Vol. 293, Issue 1, 113-120, April 2000

Selected Cysteine Residues in Transmembrane Domains of µ-Opioid Receptor Are Critical for Effects of Sulfhydryl Reagents

Hong Bing Deng, Wei Guang and Jia Bei Wang

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland

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 (B_max) and the binding affinity (K_d) 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.