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



MOLECULAR BIOLOGY OF THE GABAA RECEPTOR: FUNCTIONAL DOMAINS IMPLICATED BY MUTATIONAL ANALYSIS

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

4. MUTATIONAL ANALYSIS OF GABAA RECEPTOR BINDING SITES

4.1. The Nicotinic Acetylcholine Receptor: A Receptor Prototype

The nAChR is the best characterized of the LGICs, due largely to its abundance in the electric organs of Torpedo species. Over the last twenty years, a wealth of information about the nature of the ligand binding domains of this receptor has been accumulating from multidisciplinary studies. Several excellent reviews on this subject have appeared recently (e.g. 40, 41). Since many of the GABAAR mutations described below were designed on the basis of information from the nAChR studies, pertinent results from these earlier experiments will be discussed very briefly here.

One of the more widely discussed models proposed to explain the structure of the ligand binding domains of nAChR is the loop model developed by Changeux and colleagues (see 41). By integrating information from affinity and photoaffinity labelling studies, protein sequencing, receptor expression and mutational analysis, this model proposes that each of the two high affinity ligand binding sites is made up by four distinct, discontinuous protein loops. Three of these loops (termed A, B and C) occur within the large extracellular N-terminal of the alpha subunit, and the fourth (loop D) is part of the extracellular domain of a neighbouring alpha or delta subunit. Specific amino acids that have been identified in each of these putative loops are illustrated in Figure 1. The emerging theme is that the high affinity sites for agonists and competitive antagonists lie at the interfaces between the alpha-gamma and alpha-delta subunits. Since the interfaces are non-identical, this model can explain the non-equivalence that is seen in the binding of some nAChR ligands, such as d-tubocurarine and alpha-conotoxin (42-46).

Figure 1 Schematic illustration depicting the hypothetical arrangement of the four loops proposed to form the ligand-binding pocket at subunit-subunit interfaces in members of the LGIC family of receptors. Loops A to C on subunit 1 and loop D on subunit 2 represent the four loops that have been demonstrated by multi-disciplinary approaches to contain residues necessary for ligand-receptor interaction (see Figure 2 and text for details). Subunit 1 in the foreground is shown in cross-section to reveal the contribution made by subunit 2 to the binding pocket at the subunit-subunit interface.

Based on much previous work on multisubunit enzymes (47) and the evidence supporting the above model for ligand binding sites in nAChR, it may be predicted that the occurrence of binding sites at subunit-subunit interfaces is a common feature of multimeric proteins. As described below, this model appears to be relevant also to the location of binding sites in the GABAAR (and also the GlyR). Most of the residues identified to date as important determinants for the recognition of GABA and benzodiazepines lie within one of the four loops predicted by the above nAChR model.

4.2. GABA Recognition Site(s)

4.2.1. Residues implicated in GABA binding

The first hints concerning the location of the GABA binding site on the GABAA receptor were obtained from biochemical studies. In early photoaffinity labelling experiments, the subunit was identified as the major site of incorporation of the GABA analogue, [3H]muscimol (12, 13). The ß subunit is not, however, the only determinant of agonist binding. In expression studies, it has been shown that individual alpha, ß, gamma and delta subunits are capable of forming homo-oligomeric GABA-gated chloride channels when expressed alone in vitro (48, 49). This suggests that most, if not all, GABAAR subunits can form functional GABA binding sites which, when occupied by agonists, mediate channel opening. However, since the properties of the homo-oligomeric channels are not entirely consistent with the native receptor, it is likely that the contributions of residues from other subunits are important for conferring the properties of native receptors.

Mutational analysis of the ß subunit has provided evidence for the involvement of tyrosine and threonine residues in GABA recognition. Using rat clones of alpha1, ß2, and gamma2 subunits expressed in Xenopus oocytes, Amin and Weiss identified two domains in the ß subunit which appear to be crucial for the activation of the receptor by GABA (50). Their analysis targeted regions of the ß2 subunit which correspond to loops B and C of the nicotinic four loop model (see Figure 1 and Figure 2). The two domains consist of inverted repeats of four amino acids, YGYT and TGSY (Figure 2) at positions 157-160 and 202-205, respectively. The conservative mutations Y157F and Y205F each produced a 50-fold increase in EC50, whereas mutations T160S and T202S produced 20-fold increases. Mutation of other residues in these two domains had little or no effect on the EC50 for GABA. Less conservative substitutions of the tyrosine residues produced even more dramatic shifts in the EC50, demonstrating the importance of the aromatic ring structure in these positions. The observed effects were relatively specific for the ß subunits since similar changes introduced into homologous residues in the alpha or gamma subunits resulted in rather modest shifts in EC50 values. As noted above (Section 2), the interpretation of the consequences of mutations is often complex. However, in this study, the authors provide a convincing argument that the mutations affected agonist binding and not channel gating, since it was always possible to overcome the reduction in ligand recognition and reach the same maximum current seen in wild-type receptors by using an increased concentration of GABA (see 51).

