Wild-type a1b2g2 g-aminobutyric acid (GABA)A receptors and receptors containing a point-mutated subunit g2F77Y were expressed by transient transfection in human embryonic kidney 293 cells. Mutant receptors bound the benzodiazepine binding site ligand [H]flumazenil with similar, subnanomolar affinity as wild-type receptor. Displacement studies with diazepam showed that the affinity for this compound was reduced 250fold on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chemical entity presumably interacting with the phenyalanine residue in position 77 of the g2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substituent of diazepam is located close to gF77. Similarly, we investigated the possible location of a1T206 and g2M130. Electrophysiological data obtained with the wild-type receptor furthermore suggest a simple overlap between positive allosteric modulators acting at the benzodiazepine binding site with its antagonists. The GABAA receptor is the most important ion channel conferring fast synaptic inhibition in the mammalian nervous system. Initially, two subunits of the receptor have been purified (Sigel et al., 1983), and its coding DNAs have been cloned (Schofield et al., 1987). Eighteen subunits from mammalian tissue have been cloned: 6a, 3b, 3g, 1d, 1e, 3r, and 1p (for reviews, see Macdonald and Olsen, 1994; Rabow et al., 1995). The major adult isoform is most likely a1b2g2 (McKernan and Whiting, 1996). The receptor channel is modulated by numerous drugs (Sieghart, 1995). Among these, some compounds act at the binding site for benzodiazepines. They act as anxiolytics, sedatives, muscle relaxants, and anticonvulsives and exert a positive allosteric effect on the channel. An antagonist acting at this site also is in clinical use (Hunkeler et al., 1981), whereas negative allosteric modulators such as DMCM are investigational tools. Amino acid residues H101, Y159, G200, T206, and Y209 on the a1 subunit and F77 and M130 on the g2 subunit have been identified as putative parts forming the binding pocket for the ligands of the benzodiazepine binding site (Pritchett et al., 1991; Wieland et al., 1992; Buhr et al., 1996, 1997a, 1997b; Amin et al., 1997; Buhr and Sigel, 1997; Wingrove et al., 1997; Schaerer et al., 1998). They are highly homologous to amino acids F64 and I120 on the a1 subunit and Y157, T160, T202, and Y205 on the b2 subunit that take part in the formation of the binding site for the channel agonist GABA (Sigel et al., 1992; Amin and Weiss, 1993; Smith and Olsen, 1994; Westh-Hansen et al., 1997). Thus, the channel agonist and allosteric modulators of the channel seem to bind to pseudosymmetrical structures (Galzi and Changeux, 1994; Sigel and Buhr, 1997). Many attempts (Borea et al., 1987; Villar et al., 1989; Schove et al., 1994; Zhang et al., 1995) have been made to characterize spatial properties of the benzodiazepine binding pocket. These studies used either in vivo effects or chloride flux experiments in combination with radioligand binding studies on brain membranes of a large number of structurally related compounds. Derived models for the binding pocket are complex and suggest distinct but partially overlapping binding sites for ligands differing in their allosteric effect. Considering the variety of GABAA/benzodiazepine receptors present in brain, it is not surprising that a model that satisfactorily explains all observations is still missing. It obviously is important to map all the amino acid residues participating in the formation of the benzodiazepine pocket relative to the ligands of this site. Currently, we limit ourselves to a206, g77, and g130, which are available in our laboratory, show a high level of expression in HEK 293 cells, This study was supported by grants 31–37192.93 from the Swiss National Science Foundation and EU Grant BIO4-CT96–0585 (BBW 96.0010). ABBREVIATIONS: GABA, g-aminobutyric acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMCM, methyl-6,7-dimethoxy-4ethyl-b-carboline-3-carboxylate; HEK, human embryonic kidney. 0026-895X/98/061097-09$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:1097–1105 (1998). 1097 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from and display high affinity to a commercially available radioligand. We investigated binding affinities of a variety of ligands of this binding site in the receptor of the defined recombinant subunit composition a1b2g2 and point mutants of the three corresponding residues. Based on our observations, we also propose a model of the interaction of positive allosteric modulators and antagonists with the receptor. Materials and Methods Substances. [H]Flumazenil (87 Ci/mmol) was from DuPont-New England Nuclear (Boston, MA). All nonradioactive ligands of the benzodiazepine binding site were obtained as a kind gift of Hoffmann-La Roche (Basel, Switzerland). Construction of receptor subunits. The cDNAs coding for the a1, b2, and g2S subunits of the rat GABAA receptor channel have been described elsewhere (Lolait et al., 1989; Malherbe et al., 1990a, 1990b). The mutant gF77Y has a phenylalanine-to-tyrosine substitution at position 77 of the mature peptide and has been described previously (Buhr et al., 1996, 1997a). The same is true for gM130L, which contains a methionine-to-leucine substitution (Buhr and Sigel, 1997). aT206V was prepared using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). In vitro