Accepted

Transient receptor potential melastatin 3 (TRPM3) is a non-selective 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 heterologously expressed TRPM3 in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C-terminus of β-adrenergic receptor kinase-1) was co-expressed with TRPM3. In neurons isolated from mouse dorsal root ganglia (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 GPCR activation. When DRG neurons were dialysed 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 wildtype 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 native sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- 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 shared by the major Gα subunits.

Neonatal tissue injury disrupts the balance between primary afferent-evoked excitation and inhibition onto adult spinal projection neurons. However, whether this reflects cell type-specific alterations at synapses onto ascending projection neurons, or rather is indicative of global changes in synaptic signaling across the mature superficial dorsal horn (SDH), remains unknown. Therefore the present study investigated the effects of neonatal surgical injury on primary afferent synaptic input to adult mouse SDH interneurons using in vitro patch clamp techniques. Hindpaw incision at postnatal day (P) 3 significantly diminished total primary afferent-evoked glutamatergic drive to adult Gad67-GFP and non-GFP neurons, and reduced their firing in response to sensory input, in both males and females. Early tissue damage also shaped the relative prevalence of monosynaptic A- vs. C-fiber mediated input to mature GABAergic neurons, with an increased prevalence of Aβ- and Aδ-fiber input observed in neonatally-incised mice compared to naïve littermate controls. Paired presynaptic and postsynaptic stimulation at an interval that exclusively produced spike timing-dependent long-term potentiation (t-LTP) in projection neurons predominantly evoked NMDAR-dependent long-term depression (t-LTD) in naïve Gad67-GFP interneurons. Meanwhile, P3 tissue damage enhanced the likelihood of t-LTP generation at sensory synapses onto the mature GABAergic population, and increased the contribution of Ca-permeable AMPARs to the overall glutamatergic response. Collectively, the results indicate that neonatal injury suppresses sensory drive to multiple subpopulations of interneurons in the adult SDH, which likely represents one mechanism contributing to reduced feedforward inhibition of ascending projection neurons, and the priming of developing pain pathways, following early life trauma.Mounting clinical and preclinical evidence suggests that neonatal tissue damage can result in long-term changes in nociceptive processing within the CNS. While recent work has demonstrated that early life injury weakens the ability of sensory afferents to evoke feedforward inhibition of adult spinal projection neurons, the underlying circuit mechanisms remain poorly understood. Here we demonstrate that neonatal surgical injury leads to persistent deficits in primary afferent drive to both GABAergic and presumed glutamatergic neurons in the mature superficial dorsal horn (SDH), and modifies activity-dependent plasticity at sensory synapses onto the GABAergic population. The functional denervation of spinal interneurons within the mature SDH may contribute to the 'priming' of developing pain pathways following early life injury.