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• W2014342273 abstract "Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the γ-aminobutyric acid type A (GABAA) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABAA receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABAA receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly α1H101C and also three other mutants (α1G157C, α1V202C, and α1V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (–NCS) replaces the azide group (–N3) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, α1H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed. Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the γ-aminobutyric acid type A (GABAA) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABAA receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABAA receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly α1H101C and also three other mutants (α1G157C, α1V202C, and α1V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (–NCS) replaces the azide group (–N3) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, α1H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed. The fast neuronal action of the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA), 2The abbreviations used are: GABAγ-aminobutyric acidGABAAγ-aminobutyric acid type A. is mediated by GABA type A (GABAA) receptors. They are composed of five subunits and form a chloride-selective ion channel (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar). A variety of subunit isoforms have been cloned, leading to a multiplicity of receptor subtypes (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (458) Google Scholar, 3Barnard E.A. Skolnick P. Olsen R.W. Möhler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). The major receptor isoform in mammalian brain consists of α1, β2, and γ2 subunits (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (458) Google Scholar, 4McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). Different approaches have indicated a 2α:2β:1γ subunit stoichiometry for this receptor (5Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar, 6Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar, 7Farrar S.J. Whiting P.J. Bonnert T.P. McKernan R.M. J. Biol. Chem. 1999; 274: 10100-10104Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 8Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 9Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2002; 277: 46020-46025Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 10Baur R. Minier F. Sigel E. FEBS Lett. 2006; 580: 1616-1620Crossref PubMed Scopus (79) Google Scholar). γ-aminobutyric acid γ-aminobutyric acid type A. Benzodiazepines, which act at GABAA receptors, are among the most widely used drugs. Ligands of the benzodiazepine-binding site have been classified into positive and negative allosteric modulators and antagonists. To synthesize ligands of the benzodiazepine-binding site specific for different GABAA receptor isoforms, it is desirable to understand the relative positioning and overlap of different allosteric modulators. Pharmacophore/receptor models for positive allosteric modulators and antagonists have been developed to identify the molecular properties and mode of ligand/receptor interaction (e.g. Refs. 11Huang Q. Liu R. Zhang P. McKernan R. Gan T. Bennet D.W. Cook J.M. J. Med. Chem. 1998; 41: 4130-4142Crossref PubMed Scopus (44) Google Scholar and 12Huang Q. He X. Ma C. Liu R. Yu S. Dayer C. Wenger G.R. McKernan R. Cook J.M. J. Med. Chem. 2000; 43: 71-95Crossref PubMed Scopus (162) Google Scholar). This work has suggested common contact points of ligands differing in their allosteric effect, but there is only speculation on corresponding amino acid residues (13He X. Zhang C. Cook J.M. Med. Chem. Res. 2001; 10: 269-308Google Scholar). Photoaffinity labeling has been used to identify His101 of the α1 subunit as the target of [3H]flunitrazepam (14Duncalfe L.L. Carpenter M.R. Smillie L.B. Martin I.L. Dunn S.M.J. J. Biol. Chem. 1996; 271: 9209-9214Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and Tyr209 as the target of [3H]Ro15-4513 (15Sawyer G.W. Chiara D.C. Olsen R.W. Cohen J.B. J. Biol. Chem. 2002; 277: 50036-50045Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, these results should take into account possible formation of long-lived reactive intermediates after photoactivation (16Brunner J. Annu. Rev. Biochem. 1993; 62: 483-514Crossref PubMed Scopus (441) Google Scholar, 17Rizk M.S. Shi X. Platz M.S. Biochemistry. 