Identification in the μ-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents

Inactivation by thiol reducing and alkylating agents of ligand binding to the human μ-opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the μ receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50=20 μM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50=2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the μ-opioid receptor.