LOOP ALOOP BLOOP C

rat GABAA 1
92 KIWTPDTFFHNGKK
138 CPMHLEDFPMDAHACPLKFGSYAYTRA
196 TVDSGIVQ-SSTGEYVVMTTHFHLKR
rat GABAA 2
117 KIWTPDTFFHNGKK
163 CPMHLEDFPMDVHACPLKFGSYAYTKA
22VVGTEIIR-SSTGEYVVMTTHFHLKR
rat GABAA 6
91 KIWTPDTFFRNGKK
137 CPMRLVNFPMDGHACPLKFGSYAYPKS
195 TVSSETIK-SNTGEYVIMTVFHLORK
rat GABAA 2
90 QLWVPDTYFLNDKK
136 CMMDLRRYPLDEQNCTLEIESYGYTTD
192 KLITKKVVF-STGSYPRLSLSFKLKR
rat GABAA 2
105 KIWIPDTFFRNSKK
152 CQLQLHNFPMDEHSCPLEFSSYGYPRE
206 RNTTEVVK-TTSGDYVVMSVYFDLSR
hum GABA 1
131 KIWVPDMFFVHSKR
177 CNMDFSRFPLDTQTCSLEIESYAYTED
233 HTTTKLAFYSSTGWYNRLYINFTLRR
hum GlyR 1
92 SIWKPDLFFANEKG
138 CPMDLKNFPMDVQTCIMQLESFGYTMN
193 KDLRYCTKHYNTGKFTCIEARFHLER
hum GlyR 2
99 SIWKPDLFFANEKS
145 CPMDLKNFPMDVQTCTMQLESFEYTMN
200 KELGYCTKHYNTGKFTCIEVKFHLER
Tor nAChR
84 DVWLPDLVLYNNAD
128 CEIIVTHFPFDQQNCTMKLGIWTYDGT
185 KHWVYYTCCPD-TPYLDITYHFIMQR
chick nAChR 7
83 LIWKPDILLYNSAD
127 CYIDVRWFPFDWQKCNLKFGSWTYGGW
181 KRTESFYECCK-EPYPDITFTVTMRR

Figure 2. Sequence alignment of putative loops A, B, and C of various ligand-gated ion channels (see text for details). Blue residues: GABAA alpha1, alpha2 and alpha6, residues important in benzodiazepine recognition; GlyR alpha1, alpha2, residues involved high affinity (site I) binding of agonists; nAChR alpha and alpha7, conserved aromatic residues demonstrated to be involved in agonist and antagonist recognition. Red residues: GABAA ß2, residues involved in GABA recognition; GABA rho1, residues involved in GABA recognition; GlyR alpha2, residues involved in forming the low affinity (site II) agonist site.

It is interesting to note that in each of the above two domains identified by Amin and Weiss (50), several amino acids are conserved in nACh, glycine and 5HT3 receptors (see Figure 2). In the first domain, Y157 corresponds to W148 of the nicotinic alpha7 subunit and to W149 in the equivalent position of the Torpedo alpha subunit. These residues have been implicated in agonist recognition by both DDF photolabelling and by mutagenesis (reviewed in 52). Glycine 158 corresponds to G167 of the GlyR alpha2 subunit, a residue which has been shown to be critical in forming the glycine recognition site referred to as site I by Schmeiden et al. (53). The second tyrosine in this domain, Y160, is homologous to Y151 of the Torpedo alpha1 subunit, a residue which is also photolabelled by DDF (reviewed in 52). A homologous residue is present in all GABAAR and GlyR subunits and, in the GlyR alpha1 subunit, this residue has been implicated in the discrimination of ligands at the GlyR (54).

