title: GABA(A) receptor beta 2 Tyr97 and Leu99 line the GABA-binding site. Insights into mechanisms of agonist and antagonist actions.
abstract: The identification of residues that line neurotransmitter-binding sites and catalyze allosteric transitions that result in channel gating is crucial for understanding ligand-gated ion channel function. In this study, we used the substituted cysteine accessibility method and two-electrode voltage clamp to identify novel gamma-aminobutyric acid (GABA)-binding site residues and to elucidate the secondary structure of the Trp(92)-Asp(101) region of the beta(2) subunit. Each residue was mutated individually to cysteine and expressed with wild-type alpha(1) subunits in Xenopus oocytes. GABA-gated currents (I(GABA)) were measured before and after exposure to the sulfhydryl reagent, N-biotinylaminoethyl methanethiosulfonate (MTS). V93C, D95C, Y97C, and L99C are accessible to derivatization. This pattern of accessibility is consistent with beta(2)Val(93)-Leu(99) adopting a beta-strand conformation. Both GABA and SR95531 protect Y97C and L99C from modification, indicating that these two residues line the GABA-binding site. In D95C-containing receptors, application of MTS in the presence of SR95531 causes a greater effect on I(GABA) than MTS alone, suggesting that binding of a competitive antagonist can cause movements in the binding site. In addition, we present evidence that beta(2)L99C homomers form spontaneously open channels. Thus, mutation of a binding site residue can alter channel gating, which implies that Leu(99) may be important for coupling agonist binding to channel gating.
authors.affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Boileau
authors.forename: J Glen
authors.initials: JG
authors.lastname: Newell
authors.forename: Cynthia
authors.initials: C
authors.lastname: Czajkowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D018756:GABA Antagonists / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011724:Pyridazines / Q000494:pharmacology; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; D018076:DNA, Complementary; D018756:GABA Antagonists; D011724:Pyridazines; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; C049853:gabazine; D003545:Cysteine