The role of the hydrophilic Asn230 residue of the mu-opioid receptor in the potency of various opioid agonists.

1. To investigate the effect of the hydrophilic Asn amino acid at position 230 of the human mu-opioid receptor (hMOR230) on the potency of various agonists, we mutated this residue to Thr and Leu (hMORN230T and hMORN230L respectively). 2. Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying K(+) (GIRK1/GIRK2) channel, either the wild type hMOR or one of the mutated receptors (hMORN230L or hMORN230T) were functionally coexpressed with GIRK1/GIRK2 channels and a regulator of G-protein signalling (RGS4) in Xenopus laevis oocytes. 3. The two-microelectrode voltage clamp technique was used to measure the opioid receptor-activated GIRK1/GIRK2 channel responses. The potency of [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO), remained unaffected as measured via hMORN230T and hMORN230L, while the potency of fentanyl and morphine significantly increased via these mutated receptors. 4. Our results are indicative for the existence of hydrophobic interactions between a methyl-group of the side chain of Thr or Leu on the one hand and the piperidine-ring of fentanyl and the hexene-ring of morphine on the other. The mutations also had no influence on the potency of morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G). 5. We conclude that the hydrophilic side chain of Asn in position 230 is not involved in the formation of a H-bond with the aliphatic alcohol of morphine and that an enhancement of the potency of morphine and fentanyl can be explained by mutating this residue towards more hydrophobic amino acids.