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

Abstract

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by G␤␥ subunits liberated following activation of G␣ i/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by G␤␥ released from G␣ s and G␣ q. Activation of the G s-coupled adenosine 2B receptor and the G q-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the G␤␥ sink ␤ARK1-ct (C terminus of ␤-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 s-coupled prostaglandin-EP2 and G q-coupled bradykinin B2 (BK2) receptors. The G i/o 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 G␣ i/o 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 G␤␥ 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 Trpm3 ؊/؊ mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by G␤␥ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.

Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue

“…Instead, these data raise the possibility that increased molecular and functional expression of TRPM3 in neurons innervating inflamed tissue increases the excitability of nociceptors co-expressing TRPA1 and TRPV1, contributing to the augmented responses to agonist stimulation. This interpretation also provides a straightforward mechanism for the observation that heat hyperalgesia does not develop in TRPM3-deficient mice and is fully alleviated by TRPM3 antagonists (14,16). Since heat hyperalgesia is also strongly attenuated by pharmacological inhibition or genetic ablation of TRPV1 (10-13), we hypothesize that those DRG neurons that gain functional coexpression of TRPM3 and TRPV1 under inflammatory conditions play a central role in the development of heat hypersensitivity.…”

“…In recent work, we demonstrated that three TRP channels (TRPM3, TRPV1 and TRPA1) act as redundant sensors of acute heat: neuronal heat responses and heat-induced pain are preserved in mice in which two of these three TRP channels were genetically ablated, but combined elimination of all three channels fully abolishes the withdrawal reflex from a noxious heat stimulus (9). Notably, earlier work had also revealed an absolute requirement of both TRPV1 and TRPM3 for the development of inflammatory heat hypersensitivity, since genetic ablation or pharmacological inhibition of either channel individually fully abrogates heat hyperalgesia in the rodent CFA model (10)(11)(12)(13)(14)(15)(16). These combined findings suggested that inflammation modulates the functional interplay between these heat-activated TRP channels leading to pathological heat hypersensitivity, but the underlying processes and mechanisms remained unclear.…”

“…Intriguingly, however, there apparently is no such redundancy for the development of heat hypersensitivity in inflammatory conditions. Indeed, genetic ablation or pharmacological inhibition of either only TRPV1 (10)(11)(12)(13) or only TRPM3 (14)(15)(16) is sufficient to fully suppress inflammatory heat hyperalgesia . However, the precise mechanisms and the relative contributions of the heat-activated TRP channels to nociceptor sensitization under inflammatory conditions remains incompletely understood.…”

TRPM3_miR-204: a complex locus for eye development and disease

“…In the case of heat-sensing domains, it is notable that PS-activation of a truncated hTRPM3 channel (hTRPM3 1325 ) was not enhanced by heat [68] raising the possibility that C-terminal sequences may be involved in heat sensitivity. Other cytoplasmic sequences including those at the C-terminus may also serve as interaction sites for TRPM3 channel inhibition by Gβγ sub-units [76][77][78][79].…”

“…In vitro functional expression studies have determined that TRPM3 channels are activated by several structurally unrelated agonists including (1) the endogenous excitatory neurosteroid pregnenolone sulfate, which can also weakly activate TRPM1 channels [51,[57][58][59][60][61][62][63]; (2) cell-membrane phosphoinositol phosphates [64][65][66]; and (3) noxious heat [46][47][48][49][50]. Conversely, they are inhibited by (1) certain clinically approved drugs including nonsteroidal anti-inflammatory drugs (NSAIDs, e.g., mefenamic acid), antibiotics (e.g., voriconazole, which also inhibits TRPM1 channels), sex-steroids (e.g., progesterone), and synthetic drug-like antagonists [40,41,51,61,[67][68][69][70][71][72]; (2) naturally occurring plant-derived secondary metabolites (e.g., citrus flavanones) [73][74][75]; and (3) Gprotein coupled receptor βγ subunits [76][77][78][79]. By contrast, TRPM1 channels were found to be inhibited by interaction with both Gα or Gβγ subunits [80].…”