Abstract

The potent general anesthetic etomidate produces its effects by enhancing GABA_A receptor activation. Its photolabel analog [³H]azi-etomidate labels residues within transmembrane domains on α and β subunits: αMet236 and βMet286. We hypothesized that these methionines contribute to etomidate sites formed at α-β subunit interfaces and that increasing side-chain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in α₁β₂γ_2L receptors with α₁M236W or β₂M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC₅₀, etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both α₁M236W and β₂M286W mutations induced spontaneous channel opening, lowered GABA EC₅₀, increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on α₁β₂γ_2L channels. These changes were larger with α₁M236W than with β₂M286W. Etomidate (3.2 μM) reduced GABA EC₅₀ much less in α₁M236Wβ₂γ_2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating α₁M236Wβ₂γ_2L compared with wild type. In α₁β₂M286Wγ_2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that α₁Met236 and β₂Met286 are within etomidate sites that allosterically link to channel gating. Although α₁M236W produced the larger impact on channel gating, β₂M286W produced more profound changes in etomidate sensitivity, suggesting a dominant role in drug binding. Furthermore, quantitative mechanistic analysis demonstrated that wild-type and mutant results are consistent with the presence of only one class of etomidate sites mediating both agonist potentiation and direct activation.