2006; 45: 543-551Crossref PubMed Scopus (29) Google Scholar). An alternative approach studied consequences of single point mutation in GABAA receptors using radioactive ligand binding assay and/or electrophysiological measurements; by this, a large number of amino acid residues were identified on both α and γ subunits to build up the benzodiazepine-binding site (18Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 19Sigel E. Curr. Top. Med. Chem. 2002; 2: 833-839Crossref PubMed Scopus (190) Google Scholar). Our approach is based ultimately on the work of Karlin and Akabas (20Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar), who used site-specific mutations to cysteine in combination with a water-soluble nonspecific cysteine-reactive reagent. Covalent reaction was taken as evidence for water accessibility of the corresponding residue. This method has been extended to site-specific investigations by replacing high concentrations of the nonspecific agents with low concentrations of ligands of a defined binding site, carrying a reactive substituent. Direct apposition of such reaction partners is expected to lead to a covalent reaction (21Foucaud B. Perret P. Grutter T. Goeldner M. Trends Pharmacol. Sci. 2001; 22: 170-173Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In the benzodiazepine-binding site, a covalent interaction of α1H101C with a reactive group attached to the –C atom in diazepam normally carrying a –Cl atom was found (22Berezhnoy D. Nyfeler Y. Gonthier A. Schwob H. Goeldner M. Sigel E. J. Biol. Chem. 2004; 279: 3160-3168Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We show here covalent interaction of the same residue, plus three additional residues, with the reactive group attached to the position normally occupied by–N3 in the partial negative allosteric modulator Ro15-4513. Our results identify four contact points of this ligand with the receptor. On the basis of this result and earlier observations (22Berezhnoy D. Nyfeler Y. Gonthier A. Schwob H. Goeldner M. Sigel E. J. Biol. Chem. 2004; 279: 3160-3168Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 41Sigel E. Schaerer M.T. Buhr A. Baur R. Mol. Pharmacol. 1998; 54: 1097-1105Crossref PubMed Scopus (55) Google Scholar) we suggest a similar orientation of classical benzodiazepines and imidazobenzodiazepines bound to the receptor. Synthesis—We used a substance in the imidazobenzodiazepine series we call the Imid-NCS compound, which is similar to Ro15-4513 except that the–N3 group is replaced with–NCS (see Fig. 1). The Imid-NCS derivative was synthesized from the amine precursor, which was a gift from Hoffmann-La Roche. This precursor (200 mg, 0.67 mmol) and ammonium hydrogenocarbonate (224 mg, 1.34 mmol, 2 eq) were diluted in a mixture of chloroform (34 ml) and water (13 ml). After 20 min of vigorous stirring, thiophosgene (61 μl, 0.80 mmol, 1.2 eq) was added to the solution. The reaction mixture was stirred for 1.5 h at room temperature. 20 ml of a 5% NaHCO3 aqueous solution were then added to neutralize excess thiophosgene. The mixture was extracted with dichloromethane. After filtration and drying on Na2SO4, the organic layer was evaporated under reduced pressure. The crude Imid-NCS product was rapidly purified on a neutral alumina column eluted with a mixture of heptane/ethyl acetate (2:1), leading to quantitative formation of the Imid-NCS compound. 1H NMR (CDCl3 - 200 MHz), δ (ppm) 7.97 (m, 2H, H7 + H9), 747 (m, 2H, H10 + H1), 5.21 (m, 1H, H4(1)), 7.44 (m, 3H, H4(2) + CH2), 3.25 (3H, N-CH3), and 1.45 (t, 3H, CH3, J = 7.1 Hz); 13C NMR (CDCl3 - 75 MHz), δ (ppm) 165.7 (C=O(6)), 163.2 (EtO-C=O), 140.0 (C11), 135.8 (–NCS), 135.6 (C1), 133 (C3), 131.2 (C13), 130.7 (C8), 130.5 (C9), 130.0 (C7), 129.3 (C12), 124.1 (C10), 61.9 (O-CH2(1′)), 42.9 (CH2(4)), 36.6 (N-CH3), and 15.0 (CH3(2′)); IR (CsI), ν (cm-1) 2113 (–NCS) and 1645 (C=O). This Imid-NCS compound was dissolved in Me2SO at a concentration of 10 mm and kept at -80 °C at most for 1 month. Final dilutions in assay medium were prepared immediately before each experiment. Construction of Receptor Subunits—The cDNAs coding for the α1, β2, and γ2S subunits of the rat GABAA receptor channel have been described (23Lolait S.J. O'Carroll A.M. Kusano K. Muller J.M. Brownstein M.J. Mahan L.C. FEBS Lett. 1989; 246: 145-148Crossref PubMed Scopus (81) Google Scholar, 24Malherbe P. Draguhn A. Multhaup G. Beyreuther K. Möhler H. Mol. Brain Res. 1990; 8: 199-208Crossref PubMed