Halothane Binding to a G Protein Coupled Receptor in Retinal Membranes by Photoaffinity Labeling

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Abstract
General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [14C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [14C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC50 of 0.9 mM and a Hill coefficient of −0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC50 values of isoflurane and 1-chloro-1,2,2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1,2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.