Sulfhydryl groups on opioid receptors revisited. Evidence for two sulfhydryl groups at or near the active site of the mu opioid receptor.

Sulfhydryl groups were studied in opioid receptors solubilized from bovine striatal membranes and reconstituted into liposomes. This system has the advantage of permitting the complete uncoupling of tightly coupled opioid binding sites from guanine nucleotide binding proteins. Sensitivity of opioid receptors to N-ethylmaleimide (NEM) inactivation, as measured by [3H]bremazocine binding, was similar whether coupled or uncoupled from the G protein. Moreover, the binding of uncoupled receptors could be protected from NEM inactivation by preincubation with a ligand, as previously observed in coupled, membrane-bound receptors. These findings provide strong support of earlier results suggesting the presence of sulfhydryl groups on opioid binding sites. An examination of the major receptor types provided the following decreasing order of sensitivity to NEM: mu > delta > kappa. Mu agonist binding was found to be much more sensitive to NEM than antagonist binding, especially in the presence of NaCl, which affects the binding of the two types of ligands in opposite directions, as previously reported for membrane-bound receptors. At 100 microM NEM in the presence of 100 mM NaCl, [3H] (D-Ala2,N-methyl-Phe4,Gly-ol)-enkephalin (DAGO) binding is essentially eliminated, whereas [3H]bremazocine or [3H]naloxone binding is virtually unaffected. These results are most readily explained by the hypothesis that there are two sulfhydryl groups at or near the mu binding site; one essential for agonist (but not antagonist) binding, the other essential for antagonist and perhaps, also agonist binding. The sodium effect on NEM inactivation of antagonist binding was maintained in the uncoupled state indicating that this effect occurs at the level of the receptor protein.