Identification of Benzodiazepine Binding Site Residues in the g2 Subunit of the g-Aminobutyric AcidA Receptor

g-Aminobutyric acidA receptor g-subunits are important for benzodiazepine (BZD) binding and modulation of the g-aminobutyric acid-mediated Cl current. Previously, by using g2/a1 chimeric subunits, we identified two domains of the g2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional g2/a1 chimeric subunits and g2 point mutants to identify specific residues within the g2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant g2 and g2/a1 chimeric subunits were expressed with wild-type a1 and b2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the g2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, g2 Met-57 and g2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, g2 Ala79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of g2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds. g-Aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the mammalian brain. The native receptor is likely to be a heteropentameric protein (Nayeem et al., 1994), assembled from multiple subunit subtypes: 6 a, 4 b, 3 g, 1 d, 1 e, 3 r, and 1 p (Barnard et al., 1998). The receptor contains an integral chloride-selective channel with specific binding sites for GABA and a variety of neuroactive drugs, including benzodiazepines (BZDs), barbiturates, neurosteroids, and anesthetics (Sieghart, 1995; Smith and Olsen, 1995). BZDs, clinically used for their anxiolytic, muscle relaxant, sedative, and antiepileptic actions, exert their therapeutic effects by allosterically modulating the activation of the GABAA receptor. Because of their clinical usefulness, a substantial effort has been made to understand the structural determinants of BZD binding in this receptor. A variety of structurally diverse ligands bind with high affinity to the BZD binding site. These compounds include classic benzodiazepines, triazolopyridazines, imidazopyridines, cyclopyrrolones, pyrazoloquinolinones, and b-carbolines (Barnard et al., 1998). Depending on the ligand and the subunit composition of the GABAA receptor, the modulatory actions of these compounds range from full agonist (positive modulator) to inverse agonist (negative modulator). BZD positive modulators decrease the GABA concentration needed to elicit half-maximal channel activity (EC50), whereas BZD negative modulators increase the GABA EC50 value. BZD antagonists block the effect of both positive and negative modulators. Although all BZD binding site ligands appear to compete for a common binding site (McKernan et al., 1998), it is likely that different microdomains within the site interact with different subsets of BZD ligands (Davies et al., 1996). The BZD binding site of the GABAA receptor has been proposed to lie at the interface between the aand g-subunits, with residues from each subunit contributing to the binding site (Smith and Olsen, 1995; Sigel and Buhr, 1997). In the a1 subunit, photoaffinity-labeling (Duncalfe et al., 1996) and mutagenesis (Wieland et al., 1992; Davies et al., 1998) experiments have identified histidine at position 101 (a1 H101) as forming part of the BZD binding site. Other a1 residues implicated in BZD binding include Tyr-159, Thr162, Gly-200, Thr-206, Tyr-209, and Val-211 (Pritchett and Seeburg, 1991; Wieland and Luddens, 1994; Amin et al., 1997; Buhr et al., 1997b). In the g2 subunit, only two residues This work was supported in part by grants to C.C. from the Alcoholic Beverage Association and NINDS-the National Institutes of Health. C.C. is a recipient of the Burroughs Wellcome Fund New Investigator Award in the Basic Pharmacological Sciences. ABBREVIATIONS: GABA, g-aminobutyric acid; PCR, polymerase chain reaction; BZD, benzodiazepine. 