Mapping the Agonist Binding Site of the GABAAReceptor: Evidence for a β-Strand

Abstract

GABA_A receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from α₁ Y59 to K70 was mutated to cysteine and expressed with wild-type β₂ subunits inXenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions α₁ T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and α₁ F64C, R66C, and S68C were protected from reaction by agonist. We conclude that α₁ F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from α₁ Y59 to S68 is a β-strand.