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 GABAA receptor in the presence of ligands that elicit different pharmacological effects. Residues in the 1Met113-Leu132 region of the GABA binding site were individually mutated to cysteine and expressed with wild-type 2 and 2 subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methanethiosulfonate (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). 1N115C, 1L117C, 1T129C, and 1R131C 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 1E122C was decreased by either GABA or pentobarbital but was unaltered by SR- 95531 binding, whereas 1L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital. Furthermore, 1E122C, 1L127C, and 1R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation.
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