Structural Elements of the gamma -Aminobutyric Acid Type A Receptor Conferring Subtype Selectivity for Benzodiazepine Site Ligands*

Stéphane RenardDagger , Anne Olivier§, Patrick Granger§, Patrick Avenet§, David GrahamDagger , Mireille Sevrin§, Pascal George§, and François BesnardDagger

From the Dagger  Department of Genomic Biology, Synthélabo, 10 rue des Carrières, 92500 Rueil-Malmaison, France and § CNS Research Department, 31 av Paul Vaillant Couturier, BP110, 92225 Bagneux, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

gamma -aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha 5 subunit were substituted by their corresponding alpha 1 residues. Given the high affinity and selectivity of alpha 1-containing compared with alpha 5-containing GABAA receptors for zolpidem, mutated alpha 5 subunits were co-expressed with beta 2 and gamma 2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha 5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha 1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha 5P162T and alpha 5E200G, might alter binding pocket conformation, whereas alpha 5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha 1 subunit, particularly alpha 1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha 1-containing receptors, and a model of this binding pocket is presented.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gamma -aminobutyric acid type A (GABAA)1 receptor is the major inhibitory ligand-gated ion channel in the mammalian brain (reviewed in Ref. 1). This chloride channel is a heteropentameric complex composed of different subunits that exist in many isoforms, alpha 1-6, beta 1-3, gamma 1-3, delta , epsilon , and rho 1-2. The alpha 1beta 2gamma 2 combination is thought to be the most abundant subtype of this receptor family found in the adult brain (2) and corresponds to the first pharmacological GABAA receptor subtype clearly identified (3).

Many classes of drugs interact with this receptor, including the benzodiazepines (BZ), which are the most widely used tranquilizers. The BZ binding site is located between the alpha  and gamma  subunits (4). In addition to BZs, other chemically distinct classes of molecules (5, 6) interact with this binding site, including the triazolopyridazines (CL218872), the beta -carbolines, and the imidazopyridines (zolpidem, saripidem).

Mapping the BZ binding site by alanine scanning mutagenesis (7) has led to the identification of histidine 101 of the alpha 1 subunit (alpha 1-His101) as a major component of the binding site interacting either with the nitrogen present in the core structure of most BZ site ligands (8) or with a phenyl moiety close to the core structure of these ligands (9). Several other amino acids surround the BZ binding site. For instance, alpha 1Y159S and alpha 1Y209S substitutions affect diazepam-mediated potentiation of the GABAA receptor, and these mutants also abolish [3H]Ro15-1788 binding to the receptor (10). The alpha 1Y161A, alpha 1T206A, and gamma 2F77A substitutions result in a 3-fold increase in potentiation by diazepam (11, 12).

An inherent drawback of site-directed mutagenesis for the study of receptor ligand interactions is the difficulty in determining whether the amino acid substitution introduced produced its effect directly at the level of the ligand binding site. It is still theoretically possible that the mutation could induce an allosteric conformational change of the receptor itself that indirectly affects ligand binding properties. One way of minimizing this problem is to compare GABAA receptor affinities for subtype-selective ligands before and after mutation of the subunit showing the lowest affinity for the ligand and to take into account only those mutations yielding increases in ligand affinity as indicators for the involvement of the mutated amino acid at the ligand binding site. Such studies have identified alpha 1-Thr162, alpha 1-Gly200, alpha 1-Val211, gamma 2-Phe77, and gamma 2-Met130 by comparison of the alpha 1 and alpha 3 subunits (13), the alpha 1 and alpha 6 subunits (14), or the gamma 2 and gamma 3 subunits (12, 15). For the present study, the ability of zolpidem to strongly discriminate between alpha 1beta 2gamma 2 and alpha 5beta 2gamma 2 GABAA receptor subtypes (16) was used to characterize mutated alpha 5 subunits in an attempt to identify those amino acids imparting the preference of zolpidem for the alpha 1 subunit compared with alpha 5. By swapping the domain responsible for zolpidem interaction from the alpha 5 subunit with that of the alpha 1 subunit and then serially replacing the amino acids in this alpha 5 sequence, we exhaustively searched for amino acids contributing to high affinity for zolpidem. This approach pinpointed three amino acid substitutions that are sufficient to confer an alpha 1-like zolpidem affinity on the alpha 5 subunit.

    EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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Nomenclature-- Amino acids are numbered in alpha 1 (rat sequence) with the putative signal peptide (25 amino acids) removed and in alpha 3 and alpha 5 by an alignment with their equivalent positions in alpha 1.

Compounds and Biological Materials-- Compounds were obtained from the following sources: zolpidem, saripidem (Synthélabo Recherche); CL218872 (American Cyanamide); and [3H]flumazenil (83.2 Ci/mmol) (NEN Life Science Products). Cloned cDNAs encoding the alpha 1, alpha 5, beta 2, and gamma 2 subunits of rat GABAA receptor were supplied by Prof. P. H. Seeburg (University of Heidelberg) and were subcloned into expression vectors as described previously (17).

Mutagenesis-- Chimeras were generated using the Higuchi method (18). 50-Mer polymerase chain reaction primers, overlapping in the chimeric area with 15 pairing nucleotides in 5' and 3', were used to generate chimeric polymerase chain reaction products to be subcloned into the BsmBI and EcoRI sites of the alpha 5 cDNA. Site-directed mutagenesis was obtained with the unique site elimination method (19). Selection primer was designed to disrupt a ScaI site in the ampicillin resistance. Mutagenic primer was always designed with at least 10 bases before and after the mutated codons, ending with a G or C. For mutant alpha 5m10, two mutagenic primers were used. Plasmids were purified by ion exchange chromatography (Qiagen). All mutants were checked by sequencing.

Protein Expression-- alpha xbeta 2gamma 2 combinations of rat GABAA receptor subunits (where alpha x is alpha 1, alpha 5, or any chimera between the two) were transiently transfected in a 1:4:0.2 ratio using the Chen and Okayama procedure (20) on HEK293 cells seeded at 3 × 106/100-mm dish in minimum essential medium (Life Technologies, Inc.). Cells were collected 2 days post-transfection for binding assays. For electrophysiological assays, cells were transfected with the Fugene reagent (Boehringer Mannheim) according to the manufacturer's protocol.

[3H]Flumazenil Equilibrium Binding Assays-- Binding assays were performed as described previously (17). Briefly, 50 µg of membrane preparation from the transfected cells were incubated with different concentrations of [3H]flumazenil for the equilibrium saturation analysis. For competitive inhibition experiments, 50 µg of membrane preparation of the transfected cells were incubated with [3H]flumazenil at a concentration of 4 nM in the presence of various concentrations of the competing ligand. The concentration of ligand that produced half-maximal inhibition of [3H]flumazenil binding (IC50 value) was established by nonlinear regression analysis using Origin 3.0 software (Microcal Inc.) for each of the GABAA receptor combinations and a Ki value deduced using the Cheng and Prusoff equation (21).

Electrophysiology-- We used the whole cell configuration of the patch-clamp technique with symmetrical chloride conditions (pipette solution composition was 140 mM CsCl, 1 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, 10 mM Hepes/Tris-OH, pH 7.2, 4 mM Na2ATP; external solution was 147 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes/Tris-OH, pH 7.4). Currents were recorded using an Axopatch 1D amplifier (Axon Instrument) connected to a personal computer driven by the Clampex7 software. For EC50 determinations, the nonlinear curve fitting routine of the Origin software (Microcal Software Inc.) was used.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding Properties of alpha 5 versus alpha 1 Subunit-containing Receptors-- The alpha 1 or alpha 5 subunits were co-expressed together with beta 2 and gamma 2 in HEK 293 cells, and the parameters of [3H]flumazenil binding to membrane preparations of these transfected cells were evaluated in equilibrium binding assays. Transfected receptors displayed a similar affinity for several benzodiazepine site ligands such as flumazenil but differed remarkably in their affinity for zolpidem and CL218872 (Table I). CL218872 is 3-10 times more selective for alpha 1- than for alpha 5-containing subtypes, and zolpidem is over 300 times more selective (Table I) (17, 22, 23).

                              
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Table I
Zolpidem and CL218872 affinity for GABAA receptors with alpha xbeta 2gamma 2 combinations
Kd values (flumazenil) were assessed by [3H]flumazenil binding equilibrium saturation analyses, and Ki values (zolpidem, CL218872) were determined using [3H]flumazenil competitive inhibition experiments. Ki values represent the mean ± S.D. of three independent experiments.

Binding Properties of Chimeras-- Three chimeras (alpha 5C1, alpha 5C2, and alpha 5C3) of alpha 1 and alpha 5 were constructed in the putative extracellular N-terminal domain (Fig. 1). The alpha 5C1 subunit corresponds to an alpha 5 subunit in which the 198-204 region (GTENIST) is replaced by the corresponding amino acids of alpha 1 (DSGIVQS). The alpha 5C2 subunit has an additional 172-176 region (NGSTK) of alpha 5 replaced by the corresponding amino acids of alpha 1 (REPAR). The alpha 5C3 subunit has an additional 162-164 region (TRA) of alpha 5 replaced by the corresponding amino acids of alpha 1 (PNS). Each of these chimeras was transfected with beta 2 and gamma 2 subunits in HEK293 cells, and the affinity of the corresponding combination for [3H]flumazenil was determined. Both wild type and chimeric receptors displayed similar high affinity for [3H]flumazenil (Table II), demonstrating that the pharmacological profile of this BZ antagonist at its binding site was not affected by these mutations.


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Fig. 1.   Alignment of partial protein sequence of alpha 1 (top lane) and alpha 5 subunits (bottom lane). Regions replaced in chimeras C1 to C3 are lettered in black, and the three alpha 1 amino acids essential for zolpidem binding are enclosed in a gray circle.

                              
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Table II
Mutant receptor affinity for zolpidem
Chimeric subunits were constructed by exchanging various parts of the N-terminal extracellular domain between alpha 5 and alpha 1. Single amino acid substitutions were realized by exchanging various amino acids of the N-terminal extracellular domain between alpha 5 and alpha 1. Kd values (flumazenil) were assessed by [3H]flumazenil binding equilibrium saturation analyses, and Ki values (zolpidem) were assessed using [3H]flumazenil competitive inhibition experiments. Values represent the mean ± S.D. of three independent experiments for the chimeras and two independent experiments for the mutants m1-m7. For m1-m7, the Kd value for [3H]flumazenil was assumed to be the same as that exhibited by chimera alpha 5C1. Amino acids underlined are the same as in alpha 1 sequence.

The alpha 5C1beta 2gamma 2 receptor showed a Ki value of 244 ± 93 nM for zolpidem, representing an increase in affinity of at least 100-fold for zolpidem compared with that of the wild type alpha 5beta 2gamma 2. The Ki values of alpha 5C2beta 2gamma 2 and alpha 5C3beta 2gamma 2 for zolpidem were 210 ± 52 and 99.2 ± 53.1 nM, respectively. The alpha 5C3beta 2gamma 2 combination exhibited an affinity for zolpidem close to that exhibited by the alpha 1beta 2gamma 2 combination.

Regions 162-164 and 198-204 of alpha 1 are therefore of crucial importance for zolpidem affinity. To determine the precise amino acids involved in high affinity zolpidem binding, combinations of amino acids were then substituted between alpha 5 and alpha 1 in the 162-164 and 198-204 regions of alpha 5.

Single Amino Acid Substitutions (Table II)-- Seven amino acids are different from alpha 1 in the 198-204 region of alpha 5. Each combination of six amino acids in this region was substituted by their corresponding residues found in alpha 1. In other terms, mutants alpha 5m1 to alpha 5m7 correspond to chimera alpha 5C1 except for a single residue in the 198-204 region that is conserved as in the wild-type alpha 5.

The various alpha 5 mutants in combination with beta 2 and gamma 2 retained their capacity to bind [3H]flumazenil with the exception of mutant alpha 5m4, which we chose not to analyze further. Mutants alpha 5m3 and alpha 5m7 lost part of their affinity for zolpidem compared with alpha 5C1; the amino acids differing in these mutants compared with alpha 5C1 were Gly200 and Ser204, respectively. To confirm that these amino acids were indeed responsible for zolpidem affinity, an alpha 5m9 (E200G/T204S) that incorporates these two amino acids in alpha 5 was constructed (Table I). The alpha 5m9 subunit in combination with beta 2 and gamma 2 had a zolpidem affinity of 829 ± 197 nM, nearly the same as chimera alpha 5C1. This result pinpoints the determinant roles that alpha 1-Gly200 and alpha 1-Ser204 play in zolpidem binding.

