Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity.

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties of receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and the resulting effects is important. Here, we calculated the respective binding poses for several opioids and analyzed interaction fingerprints between ligand and receptor. We further corroborated the interactions experimentally by cellular assays. As MOR was observed to display ligand-induced modulation of activity due to changes in membrane potential, we further analyzed the effects of voltage sensitivity on this receptor. Combining in silico and in vitro approaches, we defined discriminating interaction patterns responsible for ligand-specific voltage sensitivity and present new insights into their specific effects on activation of the MOR.

Voltage modulates the effect of µ-receptor activation in a ligand-dependent manner.

BACKGROUND AND PURPOSE: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of µ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on µ receptor signalling. EXPERIMENTAL APPROACH: We investigated potential voltage sensitivity of µ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials. KEY RESULTS: Studying µ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (VM ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (Kir 3.X) currents and binding of GPCR kinases and arrestin3 to µ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced µ receptor activation was also observed at the level of Kir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate µ receptors was not (Met-enkephalin) or only moderately ([D-Ala2 , N-Me-Phe4 , Gly5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate µ receptors. CONCLUSION AND IMPLICATIONS: Our results indicate a strong ligand-dependent modulation of µ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.

Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid µ-opioid receptor desensitization.

G protein receptor kinases (GRKs) and ß-arrestins are key regulators of µ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and ß-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was ß-arrestin to a DAMGO-activated MOR. ß-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and ß-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.