MTS-Etomidate Selectively Reacts Within the GABAA Receptor Etomidate Binding Site

Abstract

The general anesthetic etomidate exerts its major clinical actions through potentiation of GABAA receptor activation. GABAA receptors are pentameric, usually consisting of combinations of alpha, beta, and gamma subunits. A photoreactive structural analog of etomdiate ([3H]azietomidate) labels amino acids on transmembrane domains in both alpha (M236) and beta (M286) subunits. This suggests the presence of two interfacial anesthetic binding sites per GABAA receptor, consistent with receptor structural homology models based on Torpedo nicotinic acetylcholine receptor. To further characterize the etomidate binding site, we have created a number of Cys substitutions within the α/β intersubunit region and have synthesized a novel etomidate derivative, 2-(methylsulfonyl) thio-etomidate (MTS-etomidate), designed to covalently modify cysteines. Human GABAA receptors (α1, β2, γ2L) were expressed in Xenopus oocytes and current responses were measured using two-electrode voltage clamp. Using pCMBS and MTSEA, we found that cysteine substitutions at both α1M236 and β2M286, were accessible to modification. Cysteine modification was also evident with pCMBS at α1L232C, one helical turn above α1M236 in TM1. In contrast, modification by MTS-etomidate was evident only at α1M236C, but not at β2M286C or, α1L232C. These results suggest that MTS-etomidate orients itself in a precise conformation within its binding pocket and acts as a highly selective structural probe, yielding information not only about the residues with which it interacts but also about the orientation of etomidate within the biding pocket consistent with its mechanism of action.