Abstract

To investigate the effect of the hydrophilic Ser amino acid in position 329 of the human μ-opioid receptor (hMORwt) on the potency of various agonists, we mutated this residue to Ala (hMORS329A). Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying potassium channel (GIRK1/GIRK2), either the wild-type hMOR or the mutated receptor (hMORS329A) was functionally coexpressed with GIRK1 and GIRK2 channels together with a regulator of G-protein signaling (RGS4) inXenopus laevis oocytes. The two-microelectrode voltage-clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. The potency of the peptide agonist [d-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAMGO) decreased as measured via hMORS329A, whereas the potency of nonpeptide agonists like morphine, fentanyl, and β-hydroxyfentanyl (R004333) increased via the mutated receptor. Our results are indicative for the existence of hydrophilic interactions between Ser³²⁹ and DAMGO, thereby decreasing the potency of DAMGO via the mutated receptor, whereas hydrophobic interactions between the mutated receptor and the N-phenylethyl of morphine and fentanyl can explain the increased potency. We conclude that the hydroxyl group of Ser³²⁹ is not involved in the formation of a hydrogen bond with the β-hydroxy group of fentanyl and that mutation of this residue to alanine caused dual effects depending on the nature of the ligand.