Received for publication July 14, 2008.
Revised September 18, 2008.
Accepted for publication September 18, 2008.

Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on ₁ or ₂ Subunits Enhance GABA_A Receptor Gating and Reduce Etomidate Modulation

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 and subunits: M236 and M286. We hypothesized that these methionines contribute to etomidate sites formed at - 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 _122L receptors with ₁M236W or ₂M286W mutations, both in the absence and presence of etomidate. Measurements included spontaneous activation, GABA EC₅₀, etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both ₁M236W and ₂M286W mutations induced spontaneous channel opening, lowered GABA EC₅₀, increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on ₁22L channels. These changes were larger with ₁M236W than with ₂M286W. Etomidate (3.2 µM) reduced GABA EC₅₀ much less in ₁M236W_22L receptors (2-fold) than in wild-type (23-fold). However, etomidate was more potent and efficacious in directly activating ₁₂M286W_2L compared to wild-type. In ₁₂M286W_2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that ₁M236 and ₂M286 are within etomidate sites that allosterically link to channel gating. While ₁M236W produced the larger impact on channel gating, ₂M286W 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.

Key words: GABAA, GABAC, Ion channel regulation, Thermodynamic and kinetic processes and modeling, Mutagenesis/Chimeric approaches, Barbiturates, Gases/general anesthetics

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