In the second domain identified by Amin and Weiss (50), the tyrosine at position 205 is conserved in several nAChR subunits. Photolabelling and mutagenesis studies indicate that this residue plays an important role in ligand binding and possibly also in the coupling of ligand binding to channel gating in the nAChR (reviewed in 52). The homologous position in the 5HT3 and GlyR ß receptor subunits is also a tyrosine, while in GlyR subunits, the residue in this position is a phenylalanine. Mutation of this residue in GlyR alpha subunit results in changes in agonist recognition (55). Thus, it would appear that these two domains are conserved throughout the family of LGICs as important determinants of ligand/receptor interaction.

The findings discussed above illustrate that discontinuous regions of the ß subunit are important for GABA binding. As previously noted, the two domains targeted by Amin and Weiss (50) for mutagenesis are homologous to two of the three loops involved in agonist recognition in the nAChR alpha subunit. In the nAcChR, specific residues in the gamma and delta subunits have also been implicated in forming the fourth loop (loop D; see Figure 1). Photoaffinity labelling and protein sequencing studies of Torpedo nAChR have shown that the competitive antagonist, [3H]d-tubocurarine is incorporated not only into the alpha subunits, but also into W55 of the gamma subunit and W57 in the homologous position of the delta subunit (56, 57). In early equilibrium binding studies, the two binding sites for d-tubocurarine in nAChR were shown to be non-equivalent, being characterized by a 200-fold difference in affinities (58). Site-directed mutagenesis of these residues has more recently suggested that gammaW55 contributes to the high affinity binding site for this ligand, while deltaW57 is involved in forming its lower affinity site (59).

In support of the notion that agonist binding sites occur at subunit-subunit interfaces, a homologous residue to those labelled by [3H]d-tubocurarine in the nAChRgamma and delta subunits has been implicated in the agonist binding site of the GABAAR. As discussed below, this residue is found in the alpha subunit suggesting that the GABA binding site(s) may be located at the ß-alpha interface of this receptor. Sigel et al. (60) identified a leucine at position 64 in the rat alpha1 subunit whereas, in other published sequences, a phenylalanine had been reported in this position (61, 62). When this unique clone was coexpressed with ß2 and gamma2 subunits in oocytes, the EC50 value for GABA was much greater than that reported by others. In addition, the Hill coefficient was close to 1, suggesting a loss of cooperativity of channel activation. Further investigation of the properties of this unique subunit revealed that its inclusion in receptor oligomers caused profound changes in the responses to the competitive antagonists, bicuculline methiodide and SR95531. Thus this residue in the alpha subunit appears to play an important role in the recognition of both agonists and antagonists. The mutation of adjacent residues produced only moderate changes in EC50 values for GABA. Homologous residues in the ß and gamma subunits were mutated to leucine, and this substitution resulted in similar, though smaller, shifts in EC50 values to the right. Additional evidence for the involvement of alphaF64 in agonist binding has since come from photoaffinity labelling studies in which this residue in the rat alpha1 subunit was identified as a site of labelling by [3H]muscimol (63).

The results of the above studies suggest that, as in the nAChR, the agonist binding site(s) for the GABAAR occurs at subunit-subunit interfaces, specifically between the and subunits. Domains in the ß subunit of the GABAAR, which correspond to loops B and C of the nAChR four loop model (see above), appear to play a major role in ligand recognition. In addition, a residue (F64) in the alpha1 subunit of the GABAAR corresponds to homologous residues in the gamma and delta subunits of Torpedo nAChR which appear to participate in agonist and antagonist binding (loop D; see Figure 1). By analogy with the nAChR, it may be predicted that the region of the ß subunit which corresponds to the A loop of the nAChR alpha subunit may also contain important determinants for GABA recognition. As yet, there have been no published studies to support this prediction.

4.2.2 Investigation of a role for a conserved disulfide loop

A common feature of LGICs is a disulfide bridge between cysteine residues in the extracellular N-terminal domain of each subunit (reviewed in 1). These cysteines lie within loop B in the ligand binding model illustrated in Figure 1 and Figure 2. This disulfide loop is thought to stabilize the tertiary structure of the extracellular domain and to be important for receptor assembly and expression. In studies of the Torpedo nAChR, for example, Sumikawa and Gehle found that the main effect of eliminating the disulfide loop from either the alpha or ß subunit was retention of the altered subunits in the cytoplasm of the oocytes in which they were expressed (64). However, there was also some indication that the disulfide bridge may have been involved in the formation of the alpha-bungarotoxin binding site.

