ABSTRACT
Although gamma-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from alpha(1)T60 to alpha(1)K70 was mutated to cysteine and expressed with wild-type beta(2) subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with alpha(1)T60C, alpha(1)D62C, alpha(1)F64C, alpha(1)R66C, alpha(1)S68C, and alpha(1)K70C. gamma-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of alpha(1)F64C, alpha(1)R66C, and alpha(1)S68C, whereas SR-95531 slowed modification of alpha(1)D62C, alpha(1)F64C, and alpha(1)R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for alpha(1)F64C and alpha(1)R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized alpha(1)F64C and alpha(1)R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of alpha(1)D62C and alpha(1)S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.
