Etomidate Binding Interactions in GABA A Receptor Defined with Cysteine Substitutions

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 α (Met236) and β (Met286) subunits. Consistent with receptor structural homology models based on Torpedo nicotinic acetylcholine receptor, the labeling results suggests the presence of two interfacial anesthetic binding sites per GABAA receptor located at the transmembrane β/α subunit interface. However, azietomidate may fail to label other important binding determinants. In particular, β subunit reside 265 is predicted to be near the etomidate site, and β2/β3Asn265 mutations profoundly affect etomidate sensitivity. To determine if specific residues are within the etomidate site, we tested whether etomidate protects Cysteines substituted at these residues from sulfhydryl modifying reagents. In human α1β2γ2L background, GABAA receptors with the α1M236C and α1L232C mutations displayed etomidate sensitivity similar to wild-type in electrophysiological experiments, while receptors with β2M286C and β2N265C mutations largely eliminated etomidate effects. All of these Cysteine substituted receptors were modified by p-chloromercuribenzenesulfonate (pCMBS), which altered electrophysiological properties. Using allosteric GABA/etomidate co-agonist gating models for each mutant, we identified protection conditions where the mix of receptor states was similar to control modification conditions and etomidate site occupancy was expected to be high. In the presence of etomidate, the apparent rate of pCMBS modification at both α1M236C and β2M286C was reduced more than ten-fold and was reduced two-fold at α1L232C. In contrast, pCMBS modification of β2N265C was unaffected by high concentrations of etomidate. These results suggest that etomidate sterically interacts strongly with α1Met236 and β2Met286 and weakly with α1Leu232, but does not interact with β2Asn265.