Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on α1 or β2 Subunits Enhance GABAA Receptor Gating and Reduce Etomidate Modulation
- 19 September 2008
- journal article
- Published by American Society for Pharmacology & Experimental Therapeutics (ASPET) in Molecular Pharmacology
- Vol. 74 (6), 1687-1695
- https://doi.org/10.1124/mol.108.050500
Abstract
The potent general anesthetic etomidate produces its effects by enhancing GABAA 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 α1β2γ2L 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 α1β2γ2L channels. These changes were larger with α1M236W than with β2M286W. Etomidate (3.2 μM) reduced GABA EC50 much less in α1M236Wβ2γ2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating α1M236Wβ2γ2L compared with wild type. In α1β2M286Wγ2L 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.Keywords
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