The contribution of amino acids 162-164 of alpha 1 is shown by chimera alpha 5C3, whose affinity for zolpidem could be explained by a P162T mutation (14). The alpha 5m10 (P162T/E200G/T204S) mutant that integrates both E200G and T204S and this new substitution was constructed (Table I). The resultant alpha 5m10beta 2gamma 2 combination had an affinity of 105 ± 17 nM for zolpidem, corresponding to more than a 100-fold increase in affinity for this compound compared with that of alpha 5beta 2gamma 2. A small 3-fold difference of affinity for zolpidem remained compared with that of alpha 1beta 2gamma 2, but no other substitution appeared obvious that might account for this subtle change.

CL218872 Binding (Table I)-- CL218872 displayed a 3-fold selectivity for the alpha 1beta 2gamma 2 compared with the alpha 5beta 2gamma 2 subunit combination (Ki values of 137 ± 25 and 333 ± 17 nM, respectively). The affinities of the alpha 5m8beta 2gamma 2, alpha 5m9beta 2gamma 2, and alpha 5m10beta 2gamma 2 combinations for CL218872 were examined in [3H]flumazenil competitive inhibition experiments to check if the same amino acids involved in high affinity binding for zolpidem would also explain the more limited subtype selectivity observed with CL218872. Mutant alpha 5m8 (E200G) gave a Ki value of 157 ± 23 nM for CL218872. Also, alpha 5m10 (P162T, E200G, T204S) gave a Ki value of 127 ± 9 nM for CL218872, again an affinity close to that displayed by alpha 1beta 2gamma 2 for this compound. In contrast, alpha 5m9 (E200G/T204S) exhibited a significantly lower affinity for CL218872 with a Ki value of 447 ± 62 nM.

Functional Characterization of alpha 5m10beta 2gamma 2 Receptor-- Agonists of the BZ site allosterically increase the apparent affinity of GABAA receptors for GABA (5) and thereby potentiate chloride currents obtained with submaximal GABA concentrations. Zolpidem has been shown to maximally potentiate GABA-induced currents at 1 µM by as much as 384% at alpha 1beta 2gamma 2 receptors stably expressed in HEK293 cells, but 10 µM zolpidem is without effect at alpha 5beta 2gamma 2 receptors (17). To study the functional relevance of the three mutations introduced into the alpha 5 subunit, a whole cell patch clamp analysis of HEK cells cotransfected with alpha 5m10beta 2gamma 2 was performed. We wanted to verify not only the responsiveness of the mutant receptor to GABA but also check whether the gain in affinity for zolpidem observed in this mutated receptor translated into a potentiation of GABA-induced currents. The EC50 value for GABA measured on two cells was 4.2 ± 0.4 µM, a value that compares well with those of 4.5 and 4.2 µM observed for recombinant alpha 1beta 2gamma 2 and alpha 5beta 2gamma 2 receptors, respectively (24). As shown in Fig. 2, both diazepam and zolpidem potentiated the Cl- current induced by GABA at the mutated alpha 5m10beta 2gamma 2 receptor. The mean potentiation values recorded from six cells were 108 ± 28 and 139 ± 31% for 1 µM of zolpidem and diazepam, respectively. The EC50 value of zolpidem (measured on four cells) was 192 ± 5.2 nM.


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Fig. 2.   Whole cell recording of HEK293 cells transfected with the alpha 5m10beta 2gamma 2 mutant. Currents were recorded using a whole cell protocol upon application of GABA (1 µM; black bars) and subsequently GABA in the presence of diazepam (open bars) or zolpidem (hatched bars).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

All mutations in the alpha 5 subunit were designed to substitute alpha 5 residues by amino acids in their equivalent positions in alpha 1. None of these mutations (except mutant alpha 5m4) affected the affinity of the resultant recombinant GABAA receptor for [3H]flumazenil, the ligand used as a probe (see Table II). Moreover, all alpha 5 mutants in this study (except mutant of alpha 5m4) showed an increase in affinity for zolpidem compared with that of the wild type alpha 5 subunit. This gain of function strategy strongly suggests that the amino acids identified are involved in zolpidem binding as opposed to an indirect effect resulting from a conformational modification. The only exception is mutant alpha 5m4, which unexpectedly conferred no [3H]flumazenil binding on transfected cells; we currently do not know with this particular mutant whether the alpha 5m4 polypeptide is destabilized or if an alpha 5m4beta 2gamma 2 heteromeric receptor is indeed formed but with conformational change impeding [3H]flumazenil binding.

