Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on α1 or β2 Subunits Enhance GABAA Receptor Gating and Reduce Etomidate Modulation

The potent general anesthetic etomidate produces its effects by enhancing GABA A receptor activation. Its photolabel analog [ 3 H]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 α 1 β 2 γ 2L receptors with α 1 M236W or β 2 M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC 50 , etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both α 1 M236W and β 2 M286W mutations induced spontaneous channel opening, lowered GABA EC 50 , increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on α 1 β 2 γ 2L channels. These changes were larger with α 1 M236W than with β 2 M286W. Etomidate (3.2 μM) reduced GABA EC 50 much less in α 1 M236Wβ 2 γ 2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating α 1 M236Wβ 2 γ 2L compared with wild type. In α 1 β 2 M286Wγ 2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that α 1 Met236 and β 2 Met286 are within etomidate sites that allosterically link to channel gating. Although α 1 M236W produced the larger impact on channel gating, β 2 M286W 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.