Abstract

The structural basis by which agonists, antagonists, and allosteric modulators exert their distinct actions on ligand-gated ion channels is poorly understood. We used the substituted cysteine accessibility method to probe the structure of the GABA_A receptor in the presence of ligands that elicit different pharmacological effects. Residues in the α₁ Met¹¹³-Leu¹³² region of the GABA binding site were individually mutated to cysteine and expressed with wild-type β₂ and γ₂ subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methaneth-iosulfonate (MTSEA-biotin) with the introduced cysteines in the resting (unliganded) state and compared them with rates determined in the presence of GABA (agonist), 4-[6-imino-3-(4-methoxyphenyl)pyridazin-1-yl]butanoic acid hydrobromide (SR-95531; antagonist), pentobarbital (allosteric modulator), and flurazepam (allosteric modulator). α₁N115C, α₁L117C, α₁T129C, and α₁R131C are predicted to line the GABA binding pocket because MTSEA-biotin modification of these residues decreased the amount of current elicited by GABA, and the rates/extents of modification were decreased both by GABA and SR-95531. Reaction rates of some substituted cysteines were different depending on the ligand, indicating that barbiturate- and GABA-induced channel gating, antagonist binding, and benzodiazepine modulation induce specific structural rearrangements. Chemical reactivity of α₁E122C was decreased by either GABA or pentobarbital but was unaltered by SR-95531 binding, whereas α₁L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital. Furthermore, α₁E122C, α₁L127C, and α₁R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation.