Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effec ts might be mediated by direct binding interactions with the GPCR or its as sociated G protein, we studied the binding character of halothane on mammal ian rhodopsin, structurally the best understood GPCR, by using direct photo affinity labeling with [C-14]halothane. In the bleached bovine rod disk mem branes (RDM), opsin and membrane lipids were dominantly photolabeled with [ C-14]halothane, but none of the three G protein subunits were labeled. In o psin itself, halothane labeling was inhibited by unlabeled halothane with a n 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-chlo ro-1,2,2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1,2-dichlorohe xafluorocyclobutane. inhibited halothane labeling by 50% at 0.05 mM. The pr esent results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G prot ein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor pr oteins.