Different Residues in the GABAA Receptor

Although -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 1T60 to 1K70 was mutated to cysteine and expressed with wild-type 2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with 1T60C, 1D62C, 1F64C, 1R66C, 1S68C, and 1K70C. -Aminobutyric acid (GABA) slowed methanethiosulfonate modification of 1F64C, 1R66C, and 1S68C, whereas SR-95531 slowed modification of 1D62C, 1F64C, and 1R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for 1F64C and 1R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized 1F64C and 1R66C 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 1D62C and 1S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.