Vol. 294, Issue 3, 1024-1033, September 2000

Interaction of p-Fluorofentanyl on Cloned Human Opioid Receptors and Exploration of the Role of Trp-318 and His-319 in µ-Opioid Receptor Selectivity

Chris Ulens, Maurits Van Boven, Paul Daenens and Jan Tytgat

Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium

In this study, we investigated the interactions of p-fluorofentanyl, an opioid designer drug, fentanyl, sufentanyl, and morphine on cloned human µ-, -, and -opioid receptors coexpressed with heteromultimeric G protein-coupled inwardly rectifying K⁺ channels (GIRK1/GIRK2) and a regulator of G protein signaling (RGS4) in Xenopus oocytes. We demonstrate that p-fluorofentanyl more potently activates GIRK1/GIRK2 channels through opioid receptors than fentanyl and that the p-fluoro substitution also changes the potency profile from µ >  >  (fentanyl) to µ >    (p-fluorofentanyl). A comparison of ligand efficacy revealed that morphine, fentanyl, and its analogs less efficiently activate GIRK1/GIRK2 channels through human µ-opioid receptor than [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin. Using site-directed mutagenesis, we investigated whether mutating residues Trp-318 and His-319 to their corresponding residues in - and -opioid receptors provides the molecular basis for µ/ selectivity and µ/ selectivity. Changes in EC₅₀ values for the W318L and W318Y/H319Y µ-opioid receptors show a partial contribution of these residues to the decreased GIRK1/GIRK2 channel activation by fentanyl analogs through - and -opioid receptors. The most pronounced effect was observed for p-fluorofentanyl, suggesting that an interaction between the 4-fluorophenylpropanamide moiety of the drug and residues Trp-318 and His-319 is important for the resulting enhanced GIRK1/GIRK2 channel activation through the µ-opioid receptor. Finally, we demonstrate that mutation of W318L confers -like potency for morphine on the mutant µ-opioid receptor.