Scopus (85) Google Scholar). All mutant subunits were prepared using the QuikChange™ mutagenesis kit (Stratagene). The cDNAs were subcloned into the polylinker of pBC/CMV (25Bertocci B. Miggiano V. Da Prada M. Dembic Z. Lahm H.W. Malherbe P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1416-1420Crossref PubMed Scopus (185) Google Scholar) for cell transfection. This expression vector allows high level expression of a foreign gene under the control of the cyto-megalovirus promoter. The α subunit was cloned into the EcoRI site and the β and γ subunits into the SmaI site of the polylinker using standard techniques. Transfection of Recombinant GABAA Receptors in Human Embryonic Kidney 293 Cells—Human Embryonic kidney 293 cells (CRL1573, American Type Culture Collection, Manassas, VA) were grown in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mm glutamine, 50 units/ml penicillin, and 50 μl/ml streptomycin. The cells were plated on 90-mm dishes and transfected with a total amount of 20 μg of DNA (plasmid ratio of 1:1:1)/dish by the calcium phosphate precipitation method (26Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar). After overnight incubation (3% CO2), the transfected cells were washed twice with serum-free medium and refed with supplemented medium. Membrane Preparation—Approximately 60 h after transfection, the cells were harvested by washing with ice-cold phosphate-buffered saline (130 mm NaCl, 20 mm Na2HPO4, and 4 mm KH2PO4, pH 7.4) and centrifuged at 560 × g. Two more washing steps were performed with phosphate buffer (10 mm KH2PO4, 100 mm KCl, and 0.1 mm K-EDTA, pH 7.4). Cells were sonicated in the presence of 700 μm phenylmethylsulfonyl fluoride and 1 mm EDTA. The membranes collected by three centrifugation resuspension cycles (100,000 × g for 20 min) were then used for ligand binding or were shock-frozen in liquid nitrogen and stored at -80 °C. Radioactive Ligand Binding Assay—The properties of the recombinant mutant receptors were only estimated because a complete characterization of all membranes expressing mutant receptors would have exceeded our working capacity. For this affinity estimate, membranes were resuspended in phosphate buffer using a Teflon homogenizer. They were incubated in a total volume of 360 μl for 1 h on ice in the presence of [3H]Ro15-1788 (78.6 Ci/mmol; PerkinElmer Life Sciences) or [3H]flunitrazepam (71–84 Ci/mmol; PerkinElmer Life Sciences). The final protein concentration was 0.1–1 mg of protein/ml. Total binding was measured at 2 and 20 nm [3H]Ro15-1788 or [3H]flunitrazepam. Nonspecific binding was determined under the same condition but in the presence of 100 μm unlabeled Ro15-1788 or flunitrazepam, respectively. Membranes were collected by rapid filtration on GF/C filters presoaked in 0.3% polyethyleneimine. After three washing steps with 5 ml of phosphate buffer, the filter-retained radioactivity was determined by liquid scintillation counting. To perform displacement binding assays, various concentrations of competing ligands (Imid-NCS compound) were added. The concentration of [3H]Ro15-1788 used was ∼3 times the Kd value. On the basis of IC50 determinations, the Ki value was determined according to the Cheng-Prusoff equation (27Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12292) Google Scholar). In all experiments, each single sample was measured in triplicate. Detection of a Covalent Reaction—Detection of a covalent reaction included (a) incubation of membranes expressing wild-type or mutant receptors with the Imid-NCS compound, (b) extensive washing of the membranes to remove non-reacted Imid-NCS compound, and (c) radioactive ligand binding assay to determine residual binding. No covalent reaction would result in 100% residual binding/0% reaction, and 100% covalent reaction would result in 0% residual binding/100% reaction. 