Among benzodiazepine site ligands, zolpidem has a strong selectivity toward alpha 1-containing GABAA receptors. A chimera constructed by Wieland and Lüddens (14) comprising the N-terminal part of alpha 6 up to amino acid 158 (with R101H) together with the rest of the protein exchanged for the corresponding fragment of alpha 1 showed an excellent affinity for zolpidem (Ki = 125 ± 50 nM). The zolpidem binding site on alpha 1 could thus be expected to be localized principally between alpha 1-Ala160 and the first putative transmembrane domain of alpha 1 (amino acids 226-246). Three segments differ significantly between alpha 1 and alpha 5 in this region of the protein: 162-164, 172-176, and 198-204 of alpha 5. We thus generated three chimeras by substituting these alpha 5 segments by their alpha 1 counterpart.

The alpha 5C1beta 2gamma 2 combination gave a Ki value of 244 ± 93 nM for zolpidem. This represented a 100-fold increase in affinity compared with the wild-type alpha 5-containing combination, which might correlate with the establishment of an additional hydrogen acceptor interaction (2-3 kcal/mol). Previous data (13, 14, 25) have suggested that the alpha 1-Gly200 contained in this region is of some importance for zolpidem binding. However, Gly200 is replaced by a glutamic acid in alpha 3 and alpha 5, but alpha 3 nevertheless retains a medium affinity for zolpidem (23). Thus, Gly200 by itself is not sufficient to explain the low affinity of alpha 5-containing receptors for zolpidem. As such, a combination of amino acid substitutions in the 198-204 region of alpha 5 could play an additional role, permitting further increases in zolpidem affinity to be attained. The alpha 5C1 chimera includes this combination of amino acids and, in addition, others whose presence did not affect the parameters of zolpidem binding.

Rather than testing all possible combinations, seven mutants were generated, each having six of the 198-204 amino acids substituted with their corresponding alpha 1 counterparts. Two mutants (alpha 5m3 and alpha 5m7) did not confer an affinity for zolpidem equivalent to that of chimera alpha 5C1 (Table II). The two amino acids that were unchanged in these mutants revealed certain substitutions necessary to explain chimera alpha 5C1 properties. The generation of alpha 5m9 confirmed that two amino acids changes (E200G/T204S) are also sufficient to confer alpha 5 with high affinity zolpidem binding properties (Table I).

The alpha 5C2beta 2gamma 2 combination exhibited a Ki value of 210 ± 52 nM for zolpidem, indicating that amino acids 172-176 play no significant role in zolpidem selectivity. The alpha 5C3beta 2gamma 2 combination gave a Ki value of 99.2 ± 53.1 nM for zolpidem, indicating that amino acids 162-164 are situated at the high affinity zolpidem binding site. The mutant alpha 5m10 (P162T/E200G/T204S) confirmed our assumption (based on results from Ref. 14) that alpha 5-Pro162 could have limited influence on zolpidem affinity.

Alignment of alpha 1 and alpha 5 sequences reveals that 115 amino acids are different between alpha 1 and alpha 5. Given this difference, our present data highlight the finding that only three single amino acid substitutions are sufficient and necessary to confer an alpha 1-like affinity for zolpidem on alpha 5-containing receptors. Additional evidence discussed below may favor the hypothesis that alpha 5P162T and alpha 5E200G are involved in conformational changes and alpha 5T204S is implicated in hydrogen bonding between the hydroxyl group of the serine side chain and an hydrogen acceptor site in zolpidem (Table II).

