[Frontiers in Bioscience 1, d214-233, September 1, 1996]


Martin Davies1, Alan N. Bateson1,2 and Susan M. J. Dunn1,2

1 Department of Pharmacology

2Division of Neuroscience, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Received 07/16/96; Accepted 07/22/96; On-line 09/01/96


gamma-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the vertebrate central nervous system where it activates two major receptor types, namely GABAA and GABAB receptors. The former is a member of a ligand-gated ion channel superfamily (1) whereas the latter is believed to be a G-protein-coupled receptor, although this has yet to be confirmed by cloning and expression studies. The GABAA receptor contains an integral ion channel which opens, as a consequence of GABA binding, to allow the flux of Cl- ions. In most brain regions Cl- ions pass down a concentration gradient into the cell to cause hyperpolarisation; however, depolarizing responses have been reported in specific neuronal populations (2) and at certain stages in development (3). GABAA and GABAB receptors are most readily distinguished by the actions of bicuculline and baclofen, the former being a GABAA receptor-specific antagonist while the latter is a GABAB receptor-specific agonist (4). Other receptor-selective agents exist, one of the most widely used of which is muscimol, a specific agonist at GABAA receptors (5).

The GABAA receptor has a very rich pharmacology and this has provided the impetus both for the identification of more (or better) therapeutic agents with which to modulate receptor function and also for the use of this protein as a model system to study complex interactions between multiple binding sites. A large number of modulators, that apparently bind to discrete sites on the GABAA receptor, have been identified. These include benzodiazepines, barbiturates, neurosteroids, some anesthetics, Zn2+, avermectin and possibly also alcohols (reviewed in 6). Occupancy of one site can alter the characteristics of one or more of the others, including allosteric modulation of the binding of GABA and/or channel gating.

From a therapeutic perspective, the benzodiazepine site has proved to be the most important. Remarkably, it was some 15 years after the introduction of benzodiazepines into clinical practice that their site of action (i.e. the GABAA receptor) was elucidated (reviewed in 6). While benzodiazepines have both muscle relaxant and anticonvulsant properties, they have proved to be most useful in the treatment of anxiety and insomnia. The benzodiazepines that are used therapeutically are known as "agonists". These drugs potentiate the actions of GABA by causing a shift in the GABA concentration-effect curve to lower concentrations and increasing the frequency of channel opening without affecting either the channel open time or conductance (7).

The discovery of a number of structurally unrelated compounds that appeared to recognize the benzodiazepine site led to the intriguing observation that drugs that act at this site can elicit a spectrum of pharmacological activities ranging from full agonist through antagonists to inverse agonists (8). The latter group interacts with the receptor to produce an effect that is opposite to that of the classical agonists i.e. a rightward shift in the GABA concentration-effect curve and a decrease in the frequency of channel opening. Benzodiazepine antagonists, such as Ro15-1788 (more recently known as flumazenil), compete for the same binding site and thus block the effects of both agonists and inverse agonists, although alone they have no overt effects on GABA responses. A number of compounds display either agonist or inverse agonists properties but are less efficacious than others; consequently these agents have been termed partial agonists and partial inverse agonists, respectively. As discussed below, there has been intense interest in the elucidation of the structure-function relationships that determine the pharmacological profiles of the benzodiazepines and related compounds. In this regard, two benzodiazepines that have proved to be particularly useful are the agonist, flunitrazepam, and the partial inverse agonist, Ro15-4513. Both of these drugs are available in radiolabelled form and both can be used as photoaffinity reagents to probe the environment of the benzodiazepine binding site (for example, see 9).

The first indication of the inherent complexity of the GABAA receptor came from studies of drugs that compete for the benzodiazepine site. CL218872, a triazolopyridazine, was shown to have a differential ability to displace classical benzodiazepine agonists from their binding sites in different regions of the brain (10). This compound appeared to have a higher affinity for sites in rat cerebellum than for those in the hippocampus. Thus, in these early studies, two types of benzodiazepine sites, designated BZI and BZII respectively, were pharmacologically distinguished. This provided the first evidence, later to be confirmed by molecular cloning, that GABAA receptors are heterogeneous.

Knowledge that the GABAA receptor carried a high affinity binding sites for benzodiazepines, spurred attempts to purify the receptor by benzodiazepine affinity chromatography. Early purification studies and biochemical characterization suggested that the GABAA receptor was formed by an alpha and a ß subunit which could be photoaffinity labelled, respectively, by [3H]flunitrazepam (11) and by [3H]muscimol (12, 13). In 1987, the results of a fruitful collaboration between the groups of Eric Barnard and Peter Seeburg provided the first GABAA receptor cDNA sequences (14). Sequences of an alpha and ß subunit were obtained by a cloning strategy which was based on partial amino acid sequences of the purified receptor subunits. In the same issue of Nature, the sequence of a glycine receptor (GlyR) subunit was reported (15). Comparison of these cDNA sequences led to the landmark discovery that, not only were the sequences of the two GABAAR subunits and the GlyR subunit related to each other but they were also homologous to the previously reported sequences of the four subunits of the Torpedo nicotinic acetylcholine receptor (nAChR; 16). Thus, for the first time, it was clear that these proteins belonged to a structurally related "superfamily" of ligand-gated ion channels (LGIC). More recently, another member of this family has been identified i.e. the 5-hydroxytryptamine type 3 (5HT3) receptor (17). As discussed below, cloning studies have since demonstrated the existence of a large family of GABAA receptor subunit genes and, like many other CNS receptors in the LGIC superfamily, the potential for receptor heterogeneity is much greater than had earlier been anticipated from pharmacological characterization.

