• Deirdre S Stewart, Mayo Hotta, Rooma Desai, and Stuart A Forman.
• Department of Anesthesia Critical Care & Pain Medicine, Beecher-Mallinckrodt Research Laboratories, and Department of Anesthesia Critical Care & Pain Medicine, Massachusetts General Hospital, Boston, MA, USA.
• Mol. Pharmacol. 2013 Jun 1;83(6):1200-8.

AbstractA central axiom of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. However, measuring agonist affinity in inactive receptors is confounded by concomitant activation. We identified a cysteine substituted mutant γ-aminobutyric acid type A (GABAA) receptor with unique characteristics allowing the determination of allosteric agonist site occupancy in both inactive and active receptors. Etomidate, the allosteric agonist, is an anesthetic that activates or modulates α1β2γ2L GABAA receptors via transmembrane sites near β2M286 residues in M3 domains. Voltage-clamp electrophysiology studies of α1β2M286Cγ2L receptors show that GABA is an efficacious agonist and that etomidate modulates GABA-activated activity, but direct etomidate agonism is absent. Quantitative analysis of mutant activity using an established Monod-Wyman-Changeux (MWC) allosteric model indicates that the intrinsic efficacy of etomidate, defined as its relative affinity for active versus inactive receptors, is lower than in wild-type receptors. Para-chloromercuribenzene sulfonate covalently modifies β2M286C side-chain sulfhydryls, irreversibly altering GABA-induced currents. Etomidate concentration dependently reduces the apparent rate of β2M286C-pCMBS bond formation, tracked electrophysiologically. High etomidate concentrations completely protect the β2M286C suflhydryl from covalent modification, suggesting close steric interactions. The 50% protective etomidate concentration (PC50) is 14 μM in inactive receptors and 1.1 to 2.2 μM during GABA-activation, experimentally demonstrating that activated receptors bind etomidate more avidly than do inactive receptors. The experimental PC50 values are remarkably close to, and therefore validate, MWC model predictions for etomidate dissociation constants in both inactive and active receptors. Our results support MWC models as valid frameworks for understanding the agonism, coagonism, and modulation of ligand-gated ion channels.

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