Since the disulfide loop is so highly conserved, this feature was modelled by Cockcroft et al.(65) as an important determinant in ligand recognition by all members of the receptor family (see Section 6). In testing this model, however, Amin et al., using rat cRNAs expressed in Xenopus oocytes, showed that mutations within this loop did not significantly affect ligand interaction with the GABAAR (66). They specifically targeted the residues in the subunit that were proposed to form electrostatic interactions with the charged groups present in the GABA molecule, and substituted them with either a neutral residue or a residue of the opposite charge. Using numbering that refers to the position of residues within the cystine loop, they showed that substitution of arginine at position 6 (proposed to interact with the negative site of GABA) caused a moderate (3-fold) shift in EC50 values to the right. Since this effect occurred regardless of the charge of the introduced amino acid, this residue is unlikely to be involved in electrostatic interaction with GABA. Substitution of the tyrosine residue at position 8 (proposed to confer selectivity for GABA) with phenylalanine produced a channel that was approximately 3-fold more sensitive to GABA activation, while substitution with a serine resulted in no detectable current, probably due to deficits in receptor expression. Mutation of the residue D11 (proposed to interact with the positive amino group of GABA) produced two results. Substitution with asparagine resulted in no current, while substitution with glutamate did not affect the EC50, but did decrease the Emax and Hill coefficient. The authors suggest that these effects are a result of general disruption of receptor assembly and/or gating. The most dramatic change was seen when L5 was substituted with glutamine. This mutation resulted in a twelve-fold increase in the EC50 value for GABA, and a decrease in maximum current.

Removal of the cysteine residues which form the disulfide bridge invariably led to a lack of GABA-gated currents, presumably due to much reduced receptor expression (66). In this study, the authors were able to discriminate between loss of expression and lack of agonist recognition by introducing the same mutations into the gamma subunit. Coexpression of the mutated gamma subunit with alpha and ß produced GABA-gated channels which had an EC50 closely matching that obtained when alpha and ß subunits alone were expressed. Further evidence that the mutated gamma subunit was poorly expressed, if at all, was that these receptors lacked characteristics normally attributed to the gamma subunit i.e. sensitivity to zinc and to the benzodiazepine agonist, diazepam. Thus mutational analysis of the conserved disulfide loop suggests that the loop is more important in the expression and assembly of the receptor than in agonist recognition.

4.2.3 A role for aromatic and hydroxylated amino acids in GABA recognition

The above studies show that, to date, few residues have been identified as being involved in GABA recognition. Those that have been implicated in binding tend to be either aromatic or polar in nature. One feature that is shared by the endogenous ligands of all members of the LGIC family (nAChR, GABAAR, GlyR, 5HT3R) is a positively charged nitrogen at physiological pH. In early studies, it was reasonably predicted that this positive charge would be stabilized by an acidic amino acid residue (aspartate or glutamate) within an anionic subsite of the receptor. However, this idea was challenged when the three-dimensional structure of acetylcholinesterase, at 2.8 Å resolution, showed that the quaternary ammonium moiety of ACh was bound, not to a negatively charged site, but rather to a preponderance of aromatic residues lining a deep gorge forming the binding site (67). Since then, evidence has been accumulating to suggest that aromatic residues are involved in the agonist binding domains of the nAChR, GlyR and GABAAR.

In the case of the nAChR, aromatic residues have been suggested either to form a negative subsite in the binding pocket which interacts with the quaternary ammonium group of ACh or to be involved in hydrophobic interactions with this portion of the ligand (reviewed in 40, 68). It has also been suggested that the most important factors for ACh recognition are the hydroxyl groups of the highly conserved tyrosines in the nAChR binding site (69). In the case of the GABAAR, the hydroxyl groups of residues Y157 and Y205 appear to be important in ligand recognition, but perhaps not as important as the aromatic side chain itself (50). The size of the residue and the presence of a hydroxyl group at position 202 of the ß-subunit appears to be essential in mediating GABA recognition. Substitution of this residue with serine shifted the dose response curve significantly, while substitution with alanine produced receptors that, although they did not respond to GABA, could be directly activated by pentobarbital, indicating that these mutations did not compromise receptor assembly (50). The phenylalanine (F64) in the alpha1 subunit that has been suggested to be important in agonist recognition (see above) has, thus far, been substituted only by a leucine in a natural mutation (60). Thus, the chemical specificity for ligand recognition in this position is unknown.

