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 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 [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) 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.
Ishizawa
Y
Y
Departments of Anesthesia, Physiology, and Biochemistry & Biophysics, University of Pennsylvania Medical Center, Philadelphia 19104-4283, USA. ishizawa@mail.med.upenn.edu
Sharp
R
R
Liebman
P A
PA
Eckenhoff
R G
RG
D000345:Affinity Labels; D018685:Anesthetics, Inhalation / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006221:Halothane / Q000378:metabolism* / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D000345:Affinity Labels; D018685:Anesthetics, Inhalation; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins; D006221:Halothane