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

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 α 1 Y59 to K70 was mutated to cysteine and expressed with wild-type β 2 subunits in Xenopus 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 α 1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and α 1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that α 1 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 α 1 Y59 to S68 is a β-strand.