As yet, although potentially important amino acid residues within the receptor have been identified, there is no good experimental evidence to indicate which parts of the GABA molecule are recognized by these residues. However, it is likely that, by analogy to the nAChR, the hydroxyl and aromatic groups of the residues identified by Amin and Weiss (50) and Sigel et al. (60) may interact with the positively charged portion of the GABA molecule.

4.2.4 Other GABA binding proteins

Analysis of the GABA recognition sites in other GABA-binding proteins may provide further information on requirements for the recognition of this molecule. The GABA binding site of the GABA receptor formed by the rho1 subunit has been examined using site directed mutagenesis. As noted in section 2, receptors formed by rho subunits have properties that distinguish them from the GABAAR subunits. Of seventeen mutations made within the proposed extracellular N-terminal domain of the rho1 subunit, only three produced significant changes in the response of the receptor to GABA as measured by electrophysiology (70). The largest change in the response to GABA came as a result of the mutation Q189H, which produced an 11-fold decrease in GABA potency, and a reduction in the Hill coefficient. This residue lies within the di-cysteine loop which is common to all LGICs (see Section 4.2.2). Further analysis prompted the authors to suggest that the effects of this mutation could be due to a change in the allosteric coupling between binding sites, and not necessarily to a change in agonist recognition. Substitution of a histidine at position 141 with an alanine also reduced the response of the receptor to GABA by three-fold and decreased the maximum current, but left the Hill coefficient unaltered. The authors conclude that the histidine residue is either involved directly in agonist binding or in stabilizing the conformation of the binding site. However, since the relationship between agonist binding and channel activation is unknown, this conclusion may be premature.

Tryptophan residues appear to play a crucial role in the agonist recognition site of the GABA transporter, GAT-1. One of these tryptophan residues is conserved throughout all identified amino acid transporters, and, as with the LGICs, has been proposed to interact with the amino group of their amino acid substrates (71). A model of the active site of GABA aminotransferase, the enzyme responsible for GABA degradation in the CNS, has been proposed (72) in which the carboxylate group of GABA interacts with an arginine and a lysine residue. It is possible that similar, as yet unidentified, residues may exist in the GABA binding pocket of the GABAAR that interact with this portion of the agonist.

4.3. Benzodiazepine Recognition Site(s)

4.3.1. Importance of the alpha subunit(s)

An understanding of the manner in which the benzodiazepines interact with the GABAAR has been of great interest because of the clinical importance of these compounds. Initial studies of native receptors (see above) revealed a heterogeneity in benzodiazepine binding that was thought to result from the presence of more than one type of site for these drugs in the mammalian brain. Based on differential affinity for the specific ligand, CL218-872, two pharmacologically distinct receptor subtypes, BZI and BZII, were postulated to exist (for review, see 8). More recently, cloning of multiple GABAAR isoforms of several different subunit classes, and the subsequent expression of different combinations of subunits in heterologous expression systems has confirmed that the differences in native receptor pharmacology can be attributed to the presence of multiple receptor subtypes (for review, see 6).

Early photoaffinity labelling studies suggested that the major site of incorporation of [3H]flunitrazepam in bovine brain was the subunit(s) of the GABAAR (11, 73). This has since been confirmed using subunit-specific antibodies (74, 75). By peptide sequencing of a fragment of photolabelled receptor, the specific site of [3H]flunitrazepam incorporation within the bovine GABAAR alpha1 subunit has recently been demonstrated to be a histidine residue in position 102 (76). As will be discussed below, mutational analysis of the GABAAR had previously suggested the importance of this and other residues in forming the benzodiazepine binding domain.