Serine alpha 1-Ser204 Interacts with the Carbonyl Group of Zolpidem (Fig. 3A)-- Serine alpha 1-Ser204 is essential for zolpidem affinity as observed when comparing mutant alpha 5m7 with alpha 5C1. Since serine residues can form hydrogen bonds with proton acceptor sites, alpha 1-Ser204 could be involved in such an interaction with zolpidem. We suggest that this tandem interaction occurs via the carbonyl group of the acetamide side chain (Fig. 3A), and other experimental evidence also seems to support this hypothesis. Thus, NMR conformational studies on zolpidem and another imidazopyridine, saripidem, has revealed that zolpidem and saripidem differ in the nature and flexibility of their side chains (26). That study reported that zolpidem can only adopt one specific conformation, whereas saripidem has two conformational states, one similar to that of zolpidem and a second in which the amidomethyl side chain of saripidem is on another plane (26). Interestingly, saripidem displays far less selectivity than zolpidem between alpha 1 and alpha 5. These observations suggest that the common conformations of zolpidem and saripidem are appropriate for maximal affinity with alpha 1, and the second conformation observed only for saripidem corresponds to a different orientation of the carbonyl that permits accommodation of the alpha 5 pocket.


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Fig. 3.   Interpretation of mutagenesis results and modeling of the BZ binding pocket interacting with zolpidem (A) or CL218872 (B). Pro162 causes displacement of a hydrophobic area. Gly200 also changes the conformation of the surrounding amino acid chain, and Ser204 in particular interacts with zolpidem (A). Solid lines represent the alpha 1 binding pocket, and dashed lines represent the alpha 5 binding pocket.

The conformation of the carbonyl group present in the zolpidem molecule indicates that this carbonyl might well interact with Ser204. If this were the case, the carbonyl of saripidem could interact with the Ser204 of alpha 1 in the conformation common to zolpidem and saripidem, whereas with alpha 5 the alternative conformational state of saripidem would interact with another acceptor site (for instance Ser203 or Thr204).

The carbonyl of zolpidem may thus be a major determinant accounting for the high selectivity of this compound for alpha 1- compared with alpha 5-containing combinations. Moreover, we suggest that this interaction might be with alpha 1-Ser204, in which case this serine would be an essential component of the binding site that predetermines alpha 1beta 2gamma 2 benzodiazepine subtype selectivity.

Glycine alpha 1-Gly200 Has a Conformational Role (Fig. 3)-- Glycine is known to confer conformational freedom to peptidic chains. A glycine residue located close to amino acid position 200 is a feature common to all alpha  subunits. Nonetheless, this residue occurs at slightly different positions on these subunits, thereby possibly contributing to different conformational states of their BZ binding sites. The alpha 1-Gly200 amino acid may be in a position that orients the pocket favorably for zolpidem binding. Since the equivalent glycine residue occurs 2 amino acids upstream in alpha 3 and alpha 5, this could probably alter the BZ binding pocket of these subunits unfavorably for high affinity zolpidem binding. Ser204 could be inappropriately oriented to interact optimally with the zolpidem carbonyl group. This could explain why alpha 3 combinations have intermediate affinity (compared with alpha 1 and alpha 5) for zolpidem, alpha 3 bearing Ser204 and not Gly200. This hypothesis would correlate with the fact that alpha 5 and alpha 3 (in which glycine residues occur at equivalent positions) exhibit similar affinity for CL218872 (23).

On the basis of this potential conformational role of alpha 1-Gly200, one can speculate on reasons behind the loss of flumazenil binding observed with the mutant alpha 5m4. Since the mutant alpha 5m4 contains an asparagine from alpha 5 adjacent to Gly200, instead of being 3 amino acids away from the glycine as in alpha 5, the close proximity of this asparagine to the glycine residue might disrupt the BZ binding site.

Proline alpha 5-Pro162 Has a Conformational Role (Fig. 3)-- The increase in affinity for zolpidem obtained with the alpha 5P162T mutation (alpha 5m10 compared with alpha 5m9) is approximately 5-fold (Table I). Although this cannot be explained by an additional hydrogen bond, it is known that proline induces turns and secondary structures. We can thus assume that this P162T mutation is responsible for a local reorganization of the zolpidem binding site, presumably allowing a better hydrophobic interaction with the pyridine heterocycle of zolpidem. Two tyrosines (alpha 1-Tyr159 and alpha 1-Tyr161) closely situated to this proline have also been reported to be implicated in benzodiazepine binding (10, 27), one affecting potentiation of GABA by benzodiazepines, the other affecting flumazenil affinity. These amino acids may interact with zolpidem, but, since they are present on alpha 1 and alpha 5, substitution mutagenesis would not help to resolve this point. In the case of CL218872 (Table I), it appears that Ser204 has a negative effect (mutant alpha 5m9) on the affinity for CL218872 when the P162 is present. This effect disappears in the presence of Thr162 (mutant alpha 5m10), thus suggesting that Pro162 may somewhat narrow the BZ binding pocket. As such, although no clear cut conclusion can be made regarding the involvement of alpha 1-Thr162 in the BZ binding site, it would appear that alpha 5-Pro162 alters the pocket configuration.

