γ-amino butyric acid type a receptor mutations at β2n265 alter etomidate efficacy while preserving basal and agonist-dependent activity

Abstract

BACKGROUND: Etomidate acts at γ-Aminobutyric acid type A (GABAA) receptors containing β2 or β3, but not β1 subunits. Mutations at β residue 265 (Ser in β1; Asn in β2 or β3) profoundly affect etomidate sensitivity. Whether these mutations alter etomidate binding remains uncertain. METHODS: Heterologously expressed α1β2γ2L GABAA receptors and receptors with β2(N265S) or β2(N265M) mutations were studied electrophysiologically in both Xenopus oocytes and HEK293 cells. Experiments quantified the impact of β2N265 mutations or substituting β1 for β2 on basal channel activation, GABA EC50, maximal GABA efficacy, etomidate-induced leftward shift in GABA responses, etomidate direct activation, and rapid macrocurrent kinetics. Results were analyzed in the context of an established allosteric coagonist mechanism. RESULTS: Mutations produced only small changes in basal channel activity, GABA EC50, maximal GABA efficacy, and macrocurrent kinetics. Relative to wild-type, β2(N265S) reduced etomidate enhancement of apparent GABA affinity six-fold, and it reduced etomidate direct activation efficacy 14-fold. β2(N265M) totally eliminated both etomidate modulation of GABA responses and direct channel activation. Mechanism-based analysis showed that the function of both mutants remains consistent with the allosteric coagonist model and that β2(N265S) reduced etomidate allosteric efficacy five-fold, whereas etomidate-binding affinity dropped threefold. Experiments swapping β2 subunits for β1 indicated that etomidate efficacy is reduced 34-fold, whereas binding affinity drops less than two-fold. CONCLUSIONS: Mutations at β2N265 profoundly alter etomidate sensitivity with only small changes in basal and GABA-dependent channel activity. Mutations at the β2N265 residue or replacement of β2 with β1 influence etomidate efficacy much more than binding to inactive receptors. © 2009, the American Society of Anesthesiologists, Inc.