One of the first studies to identify specific amino acid residues within the alpha subunit that are involved in benzodiazepine binding was that of Pritchett and Seeburg (77), who investigated the specific determinants of type I and type II benzodiazepine pharmacology. Heterologous expression studies had previously shown that GABAARs containing an alpha1 subunit display a type I BZ-binding profile, while those containing either alpha2 or alpha3 subunits display BZII-type pharmacology. Pritchett and Seeburg (77) constructed a series of -subunit chimearas consisting of sections of the alpha1 and alpha3 subunits and, after coexpression with ß2 and gamma2 subunits in HEK293 cells, assessed their benzodiazepine binding profiles. This approach, coupled with site-directed mutagenesis, led to the identification of a single glycine residue at position 225 of the alpha1 subunit which, if mutated to a glutamate (the residue which occupies the homologous position in the alpha3 subunit), produced a receptor that displayed a BZII-type pharmacology. Unfortunately, no functional assessment of these mutations has been carried out.

Although benzodiazepines are not receptor agonists but rather are modulators of GABA-gated currents, there is evidence to suggest that the benzodiazepine site shares homology with the agonist site, but lies at a different subunit-subunit interface (see 78). In addition to further information cited below, the glycine residue (G225) identified by Pritchett and Seeburg (77) is homologous to Y190 of the Torpedo nAchR alpha subunit. A number of mutational analysis studies have suggested that this residue is involved in forming the agonist recognition site (79, 69) and may be involved in both binding and gating (80). Others have instead suggested that this residue may be involved in the coupling of ligand binding to channel gating and is not directly involved in the initial binding step (81).

As noted above His-102 of the bovine GABAAR alpha1 subunit has been identified as a site of photolabelling by [3H]flunitrazepam (76). The homologous residues in rat alpha subunits appears to be critical in discriminating between the benzodiazepine agonist, diazepam, and the partial inverse agonist, Ro15-4513. Heterologous expression studies have shown that alpha6 subunit-containing receptors do not recognize the classical benzodiazepine agonist, diazepam, whereas all alpha subunits ( alpha1- alpha6) recognize the partial inverse agonist Ro15-4513. To investigate the molecular basis for this specificity, a series of chimaeras between the alpha1 and alpha6 subunits was made (82). The chimaeric alpha subunits were coexpressed in HEK293 cells with ß2 and gamma2 subunits and their benzodiazepine binding profiles were determined. The specificity of agonist recognition was narrowed down to a single amino acid residue at position 101 i.e. the same residue to that which was identified in direct photolabelling studies (see above). The residue in this position is a histidine in the alpha1 subunit and an arginine in the alpha6 subunit. Exchange of the alpha1 subunit histidine for arginine produced a receptor that displayed high affinity [3H]Ro15-4513 binding which could not be displaced by diazepam, CL218-872 or zolpidem. There was also a 200-fold decrease in the affinity of the receptor for the benzodiazepine antagonist, Ro15-1788. The converse occurred in receptors containing alpha6 subunits in which the arginine had been replaced by histidine. These receptors gained sensitivity to diazepam, Cl218-872, and zolpidem, although the ability of these ligands to displace [3H]Ro15-4513 was reduced relative to wild type alpha1 subunit-containing receptors. This indicates that, although the residue in the 101 position is a major determinant of benzodiazepine binding, other residues must be involved in forming the agonist binding pocket. Again, no functional studies were performed with the mutated receptors.

Further confirmation of the importance of this alpha subunit residue in benzodiazepine recognition was obtained when a natural mutation at position 101 of the GABAAR alpha6 subunit was found in alcohol non-tolerant rats (83). A glutamine substitution at this position occurred in the a6 subunit of these rats which, when coexpressed with ß2 and gamma2 subunits in HEK293 cells, produced a diazepam-sensitive receptor. It was further demonstrated that the allosteric effects of GABA at the receptor (inhibition of both [3H]Ro15-4513 and [35S]TBPS binding) were not affected by this substitution. This suggests that the structural requirements for allosteric modulation of the receptor by GABA are not identical to those that mediate benzodiazepine agonist binding.

It is interesting to note that the histidine residue identified in the above studies (H101) resides in a position that is homologous to Y93 of the Torpedo nAChR alpha subunit. This residue is labelled by acetylcholine mustard and DDF, and mutations introduced at this point have shown it to be involved in the binding of agonists and competitive antagonists (for review, see 41).