200 μl of diluted membranes (∼0.01–0.1 mg of protein/ml) were incubated with the Imid-NCS compound for 1 h on ice. The maximal final solvent concentration during this incubation was 1%. Subsequently, 1.8 ml of ice-cold phosphate buffer were added, and the sample was centrifuged at 14,000 × g for 1 h at 4 °C. The supernatant was removed, and 2 ml of ice-cold phosphate buffer were added to the pellets before centrifugation. The whole procedure was repeated twice. After the third centrifugation, the supernatant was removed, leaving ∼200 μl in the tube. The pellet was resuspended using a Teflon pestle, and 120 μl of ice-cold phosphate buffer were added. During this procedure, 30–50% of the protein was lost. After homogenization, a binding assay was performed in a total volume of 360 μl. Controls containing no reactive substance allowed normalization in each experiment. As the efficiency of membrane recovery during the whole procedure was found to be quite variable and differed between two experiments up to 30%, we assumed covalent reaction when residual binding was <70% of the controls. Where indicated, the Imid-NCS compound was pre-reacted by preincubation for 1 h at room temperature with 100 mm l-cysteine. To protect the binding site from covalent reaction, membranes were incubated with unlabeled Ro15-1788 or flunitrazepam for 10 min on ice. Subsequently, the Imid-NCS compound was added, and 3 min later, the first centrifugation was started. cRNA Synthesis—Capped cRNAs were synthesized (Ambion, Austin, TX) from the linearized pCMV vectors containing the different subunits. A poly(A) tail of ∼400 residues was added to each transcript using yeast poly(A) polymerase (United States Biological Inc., Cleveland, OH). The concentration of the cRNA was quantified on a formaldehyde gel using Radiant Red stain (Bio-Rad) for visualization of the RNA. Known concentrations of an RNA ladder (Invitrogen) were loaded as standards on the same gel. cRNA combinations were precipitated in ethanol/isoamyl alcohol (19:1) and stored at -20 °C. Expression and Functional Characterization in Xenopus Oocytes—Xenopus laevis oocytes were prepared, injected, and defollicated as described previously (28Sigel E. J. Physiol. (Lond.). 1987; 386: 73-90Crossref Scopus (114) Google Scholar, 29Sigel E. Minier F. Mol. Nutr. Food Res. 2005; 49: 228-234Crossref PubMed Scopus (54) Google Scholar). They were injected with 50 nl of cRNA solution containing wild-type or mutant α1, β2, and γ2 subunits at a concentration of 10:10:50 nm (30Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (102) Google Scholar) and then incubated in modified Barth's solution (10 mm HEPES, pH 7.5, 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 0.34 mm Ca(NO3)2, 0.41 mm CaCl2, 100 units/ml penicillin, and 100 μg/ml streptomycin) at +18 °C for at least 24 h before the measurements. Electrophysiological experiments were performed using the two-electrode voltage clamp method at a holding potential of -80 mV. The perfusion medium contained 90 mm NaCl, 1 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 5 mm Na-HEPES, pH 7.4. Allosteric modulation via the benzodiazepine site and covalent reaction were measured at a GABA concentration eliciting 2–5% of the maximal GABA current amplitude. GABA was applied for 20 s alone or in combination with the modulatory compound. Modulation of GABA currents was expressed as a percentage of the respective control current amplitudes determined in the absence of modulator. The perfusion system was cleaned between drug applications by washing with Me2SO to avoid contamination. Modification of Receptor Function by the Imid-NCS Compound or Iodoacetamide—Wild-type α1β2γ2 and mutant α1H101Cβ2γ2, α1G157Cβ2γ2, α1V202Cβ2γ2, and α1V211Cβ2γ2 receptors were expressed in Xenopus oocytes. After obtaining a reproducible response to the application of GABA, the Imid-NCS compound freshly diluted to 1 μm in perfusion medium was applied for 1 min (in the case of mutant receptors). Wild-type receptors were exposed for 1 or 5 min to 1 μm Imid-NCS compound or for 5 min to 10 μm Imid-NCS compound. In some cases, 10 μm Ro15-1788 was applied for a period of 30 s before, during, and for a period of 60 s after exposure to the Imid-NCS compound to protect the binding site from a covalent reaction. The maximal final solvent concentration was 0.1%. This concentration of Me2SO did not affect the response to GABA in control experiments. This treatment was followed by several GABA applications at intervals of 4 min to reach a steady level. Subsequently, 10 or 1 μm zolpidem or 1 μm Ro15-1788 was co-applied with GABA to investigate a covalent reaction with the Imid-NCS compound. The irreversible effect was then calculated as follows: stimulation ((Iafter Imid-NCS/Ibefore Imid-NCS) - 1) × 100%. Where indicated, the Imid-NCS compound was inactivated by preincubation of 500 nm Imid-NCS compound in medium containing 10 mm l-cysteine for 1 h. Control experiments showed that 10 mm l-cysteine did not significantly alter the response to GABA. The effect of the nonspecific cysteine-modifying reagent iodoacetamide was tested on wild-type α1β2γ2 and mutant α1H101Cβ2γ2 receptors. 