Imaging of the nAChR (see 18, 19) and, more recently, of the GABAA receptor (20) by electron microscopy has suggested that each member of the LGIC family is a pentamer of subunits that are assembled around a central ion channel. The mature subunits of all members of the superfamily are predicted to have a similar overall structure. Each subunit is characterized by a large extracellular N-terminal domain of 200 or more amino acids. This domain contains a conserved pair of cysteines, that are likely to be disulfide-linked, in addition to sites for N-linked glycosylation (1). Later in the primary sequence, there are four hydrophobic domains that are predicted to be transmembrane (designated TM1 - TM4), which places the C-terminus also on the extracellular side. Between TM3 and TM4, there is a large, and somewhat variable, intracellular loop which, in some subunits, contains consensus sequences for phosphorylation (21).

So far, thirteen GABAA receptor subunits have been identified in mammalian brain (reviewed in 6). These have been classified according to sequence similarity into four classes, such that there are six alpha, three ß, three gamma subunits and one delta subunit. Members of a single class share approximately 70% amino acid sequence identity, while 30-40% identity occurs between members of different classes. In addition, the mRNA which encodes the 2 subunit undergoes alternate splicing giving rise, in all vertebrate species examined to date, to two forms of the gamma2 subunit distinguished by the presence or absence of eight amino acids in the large intracellular loop between TM3 and TM4 (22-24). A similar splicing event has been shown to give rise to two forms of the gamma2 subunit in the brains of humans (25) and chickens (26) but not rodents (25-27).

In addition, two subunits, termed rho 1 and 2, have been identified. These are clearly related to GABAA receptors in terms of sequence identity but are primarily found in the retina and appear to have a different pharmacological profile to brain GABAA receptors (28, 29). Receptors that are expressed from mRNA encoding rho subunits are insensitive to both bicuculline and benzodiazepines. These pharmacological properties are not altered by co-expression with any of the GABAA receptor subunits (28, 30). It has therefore been suggested that the rho subunits form the GABAC receptor (30-32).

Heterologous expression studies have demonstrated that different subunits can combine to form receptors which display different pharmacological properties. Although homo-oligomeric GABA-gated ion channels can be formed in either the Xenopus oocyte or the human embryonic kidney (HEK) 293 cell expression systems, they do not possess the full range of functional characteristics of in vivo receptors. In order to produce GABAA receptors with pharmacological properties that resemble those found in mammalian brain at least an alpha, a ß and a gamma subunit are required (33). In particular, the pharmacology of the benzodiazepine site appears to be dependent upon which particular alpha or gamma subunit is incorporated into the receptor (see 6). It should be noted, however, that while there is evidence for the existence of a number of different in vivo GABAA receptors subtypes, not even one native receptor subtype has been unambiguously identified to date. Thus, given the existence of at least 13 subunit genes, there is a possibility of over 500,000 GABAA receptor subtypes, although current evidence suggests that the real number may be only in the tens rather than the thousands (6, 34). A further complication is our ignorance of the relative importance of these receptor subtypes to particular physiological roles. For example, while the combination of an alpha1, ß2 and gamma2 subunit appears to be the most abundant GABAA receptor subtype in mammalian brain (see 6), the precise physiological or pathological role of this receptor subtype has yet to be defined.

Of particular relevance to this review is the question of the stoichiometry of the three different subunits (alpha, ß, gamma) that are thought to occur in at least some GABAA receptor subtypes. Based upon homology with the better characterized nAChR and electron microscopic studies (see above) it is now believed that the GABAA receptor exists as a pentamer (20). There is conflicting evidence from different experimental approaches as to the stoichiometry within the receptor complex (35-37). Further, it is not clear how the subunits are oriented within the pentameric configuration. Clearly, such information is necessary for a full understanding of the nature of agonist and modulator binding sites. While the stoichiometry remains controversial, certain site-directed mutagenesis studies described below have provided some further insight, although they were not specifically designed to address this issue.

In this review, we focus on recent studies directed towards identification of structurally important domains in the GABAA receptor. We consider primarily the insights that have been provided by using mutagenesis techniques. These results are interpreted in the context of current knowledge of the structure and function of this family of ion channels, which has largely come from detailed multidisciplinary studies of the well characterized nAChR.

As discussed below, molecular biological methods have been widely used in attempts to define functional domains of the GABAAR and other LGICs. The use of domain swapping techniques and site-directed mutagenesis has provided some limited understanding of structural motifs in the GABAAR that may be involved in ligand binding, ion channel formation, and post-translational modification. Although these are powerful approaches, their use in the analysis of complex, oligomeric, membrane-bound proteins suffers from a number of potential problems. It is, therefore, important to first comment on some of the problems that arise in the interpretation of mutagenesis experiments.

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