A further investigation into the differences between the primary structures of the GABAAR alpha1 and alpha6 subunits and the resulting differences in benzodiazepine pharmacology has identified additional residues which, together with H101 and G225, appear to play roles in forming the benzodiazepine recognition site (84). Four residues in the rat alpha6 subunit (R100, P161, E199, and I211) were substituted in a stepwise fashion with the corresponding residues present in the alpha1 subunit (H101, T162, G200, and V212). As the alpha6 subunit residues were replaced by those of alpha1, there was a corresponding increase in the ability of diazepam to displace [3H]flunitrazepam from the receptors such that when all four substitutions were in place, the receptors were more sensitive to diazepam than were wild type alpha1 subunit-containing receptors. The first three substitutions had little effect on the Ki values for Ro15-4513 and Ro15-1788, but the combination of all four produced a 20-fold increase in the Ki for Ro15-4513 while producing only a minor increase (2-fold) for the antagonist.

4.3.2 The importance of the gamma subunit(s)

As discussed above, the GABAAR alpha subunit is a major determinant of benzodiazepine binding and specificity. However, the characteristic ability of these ligands to modulate GABA-gated chloride conductances in native receptors is dependent on the presence of a gamma subunit (33). Furthermore, pharmacological efficacy is determined by the type of gamma subunit that is present (for review, see 85). As yet, only one residue within the gamma2 subunit has been identified as being important in benzodiazepine recognition and efficacy (86). This residue was revealed by comparison of discrepancies in two reported sequences of the human gamma2 subunit. At position 142, a serine was present in the sequence of Pritchett et al (33), whereas a threonine occurred in the sequence determined by Whiting's group (87). When the threonine was changed to a serine and coexpressed with alpha1 and ß1 subunits, the benzodiazepine pharmacology of the receptor was significantly altered whereas there were no measured changes in responses to GABA or to the other receptor modulators, pentobarbital and alphaxalone. In the mutated receptor, Ro15-1788 and Ro15-4513 (normally an antagonist and partial inverse agonist, respectively) became potent partial agonists. In addition, type-I selective agonists became inverse agonists and there was an increased potentiation by non-selective agonists. The authors suggest that the effects of the mutation are due to differences in the ligand-induced conformational changes in the receptor rather than to differences in the binding site per se. In agreement with this possibility, unlike all other residues so far implicated in GABA and benzodiazepine interactions, threonine 142 does not lie within any of the regions corresponding to the four-loop model of the nAChR, and as yet does not have a functional homologue in any other LGIC.

Unlike the residues that have been implicated in forming the domains of the receptor that interact with GABA, no aromatic residues have yet been identified as being important determinants in benzodiazepine recognition. It is likely that some determinants remain to be revealed. In addition, as in all such studies, residues that have been implicated in binding by mutational analysis do not necessarily interact directly with the ligand but rather may stabilize a particular receptor conformation for which some ligands have greater affinity than others.

4.3.3 Other benzodiazepine binding proteins

Mutational analysis of the peripheral benzodiazepine receptor, a protein which bears no significant homology to the GABAAR but which does recognize classical benzodiazepine agonists such as flunitrazepam and diazepam, has identified residues which confer high affinity binding for the benzodiazepine Ro5-4864. In this protein, it appears that five amino acids (two threonines, a cysteine, a valine, and an arginine) play an integral role in the recognition of Ro5-4864 (88). It may be hoped that further examination of the recognition sites of other benzodiazepine-binding proteins will lead to a better understanding of the structural requirements for binding of these ligands.

4.4 Identification of a Single Residue Conferring High Affinity Loreclezole Binding

Loreclezole is a broad spectrum anticonvulsant agent that acts at the GABAAR. This compound is the only known ß subunit-selective agent, with a more than 300-fold greater affinity for ß2/ß3- than for ß1 subunit-containing receptors, a selectivity that occurs independently of the presence of specific alpha and/or gamma subunits (89). To identify the region of the ß subunits responsible for loreclezole sensitivity, chimaeras of ß1 and ß2 subunits were constructed, and it was found that the region between K237 and G334 in the ß2 subunit was necessary for loreclezole potentiation of the GABA response in oocytes (90). Mutational analysis of this region identified a single residue, N289, as being responsible for loreclezole sensitivity. The location of this residue is unusual in that it occurs in the second putative transmembrane domain, TM2 (see Section 5), a region of the receptor thought to form the lining of the ion channel. However, the authors point out that the mutated residue, rather than being part of a binding site, may affect the manner in which the binding of loreclezole modulates the gating of the channel. One might suspect the latter to be the case, since, unlike compounds which are known to directly interact with this region of LGICs, loreclezole potentiates the GABA response rather than blocks it.

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