Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gβγ Subunits

• Published in: The Journal of neuroscience 2019-10, Vol.39 (40), p.7840-7852
• Main Authors: Alkhatib, Omar, da Costa, Robson, Gentry, Clive, Quallo, Talisia, Bevan, Stuart, Andersson, David A
• Format: Article
• Language: English
• Subjects: Acetylcholine receptors (muscarinic); Activation; Adenosine; Adrenergic receptors; Agonists; Animals; Behavior, Animal; Bradykinin; Dorsal root ganglia; Female; G protein-coupled receptors; Ganglia, Spinal - cytology; Ganglia, Spinal - physiology; GTP-Binding Protein beta Subunits - antagonists & inhibitors; GTP-Binding Protein beta Subunits - physiology; GTP-Binding Protein gamma Subunits - antagonists & inhibitors; GTP-Binding Protein gamma Subunits - physiology; Heat; HEK293 Cells; Humans; Hyperalgesia - chemically induced; Hyperalgesia - physiopathology; Hyperalgesia - psychology; Hypersensitivity; Inflammation; Ion channels; Kinases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Neurons - physiology; Nociceptors - drug effects; Pertussis; Pertussis toxin; Pertussis Toxin - pharmacology; Protein kinase A; Protein kinase C; Proteins; Receptor, Adenosine A2B - physiology; Receptor, Muscarinic M1 - physiology; Receptors; Receptors, G-Protein-Coupled - antagonists & inhibitors; Receptors, G-Protein-Coupled - physiology; Signal Transduction - physiology; Toxins; Transient receptor potential proteins; TRPM Cation Channels - antagonists & inhibitors; TRPM Cation Channels - genetics; TRPM Cation Channels - physiology
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.0882-19.2019

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gα protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gα and Gα Activation of the G -coupled adenosine 2B receptor and the G -coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C terminus of β-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating G -coupled prostaglandin-EP2 and G -coupled bradykinin B2 (BK2) receptors. The G inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gα or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and , and suggest a critical role for this ion channel in inflammatory heat hypersensitivity. The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gα -coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gα , we examined whether activation of G - and G -coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of G - and G -coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that G - and G -coupled receptors inhibit TRPM3 , thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism share