TY - JOUR T1 - Selected Cysteine Residues in Transmembrane Domains of μ-Opioid Receptor Are Critical for Effects of Sulfhydryl Reagents JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 113 LP - 120 VL - 293 IS - 1 AU - Hong Bing Deng AU - Wei Guang AU - Jia Bei Wang Y1 - 2000/04/01 UR - http://jpet.aspetjournals.org/content/293/1/113.abstract N2 - The effects of sulfhydryl-specific methanethiosulfonate (MTS) derivatives on μ-opioid receptor binding were examined in Chinese hamster ovary (CHO) cells that stably express μ-opioid receptors (HμCHO). Three charged MTS derivatives inhibited the binding of [3H][d-Ala2,N-MePhe4,Gly-ol5]-enkephalin to μ-opioid receptors with IC50 values ranging from 0.12 to 13 mM. Further characterization of the μ-opioid receptor interactions with ethylammonium MTS (the most potent among tested MTS reagents) revealed that ethylammonium MTS inhibition of ligand binding to the receptor was irreversible, with both the maximal receptor binding (Bmax) and the binding affinity (Kd) being changed. Preincubation of HμCHO cells with [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin or naloxone prevented the receptor inactivation normally caused by MTS derivatives, indicating that the reactions may occur within or near the ligand-binding pocket on the receptor. To identify the susceptible sulfhydryl groups, each of the cysteine residues in the μ-receptor transmembrane domains were substituted with serine by site-directed mutagenesis. All of the mutant receptors transiently expressed in COS cells had receptor binding properties similar to the wild-type receptors. However, four mutant receptors with a serine substitution in transmembrane domain III (C161S), IV (C192S), V (C237S), or VII (C332S) displayed significant resistance against MTS inhibition compared with the wild-type receptor. We conclude that these four cysteine residues react with MTS reagents and are responsible for the effect of the MTS reagents on μ-opioid receptor binding. The American Society for Pharmacology and Experimental Therapeutics ER -