Gamma-amino butyric acid type A receptor mutations at beta2N265 alter etomidate efficacy while preserving basal and agonist-dependent activity.

摘要

BACKGROUND: Etomidate acts at gamma-Aminobutyric acid type A (GABAA) receptors containing beta2 or beta3, but not beta1 subunits. Mutations at beta residue 265 (Ser in beta1; Asn in beta2 or beta3) profoundly affect etomidate sensitivity. Whether these mutations alter etomidate binding remains uncertain. METHODS: Heterologously expressed alpha1beta2gamma2L GABAA receptors and receptors with beta2(N265S) or beta2(N265M) mutations were studied electrophysiologically in both Xenopus oocytes and HEK293 cells. Experiments quantified the impact of beta2N265 mutations or substituting beta1 for beta2 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 co-agonist mechanism. RESULTS: Mutations produced only small changes in basal channel activity, GABA EC50, maximal GABA efficacy, and macrocurrent kinetics. Relative to wild-type, beta2(N265S) reduced etomidate enhancement of apparent GABA affinity six-fold, and it reduced etomidate direct activation efficacy 14-fold. beta2(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 co-agonist model and that beta2(N265S) reduced etomidate allosteric efficacy five-fold, whereas etomidate-binding affinity dropped threefold. Experiments swapping beta2 subunits for beta1 indicated that etomidate efficacy is reduced 34-fold, whereas binding affinity drops less than two-fold. CONCLUSIONS: Mutations at beta2N265 profoundly alter etomidate sensitivity with only small changes in basal and GABA-dependent channel activity. Mutations at the beta2N265 residue or replacement of beta2 with beta1 influence etomidate efficacy much more than binding to inactive receptors.