RT Journal Article
SR Electronic
T1 Unique Pharmacological Properties of the Kappa Opioid Receptor Signaling Through Gαz as Shown with BRET
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.120.119404
DO 10.1124/mol.120.119404
A1 Miriam E. Barnett
A1 Brian I. Knapp
A1 Jean M Bidlack
YR 2020
UL http://molpharm.aspetjournals.org/content/early/2020/07/27/mol.120.119404.abstract
AB Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, Gα•βγ. Classic pharmacological methods, such as [35S]GTPγS binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous Gα proteins in a given cell type. Bioluminescent resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of Gα subunit-specific effects on opioid pharmacology. Using a Venus-tagged Gβγ and nanoluciferase-tagged truncated G-protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific Gα protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM U50,488 for the kappa OR (KOR). Opioids reached equilibrium with the KOR and concentration-response curves were generated. While the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the Gα subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through Gαz compared to other Gαi/o subunits. In contrast, the Gα subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classical mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC50 values of 7.3 nM and 110 nM and Emax values of 79% and 35% when the KOR signaled through Gαz and Gαi1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific Gα subunit coupled to the KOR.SIGNIFICANCE STATEMENT Opioid receptors couple to various heterotrimeric Gαβγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various Gα subunits within the inhibitory class influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared to other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought.