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 2 30 of the human mu -opioid receptor (hMOR230) on the potency of various ago nists, we mutated this residue to Thr and Leu (hMORN230T and hMORN230L resp ectively). 2 Taking advantage of the functional coupling of the opioid receptor with t he heteromultimeric G-protein-coupled inwardly rectifying K+ (GIRK1/GIRK2) channel, either the wild type hMOR or one of the mutated receptors (hMORN23 0L or hMORN230T) were functionally coexpressed with GIRK1/GIRK2 channels an d a regulator of G-protein signalling (RGS4) in Xenopus laevis oocytes. 3 The two-microelectrode voltage clamp technique was used to measure the op ioid receptor-activated GIRK1/GIRK2 channel responses. The potency of [D-Al a(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO), remained unaffected as measu red 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 othe r. The mutations also had no influence on the potency of morphine-6-glucuro nide (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 morphi ne and that an enhancement of the potency of morphine and fentanyl can be e xplained by mutating this residue towards more hydrophobic amino acids.