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 ␣ and ␤ subunits: ␣Met236 and ␤Met286. We hypothesized that these methionines contribute to etomidate sites formed at ␣-␤ subunit interfaces and that increasing sidechain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in ␣ 1 ␤ 2 ␥ 2L receptors with ␣ 1 M236W or ␤ 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 ␣ 1 M236W and ␤ 2 M286W mutations induced spontaneous channel opening, lowered GABA EC 50 , increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on ␣ 1 ␤ 2 ␥ 2L channels. These changes were larger with ␣ 1 M236W than with ␤ 2 M286W. Etomidate (3.2 M) reduced GABA EC 50 much less in ␣ 1 M236W␤ 2 ␥ 2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating ␣ 1 M236W␤ 2 ␥ 2L compared with wild type. In ␣ 1 ␤ 2 M286W␥ 2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that ␣ 1 Met236 and ␤ 2 Met286 are within etomidate sites that allosterically link to channel gating. Although ␣ 1 M236W produced the larger impact on channel gating, ␤ 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.

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

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