They all share a similar predicted domain structure not only between themselves but also with other members of the ligand-gated ion channel superfamily, most notably the proto- typic peripheral nicotinic acetylcholine receptor (cf. [17]). A schematic diagram of the pertinent features of a typical GABAA receptor subunit is shown in Figure 3. Thus it is predicted that each subunit has an extended, extracellular, hydrophilic N- terminal domain of the order of 220 amino acids. Within this region are consensus sequences for N-glycosylation. There are L GABc four putative hydrophobic domains, M1-M4, which are pre- GABrG ' GABm dicted to span the membrane and form the chloride ion channel. M2, again by analogy with nicotinic acetylcholine receptors and | ~~GABsB3 GABhRl also by some mutagenesis studies (see below), is thought to form GABhR2 GABcB4 the inner lining of the channel. The M I-M3 regions are adjacent ,GABrB2 to the N-terminal domain whereas M4 is at the C-terminal end GABbBl GABrBl of the proteins. This transmembrane topology model predicts 'GABhBl GABhB3 that both the N- and C-terminal regions are extracellular but, GABrB3 surprisingly, this remains unproven. Separating the M3 and M4 A GABcB3 transmembrane-spanning regions is a second hydrophilic region often referred to as the cytoplasmic

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