Selected cysteine residues in transmembrane domains of mu-opioid receptor are critical for effects of sulfhydryl reagents

The effects of sulfhydryl-specific methanethiosulfonate (MTS) derivatives o n mu-opioid receptor binding were examined in Chinese hamster ovary (CHO) c ells that stably express mu-opioid receptors (H mu CHO). Three charged MTS derivatives inhibited the binding of [H-3][D-Ala(2),N-MePhe(4),Gly-ol(5)]-e nkephalin to mu-opioid receptors with IC50 values ranging from 0.12 to 13 m M. Further characterization of the mu-opioid receptor interactions with eth ylammonium MTS (the most potent among tested MTS reagents) revealed that et hylammonium MTS inhibition of ligand binding to the receptor was irreversib le, with both the maximal receptor binding (B-max) and the binding affinity (K-d) being changed. Preincubation of H mu CHO cells with [D-Ala(2),NMePhe (4),Gly-ol5]-enkephalin or naloxone prevented the receptor inactivation nor mally caused by MTS derivatives, indicating that the reactions may occur wi thin or near the ligand-binding pocket on the receptor. To identify the sus ceptible sulfhydryl groups, each of the cysteine residues in the mu-recepto r transmembrane domains were substituted with serine by site-directed mutag enesis. All of the mutant receptors transiently expressed in COS cells had receptor binding properties similar to the wild-type receptors. However, fo ur mutant receptors with a serine substitution in transmembrane domain III (C161S), IV (C192S), V (C237S), or VII (C332S) displayed significant resist ance against MTS inhibition compared with the wild-type receptor. We conclu de that these four cysteine residues react with MTS reagents and are respon sible for the effect of the MTS reagents on mu-opioid receptor binding.