Different residues in the GABAA receptor α1T60-α1K70 region mediate GABA and SR-95531 actions

Abstract

Although γ-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 α1T60 to α1K70 was mutated to cysteine and expressed with wild-type β2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with α1T60C, α1D62C, α1F64C, α1R66C, α1S68C, and α1K70C. α-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of α1F64C, α1R66C, and α1S68C, whereas SR-95531 slowed modification of α1D62C, α1F64C, and α1R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for α1F64C and α1R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized α1F64C and α1R66C 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 α1D62C and α1S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.