2 mm iodoacetamide freshly diluted in perfusion medium was perfused for 5 min after obtaining a stable GABA response. Subsequently, GABA was applied to test whether iodoacetamide irreversibly affects GABA-elicited currents. This was followed by co-application of GABA with 1 μm zolpidem in the case of wild-type α1β2γ2 and mutant α1H101Cβ2γ2 receptors. Alternatively, mutant receptors were treated for 1 min with 1 μm Imid-NCS compound. Binding Properties of the Cysteine-reactive Compound—The–N3 group in Ro15-4513 was replaced with an–NCS group (Fig. 1) such as to become reactive with a cysteine residue. This substance, the Imid-NCS compound, was able to displace [3H]Ro15-1788 from wild-type α1β2γ2 receptors in a concentration-dependent manner. The Ki value was 10 ± 1 nm (mean ± S.D., n = 3, each point of the three displacement curves was measured in triplicate) (data not shown). Thus, the reactive molecule retained affinity for the benzodiazepine-binding site. Binding Properties of GABAA Receptors Carrying a Cysteine Point Mutation—Several amino acid residues putatively located in or near the benzodiazepine-binding pocket were individually mutated to cysteine. We focused our interest on some amino acid residues in loops A–C of the α1 subunit and loop D of the γ2 subunit. We investigated a total of 23 positions. In loops A–C of the α1 subunit, we studied His101, Gly157–Thr162, and Ile201–Met212, respectively; and in loop D of the γ2 subunit, we studied Thr73, Asp75, Ala79, and Thr81. In three of the receptors (α1G207Cβ2γ2, α1S158Cβ2γ2, and α1Y159Cβ2γ2), the mutation to cysteine drastically decreased [3H]Ro15-1788 and [3H]flunitrazepam binding affinity so that no specific binding was detected (data not shown). Fortunately, in the 20 other receptors, the mutation to cysteine did not compromise [3H]Ro15-1788 binding. All mutant receptors bound [3H]Ro15-1788 with an estimated affinity between 0.5 and 4.6 nm (data not shown). Irreversible Reaction of the Imid-NCS Compound with Mutant Receptors—The receptors were exposed to the Imid-NCS compound, and noncovalently bound reactive ligand was subsequently removed by extensive washing (see “Experimental Procedures”). In such experiments, the Imid-NCS compound is expected to first occupy its binding site reversibly. In the case of proper apposition of the–SH group of the cysteine of the mutant receptor with the–C atom of the–NCS group of the Imid-NCS compound, this is followed by the formation of a covalent bond between ligand and receptor. Fig. 2 documents the residual binding of wild-type α1β2γ2 receptors and all 20 mutant receptors after treatment with 100 nm Imid-NCS compound. 30–50% of the protein was lost during the washing steps. Therefore, each experiment included a control without reactive ligand. Residual binding in this sample was standardized to 100%, and residual binding in the other samples was expressed relative to this value. As described under “Experimental Procedures,” only receptors showing a level of residual binding lower than 70% were assumed to have reacted covalently. In receptors carrying the mutation H101C in the α1 subunit, the Imid-NCS compound led to the disappearance of ∼85% of the binding sites. α1G157Cβ2γ2, α1V202Cβ2γ2, and α1V211Cβ2γ2 receptors also reacted irreversibly with the Imid-NCS compound. In these cases, 50–74% of the binding sites disappeared. Fig. 3 (left) shows that this reaction was strongly inhibited (p < 0.001) when 30 nm Imid-NCS compound was previously reacted with 100 mm l-cysteine. Residual binding was 82 ± 9% (n = 3) with l-cysteine treatment compared with 18 ± 9% (n = 3) without treatment. In additional experiments, covalent reaction of α1H101Cβ2γ2 receptors with 10 nm Imid-NCS compound was inhibited by carrying out the reaction in the presence of 1 μm Ro15-1788 or 30 μm flunitrazepam (Fig. 3, right). Residual binding was increased from 29 ± 3% (n = 3) in the absence of both agents to 73 ± 4% (n = 3; p < 0.001) and 64 ± 6% (n = 3; p < 0.001) in the presence of Ro15-1788 and flunitrazepam, respectively. Unfortunately, the method used did not allow us to carry out similar protection experiments in α1G157Cβ2γ2, α1V202Cβ2γ2, and α1V211Cβ2γ2 receptors, as much higher concentrations of the Imid-NCS compound were required in these cases to observe covalent reaction. This would have necessitated the use of very high concentrations of Ro15-1788 or flunitrazepam, which could not have been removed efficiently