Different Residues in the GABA_A Receptor α₁T60-α₁K70 Region Mediate GABA and SR-95531 Actions*

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. γ-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.