0026-895X/00/050932-08$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:932–939, 2000 /13027/816882 932 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from have been identified as key determinants for BZD binding: g2 Phe-77 (Buhr et al., 1997a; Wingrove et al., 1997) and g2 Met-130 (Buhr and Sigel, 1997; Wingrove et al., 1997). Previously, by using g2/a1 chimeric subunits, we identified two domains of the g2 subunit, Lys-41-Trp-82 and Arg-114Asp-161, that together are necessary for high-affinity BZD binding (Boileau et al., 1998). In this study, by using g/a chimeric subunits and g2 point mutations, we focused on identifying residues within the g2 Lys-41-Trp-82 region that contribute to BZD binding. We identify three novel residues in the g2 subunit, g2 Met-57, Tyr-58, and Ala-79, that are important determinants for high-affinity BZD binding. Materials and Methods Chimera Nomenclature. All g2/a1chimeric constructs in this study contain g2 amino acids from Arg-114 to Asp-161 because this region was found to be necessary for BZD binding (Boileau et al., 1998). For ease of reading, the chimeric constructs (x) are named for the g2 residue before the junction of the first g2/a1 crossover in the mature rat protein sequence (Fig. 1). For example, x40 contains g2 sequence from Gln-1 to Asn-40 and from Arg-114 to Asp-161, whereas x82 contains g2 sequence from Gln-1 to Trp-82 and from Arg-114 to Asp-161. Mutations produced in chimeric backgrounds were named for the a1 residue mutated, the aligned position in the mature g2 subunit, and the g2 residue introduced. For instance, x56 V76I denotes that the a1 residue (Val) in the x56 subunit was mutated to the aligned residue (Ile) at position 76 of the mature g2 sequence. Mutations produced in the g2 subunit were named for the targeted g2 residue, the position in the mature g2 subunit, and the mutant amino acid. For example, g2 A79C denotes that the Ala at position 79 in g2 was mutated to Cys. Molecular Cloning. Rat cDNA clones for the a1, b2, and g2 GABAA receptor subunits were used for all molecular cloning. x40 and x82 were produced as previously described (Boileau et al., 1998). Point mutations were made in x40 using either the MORPH SiteSpecific Plasmid DNA Mutagenesis kit (5 Prime-3 Prime, Boulder, CO) or a modified form of recombinant polymerase chain reaction (PCR). In this method, a forward mutagenic oligonucleotide was paired with a reverse template-specific oligonucleotide and amplified by PCR using an appropriate template. The resulting product was purified to remove excess oligonucleotides using the High Pure PCR Product Purification kit (Boehringer-Mannheim Biochemicals, Indianapolis, IN). Using the same template, the purified primary product, now acting as a reverse primer, was paired with an upstream vector-specific oligonucleotide and amplified. After the secondary amplification, the final product was purified as earlier and subcloned into the appropriate background. x56 and x65 were produced by recombinant PCR. Then, a1-to-g2 point mutations between x65 and x82 (S67N/D68A/H69I/D70N, V76I, R79A, and S81T) were made in x65 using the modified recombinant PCR method. Single, double, and triple a1-to-g2 mutations between x56 and x65 (I57M, F58Y, T60N, F62I, I57M/F58Y, T60N/ F62I, I57M/F58Y/T60N, and F58Y/T60N/F62I) were made in x56 using the modified method. Point mutations in the g2 subunit (A79C, A79R, A79Q, A79Y, T81A, T81C, and T81S) were mutated using recombinant PCR with myc epitope-tagged g2 as template. All point mutations and chimeras were subcloned into pCEP4 (InVitrogen, Carlsbad, CA) for transient expression in HEK 293 cells (ATCC CRL 1573) and were verified by restriction enzyme analysis and double-stranded DNA sequencing. Transient Expression in HEK 293 Cells. Cells were grown in 100-mm tissue culture dishes in minimum essential medium with Earle’s salts (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) in a 37°C incubator under a 5% CO2 atmosphere. Cells were cotransfected at 50 to 60% confluency with a1-pCEP4, b2-pCEP4, and either g2pCEP4 or x-pCEP4 using a standard CaHPO4 precipitation method (Graham and van der Eb, 1973). The vector pAdVAntage (Promega, Madison, WI) was added to enhance expression levels (4 mg of each subunit and 12 mg of pAdVAntage/100-mm plate). Cells were harvested and membrane homogenates were prepared as previously described (Boileau et al., 1998). Binding Assays. Competition binding experiments with various BZD-site ligands were performed as previously described (Boileau et al., 1998). In brief, membrane homogenates (100 mg) were incubated at room temperature with [H]flunitrazepam (85 Ci/mmol; DuPontNew England Nuclear, Boston, MA) or [H]Ro15-4513 (21.7 Ci/mmol; DuPont-New England Nuclear) at a concentration slightly lower than KI and 7 to 10 concentrations of unlabeled competing ligand in a final volume of 250 ml. The unlabeled BZDs, flunitrazepam, Ro151788, and Ro15-4513 were generously supplied by Dr. Sepinwall (Hoffman-La Roche, Nutley, NJ). Data were fit by using the equation y 5 Bmax/[1 1 (x/IC50)], where y is the specifically bound dpm, Bmax is the maximal binding, and x is the concentration of displacing drug (Prism; GraphPad Software, San Diego, CA). KI was calculated according to the Cheng-Prusoff/Chou equation (Cheng and Prusoff, 1973; Chou, 1974). Immunofluorescence. x40, x56, x82, and g2 were tagged between the third and fourth residues of the mature subunit with the myc 9E10 epitope (EQKLISEEDL) using recombinant PCR and subcloned back into each respective template. The myc epitope tag had no effect on the function or expression of the subunits. Cells were grown and transfected in 12-well dishes on poly(D)-lysine (SigmaAldrich)-coated 12-mm glass coverslips. Forty-eight hours after the transfection, cells were washed and fixed in 2% paraformaldehyde. Nonspecific immunoreactivity was reduced by blocking cells with 2% BSA in PBS containing: 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 137 mM NaCl, and 14 mM Na2HPO4, pH 7.1. Antibodies were diluted in the corresponding blocking buffer. In some cases, the cells were permeabilized using PBS plus 0.1% Triton X-100 before the antibody addition. The primary antibody was an anti-myc 9E10 antibody, generously supplied by Dr. Johannes Hell (University of Wisconsin-Madison), diluted at 1:500; the secondary antibody, biotin-SP goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA), was diluted to 4.4 mg/ml. The final incubation was in Fig. 1. Constructed chimeric subunits and mutations used in the identification of g2 residues important for BZD binding. g2/a1 chimeras (x) are named for the g2 residue before the junction of the first g2/a1 crossover. All xs contain additional amino acid residues from g2 Arg-114 to Asp-161. Therefore, x40 contains the g2 sequence from Gln-1 to Asn-40 and from Arg-114 to Asp-161 (see Materials and Methods). The g2 sequence is shown in gray, the a1 sequence is shown in white, and the transmembrane segments M1 through M4 are shown in black. a1b2x82 receptors specifically bind BZDs, whereas a1b2x40 receptors do not (Boileau et al., 1998). a1 and g2 sequence homology in the region from g2 Lys-41 to Trp-82 is shown in expanded form. Identical amino acid residues are shown in light gray. Residues indicated with an asterisk were targeted for mutation. Vertical lines indicate crossover transitions for x56 and x65. Residues that were found to influence BZD binding are boxed. BZD Binding Site Residues of GABAA Receptors 933 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from Texas Red-conjugated streptavidin (Jackson ImmunoResearch), diluted to 4.2 mg/ml. After several washes, the coverslips were mounted onto slides and visualized. Fluorescent images of cells were acquired with a Zeiss 35 M inverted microscope (Carl Zeiss, Thornwood, NY), 633/1.4 NA Plan-Apochromatic objective, Texas Red filter set (Chroma Technology, Brattleboro, VT) and a Princeton Instruments MicroMax cooled CCD digital camera (Princeton Instruments, Trenton, NJ). All images were acquired at full chip resolution (Kodak KAF-1400 chip, 1035 3 1317 pixels, 6.8-mm pixel size) within the dynamic range of the camera (12-bit, 4096 gray levels) using Metamorph 4.1 imaging software (Universal Imaging, West Chester, PA). Images were scaled appropriately, converted to 8-bit images, and imported into Adobe Photoshop (ADOBE Systems, Mountview, CA). Statistical Analysis. We compared the effects of the mutations with the use of one-way ANOVA, applying Dunnett’s post-test for significance of differences (Prism; GraphPad Software).