title: Identification of benzodiazepine binding site residues in the gamma2 subunit of the gamma-aminobutyric acid(A) receptor.
abstract: gamma-Aminobutyric acid(A) receptor gamma-subunits are important for benzodiazepine (BZD) binding and modulation of the gamma-aminobutyric acid-mediated Cl(-) current. Previously, by using gamma2/alpha1 chimeric subunits, we identified two domains of the gamma2-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 gamma2/alpha1 chimeric subunits and gamma2 point mutants to identify specific residues within the gamma2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant gamma2 and gamma2/alpha1 chimeric subunits were expressed with wild-type alpha1 and beta2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the gamma2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, gamma2 Met-57 and gamma2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, gamma2 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 gamma2 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.
authors.affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Kucken
authors.forename: D A
authors.initials: DA
authors.lastname: Wagner
authors.forename: P R
authors.initials: PR
authors.lastname: Ward
authors.forename: J A
authors.initials: JA
authors.lastname: Teissére
authors.forename: A J
authors.initials: AJ
authors.lastname: Boileau
authors.forename: C
authors.initials: C
authors.lastname: Czajkowski
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001569:Benzodiazepines / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005442:Flumazenil / Q000494:pharmacology; D005445:Flunitrazepam; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011963:Receptors, GABA-A / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001386:Azides; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D001569:Benzodiazepines; D005442:Flumazenil; D005445:Flunitrazepam; C042957:Ro 15-4513