%0 Journal Article %A Amy M. Kucken %A David A. Wagner %A Peter R. Ward %A Jeremy A. Teissére %A Andrew J. Boileau %A Cynthia Czajkowski %T Identification of Benzodiazepine Binding Site Residues in the γ2 Subunit of the γ-Aminobutyric AcidA Receptor %D 2000 %J Molecular Pharmacology %P 932-939 %V 57 %N 5 %X γ-Aminobutyric acidA receptor γ-subunits are important for benzodiazepine (BZD) binding and modulation of the γ-aminobutyric acid-mediated Cl− current. Previously, by using γ2/α1 chimeric subunits, we identified two domains of the γ2-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 γ2/α1 chimeric subunits and γ2 point mutants to identify specific residues within the γ2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant γ2 and γ2/α1 chimeric subunits were expressed with wild-type α1 and β2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the γ2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, γ2 Met-57 and γ2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, γ2 Ala-79, 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 γ2 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. The American Society for Pharmacology and Experimental Therapeutics %U https://molpharm.aspetjournals.org/content/molpharm/57/5/932.full.pdf