synthesized sequences have been verified by DNA sequencing. For cell transfection, the cDNAs were subcloned into the polylinker of pBC/CMV. This expression vector allows high level expression of a foreign gene under control of the cytomegalovirus promoter. With standard techniques, the a subunit was cloned into the EcoRI site of the polylinker, and the b and g subunits were subcloned into the SmaI site. Transfection of recombinant GABAA receptor in cultured cells. The cells were maintained in minimal essential medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin by standard cell culture techniques. Equal amounts (total of 20 mg of DNA/90-mm dish) of GABA receptor subunits were transfected into HEK 293 cells (CRL 1573; American Type Culture Collection, Rockville, MD) according to the calcium phosphate precipitation method (Chen and Okayama, 1987). After overnight incubation, the cells were washed twice with serum-free medium and refed with complete medium. Membrane preparation. Approximately 60 hr after transfection, the cells were harvested by washing with ice-cold phosphatebuffered saline (130 mM NaCl, 16 mM di-sodium hydrogen phosphate, and 4 mM potassium dihydrogen phosphate) and centrifuged at 150 3 g. Cells were washed with buffer containing 10 mM potassium phosphate, 100 mM KCl, and 0.1 mM K-EDTA, pH 7.4. Cells were homogenized by sonication in the presence of 10 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. Membranes were collected through three centrifugation/resuspension cycles (100,000 3 g for 20 min) and then used for ligand binding or stored at 220°. Binding assays. Resuspended membranes (0.5 ml) obtained from cells transfected with wild-type receptor, a1b2g2F77Y, a1T206Vb2g2, or a1b2g2M130L were incubated for 90 min on ice in the presence of [H]flumazenil (87 Ci/mmol, DuPont-New England Nuclear) and varying concentrations of competing ligands. Membranes (20–50 mg of protein/filter) were collected by rapid filtration on GF/C filters presoaked in 0.3% polyethylenimine. After three washing steps with 4 ml of buffer, the filter-retained radioactivity was determined by liquid scintillation counting. Total binding was measured at 2 nM [H]flumazenil, nonspecific binding under the same condition but in the presence of 10 mM unlabeled flumazenil. Displacement curves containing numerous points for wild-type and three mutant receptors clearly would have exceeded capacity. Therefore, a simplified procedure was chosen. The affinity of a substance first was approximately estimated in a binding experiment using 1 nM and 1 mM of the displacing ligand. Displacement then was determined in at least in two independent experiments at two or three concentrations of 0.1 nM, 1 nM, 100 nM, or 10 mM depending on the initial estimates. From these data, the Ki value was calculated in each case according to the Cheng-Prusoff equation (1973). Two determinations for a Ki value do not allow application of statistical procedures; therefore, the error was estimated by the following procedure. The maximal deviation observed in a total of .200 individual Ki determinations from the corresponding mean value amounted to 28%. In most cases, the individual determination deviated far less. Using error propagation, it can be estimated that under the current conditions, the ratio Ki (mutant)/Ki (wild-type) deviates in the worst case by 1.56-fold from the true value and that the ratio is in most cases more accurate. We therefore take any value for the ratio, either .1.6 or ,0.62, as a significant change in affinity on mutation. Protein concentration was determined with the BioRad (Hercules, CA) protein assay kit with bovine serum albumin as standard. Expression and functional characterization. Xenopus laevis oocytes were prepared, injected, and defolliculated and currents were recorded as described previously (Sigel, 1987; Sigel et al., 1990). Briefly, oocytes were injected with 50 nl of cRNA dissolved in 5 mM K-HEPES, pH 6.8. This solution contained the transcripts coding for the different subunits at a concentration of 10 nM for a1, 10 nM for b2, and 100 nM for g2. Transcripts were quantified on agarose gels after staining with Radiant Red RNA Stain (BioRad) by comparing staining intensities with varying amounts of molecular weight markers (RNA-Ladder, GIBCO BRL). Electrophysiological experiments were performed by the two-electrode voltage-clamp method at a holding potential of 280 mV. The medium contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM Na-HEPES, pH 7.4. Allosteric modulation via the benzodiazepine site was measured at a GABA concentration eliciting 3–5% of the maximal GABA current amplitude by the application of GABA alone and the coapplication of GABA with the modulatory compound, usually at a fixed concentration of 100-fold Ki. Allosteric modulation is expressed as the relative current amplitude and was calculated as the modulated current amplitude, divided by the control current amplitude, and the result was multiplied by 100%. GABA was applied for 20 sec, and a washout period of 4 min was allowed to ensure full recovery from desensitization. Positive or negative modulation of GABA currents was expressed as a percentage of the respective control current amplitudes determined in the absence of modulator. To avoid contamination, the perfusion system was cleaned between drug applications by washing with dimethylsulfoxide.