to allow reversible binding experiments. Concentration Dependence of the Covalent Reaction of the Imid-NCS Compound with Wild-type α1β2γ2 and Mutant α1H101Cβ2γ2 Receptors—Fig. 4 shows the percentage of covalently reacted binding sites as a function of the concentration of the Imid-NCS compound. The apparent Kd value for mutant α1H101Cβ2γ2 receptors was 0.9 ± 0.4 nm (mean ± S.D., n = three curves, triplicates of each point). Surprisingly, wild-type α1β2γ2 receptors also reacted covalently with the compound, but with a much higher apparent Kd value of 878 ± 306 nm (mean ± S.D., n = three curves, triplicates of each point). Please note that these values due to an irreversible component do not correspond to the true Kd value of the Imid-NCS compound for the respective receptors. We hypothesized that the covalent reaction occurring with wild-type α1β2γ2 receptors could be due to an endogenous reactive amino acid residue. As there are no free cysteines in the extracellular domains of the α1 and γ2 subunits, we concentrated on Tyr and His located in the binding pocket as possible candidates and investigated mutant receptors in which the respective Tyr and His were replaced with other residues. α1Y159Fβ2γ2, α1Y161Fβ2γ2, and α1Y209Fβ2γ2 receptors bound [3H]Ro15-1788 with an estimated affinity between 0.8 and 3.6 nm, and α1H101Rβ2γ2 receptors retained an affinity for Ro15-4513 of ∼13 nm (data not shown). α1H101Rβ2γ2, α1Y159Fβ2γ2, and α1Y161Fβ2γ2 receptors showed comparable levels of covalent reaction with the Imid-NCS compound compared with wild-type α1β2γ2 receptors, whereas α1Y209Fβ2γ2 receptors reacted to a somewhat smaller extent (1 and 10 μm Imid-NCS compound for 1 h on ice) (data not shown). As all mutant receptors were still reacting covalently, the above residues can be excluded as targets of the covalent reaction in wild-type α1β2γ2 receptors. It should be pointed out here that, in the case of α1H101Cβ2γ2 receptors, a complete reaction was never reached, but only ∼85% of the receptors were observed to react covalently. After reaction of the mutant receptor with the reactive agent, the covalent adduct is slowly hydrolyzed to produce the receptor as it was prior to the reaction (31Hinman A. Chuang H. Bautista D.M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19564-19568Crossref PubMed Scopus (725) Google Scholar, 32Macpherson L.J. Dubin A.E. Evans M.J. Marr F. Schultz P.G. Cravatt B.F. Patapoutian A. Nature. 2007; 445: 541-545Crossref PubMed Scopus (907) Google Scholar). The 15% observed may correspond to an equilibrium of forward reaction and hydrolysis. The Mutation H101C in the α1 Subunit Influences Allosteric Effects of Benzodiazepine Ligands—To investigate how the mutation to cysteine influences the functional properties, wild-type α1β2γ2 and mutant α1H101Cβ2γ2 receptors were expressed in Xenopus oocytes. It has been shown previously that amino acid substitutions at position 101 of the α1 subunit can change the allosteric properties of ligands of the benzodiazepine-binding site (33Davies M. Bateson A.N. Dunn S.M.J. J. Neurochem. 1998; 70: 2188-2194Crossref PubMed Scopus (54) Google Scholar, 34Dunn S.M.J. Davies M. Muntoni A.L. Lambert J.J. Mol. Pharmacol. 1999; 56: 768-774PubMed Google Scholar). Here, we made similar observations with the substitution of α1His101 with cysteine (Fig. 5). As shown in Table 1, the antagonist Ro15-1788 had a small stimulatory effect; the partial negative allosteric modulator Ro15-4513 showed also a weak stimulating behavior. Zolpidem, which is a positive allosteric modulator, also stimulated the GABA responses, but with a 6-fold lower affinity and a 2.6-fold lower efficacy. The Imid-NCS compound potentiated the GABA-elicited current with an apparent affinity of 17 ± 1 nm and a maximal stimulation of 90 ± 7% (n = 3). Please note that the cumulative dose-response curve for the Imid-NCS compound is a mixture of noncovalent and covalent reactions. In wild-type α1β2γ2 receptors, the Imid-NCS compound showed very little stimulation, amounting maximally to 11 ± 9% (n = 5) (Fig. 5).TABLE 1Concentration-dependent modulation of the GABA EC2-5 current of wild-type and mutant GABAA receptors by zolpidem, Ro15-1788, Ro15-4513, and the Imid-NCS compound The modulatory compound was applied several times for 20 s each. The concentration of a substance at which half-maximal stimulation (EC50) was observed and the maximal stimulation (Max) a" @default.
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