The role of the hydrophilic Asn230 residue of the μ‐opioid receptor in the potency of various opioid agonists

Abstract

1 To investigate the e ect of the hydrophilic Asn amino acid at position 230 of the human mopioid 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 Gprotein-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 receptoractivated GIRK1/GIRK2 channel responses. The potency of [D-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]-enkephalin (DAMGO), remained una ected as measured via hMORN230T and hMORN230L, while the potency of fentanyl and morphine signi®cantly 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 hexenering of morphine on the other. The mutations also had no in¯uence 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.

Molecular recognition of opioid receptor ligands

“…30 Mutation of this residue to a leucine or a threonine moderately increases morphine ' s activity, thus casting serious doubts about this asparagine ' s putative role. 52 Another study used S-activated dihydromorphine derivatives, in combination with molecular mechanics, to create a pharmacophore. This model suggests that the 6 ␣ -hydroxyl group interacts with residues at the top of TM VII (near cysteine [Cys] VII:06), while the phenolic ring interacts with Tyr VI:19.…”

Clinical response to morphine in cancer patients and genetic variation in candidate genes

“…20 Genetic variation in the gene encoding the m-opioid receptor has been shown to alter the binding affinities of different opioids. [21][22][23][24] Different opioids may therefore cause differential activation and desensitisation of the m-opioid receptor.…”

Serine 329 of the μ-Opioid Receptor Interacts Differently with Agonists

“…When these four residues are engineered into the -opioid receptor, the resultant -opioid receptor mutant can bind DAMGO with high affinity (Seki et al, 1998). Mutation of the hydrophilic Asn 230 in the second extracellular loop of the -opioid receptor did not alter the potency of DAMGO (Pil and Tytgat, 2001).…”

“…Coexpression of RGS4, the brain-expressed isoform of RGS proteins, reconstitutes the native gating kinetics by accelerating GIRK1/ GIRK2 channel deactivation (Ulens et al, 2000a). This experimentally created model (Ulens et al, 2000b) provides a defined population of functionally active opioid receptor (hMORwt or hMORS329A) and an excellent tool for measuring the efficacy and potency of a ligand for a certain receptor (Pil and Tytgat, 2001). …”