State-Dependent Etomidate Binding in GABAA Receptors Probed with Cysteine Substitution and Protection from Modification

Abstract

Background: A central tenet of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. This is a difficult concept to test experimentally, because in most agonist-receptor systems, the act of assessing agonist binding affinity results in receptor activation. In α1β2γ2L GABAA receptors, the anesthetic etomidate is an allosteric agonist at high concentrations, and potentiates GABA activation at low concentrations. Sites mediating both actions were photolabeled with azi-etomidate, identifying the β2M286 sidechain as a contact point. We used a cysteine substitution and sulfhydryl modification to study etomidate interactions with this sidechain. Methods: using two-microelectrode voltage clamp electrophysiology in Xenopus oocytes, we characterized etomidate agonism and potentiation of GABA activation in α1β2M286Cγ2L GABAA receptors. We studied covalent modification of the β2M286C sidechain by a sulfhydryl-selective reagent, para-chloromercuribenzenesulfonate (pCMBS) with and without GABA. Etomidate-dependent protection of the sulfhydryl was also assessed. Results: Oocyte-expressed α1β2M286Cγ2L receptors displayed reduced sensitivity to GABA, and no agonism by etomidate. However, etomidate still enhanced GABA-activated currents from α1β2M286Cγ2L receptors. Exposure of α1β2M286Cγ2L receptors to pCMBS irreversibly increased activation by low (EC10) GABA. The apparent rate of pCMBS modification increased with addition of GABA. Etomidate in a concentration-dependent manner reduced the rate of β2M286C modification by pCMBS. Etomidate protection at β2M286C was enhanced in the presence of GABA. Conclusions: Etomidate, like azi-etomidate, binds next to the β2M286 residue. Based on its apparent protectant potency, the affinity of etomidate for GABA-bound receptors is greater than that for resting receptors, as expected. Our results have important implications for both designing and interpreting experiments that use mutations to map allosteric modulator/agonist sites.Support: NIH (R01GM89745 and P01GM58448)