Abstract

In this study, we investigated the interactions ofp-fluorofentanyl, an opioid designer drug, fentanyl, sufentanyl, and morphine on cloned human μ-, κ-, and δ-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 > κ > δ (fentanyl) to μ > δ ≥ κ (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 κ- and δ-opioid receptors provides the molecular basis for μ/δ selectivity and μ/κ 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 κ- and δ-opioid receptors. The most pronounced effect was observed forp-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 δ-like potency for morphine on the mutant μ-opioid receptor.