Key Residues Implicated in CL218872 Binding (Table I, Fig. 3B)-- CL218872, which displays a 3-fold selectivity for alpha 1 compared with alpha 3 and alpha 5, does not possess a proton acceptor comparable with the carbonyl function of zolpidem. Nevertheless, CL218872 could be expected to occupy the BZ binding pocket with a certain degree of similarity to that displayed by zolpidem, since alpha 1-Gly200 is known to affect the interaction of the BZ binding site with CL218872 (13, 14, 25). Mutant alpha 5m8 (E200G), indeed, conferred high affinity for CL218872, but the presence of Ser204 somehow affected affinity for CL218872 in mutant alpha 5m9 (E200G, T204S), and the receptor affinity for CL218872 was again restored in mutant alpha 5m10 with the P162T mutation. The alpha 5 mutants behaved as if Gly200 was essential for high affinity toward CL218872, and as if the presence of Pro162 (mutant alpha 5m9) would change the binding pocket such that Ser204 interfered with the binding of this compound. This confirms that alpha 1-Thr162 and alpha 1-Ser204 are situated close to the CL218872 binding pocket even if these residues may not interact directly with this compound.

Conclusions and Perspectives (Fig. 3)-- Both alpha  and gamma  subunits contribute to the BZ site on GABAA receptors (4), and it is generally assumed that the binding site is located at the alpha /gamma interface (4). The present study takes into account gain of affinity mutations incorporated into alpha 5 to confer an alpha 1-like affinity for some benzodiazepine site ligands. These data may be associated with previous mutagenesis studies on alpha  and gamma  subunits, mainly the identification of alpha 1-His101 (7) and gamma 2-Phe77 or gamma 2-Met130 (12, 15). The gamma 2-Phe77 substitution has the property to alter the affinity for different benzodiazepine site ligands or for only zolpidem depending on the amino acid residue substituted (15). Based upon this and the current findings of this report, we can now propose a new molecular model of the alpha 1-selective high affinity binding site occupied by zolpidem and in part by CL218872. We suggest that two regions of the binding pocket interact with proton acceptor sites of the ligand, one being alpha 1-His101 interacting with the nitrogen atom of the imidazole ring and the other alpha 1-Ser204 interacting with the carbonyl group of zolpidem. In addition, two sterically-defined hydrophobic regions of alpha 1 would interact with aromatic moieties of ligands. One hydrophobic region would be located near alpha 1-Thr162, where it would interact with the heterocycle of zolpidem. The second hydrophobic interaction might occur between gamma 2-Phe77 or gamma 2-Met130 and zolpidem; this possibly takes place through the phenyl in position 2 of the heterocycle of zolpidem, given that CL218872, which does not possess an equivalent chemical group, is not affected by changes on these amino acid residues (12, 15).

The specific moieties of zolpidem fitting into the binding pocket should be confirmed with analogs of zolpidem, whose affinities for our mutants would need to be tested in light of this new model. The three amino acids identified in this study are essential components inherent to the high affinity binding site of alpha 1 subunit-containing receptors that explain the selectivity of this binding pocket for alpha 1-specific ligands. The identification of similar residues in other alpha  subunits should help delineate other GABAA receptor subtype-specific binding sites and aid in the design of subtype-specific ligands for these subunits. The availability of such compounds is clearly of crucial importance in dissecting out the role of the different GABAA receptors in various GABAergic neuronal circuits in the brain.

    ACKNOWLEDGEMENTS

We thank Yann Even for expert technical assistance and Robert Fraser for a critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-41-39-13-59; Fax: 33-41-39-13-04.

    ABBREVIATIONS

The abbreviations used are: GABA, gamma -aminobutyric acid; BZ, benzodiazepine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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