Transient Receptor Potential Melastatin 3 Activity Research Articles

A279 EVIDENCE OF TRANSIENT RECEPTOR POTENTIAL MELASTATIN 3 (TRPM3) CHANNEL SENSITIZATION IN A MOUSE MODEL OF COLITIS

Abstract Background Abdominal pain is a primary symptom of inflammatory bowel disease (IBD). Opioids provide relief from IBD-associated pain, but they are addictive and associated with excess mortality in IBD patients. Thus, there is a need to develop novel therapeutics for IBD-associated pain. The mechanosensitive ion channel, transient receptor potential melastatin 3 (TRPM3) is upregulated in sensory neurons innervating inflamed tissue and contributes to pain from inflamed joints and cystitis. However, TRPM3’s role in abdominal pain has not been investigated. Purpose To evaluate whether TRPM3 contributes to abdominal pain using a mouse model of colitis. Method We used ratiometric Ca2+ imaging and extracellular afferent nerve recording to determine the effects of pharmacological activation or inhibition of TRPM3 on T13-L5 dorsal root ganglia (DRG) neurons and lumbar splanchnic nerves, respectively. Increased intracellular Ca2+ indicates neuronal excitation. Furthermore, the effects of TRPM3 activation in neurons and nerves from healthy mice and mice with dextran sulphate sodium-induced colitis were compared. Result(s) The TRPM3 agonists, CIM-0216 (0.1-10µM) and pregnenolone sulphate sodium (PSS; 1-300µM), concentration-dependently increased intracellular Ca2+ concentration in mouse DRG neurons and this was blocked using the TRPM3 inhibitor isosakuranetin (5µM; p<0.0001, Mann-Whitney Test). CIM-0216 (5µM)-induced increases in intracellular Ca2+ were significantly larger in neurons from mice with colitis (326±16% of baseline) compared to neurons from healthy mice (257±13% of baseline; p<0.01, Kruskal-Wallis with Dunn’s Multiple Comparison Test). The percentage of neurons responding to CIM-0216 was significantly increased in mice with colitis compared to healthy mice (79% vs 62%; p<0.001, Fischer’s Exact Test). Similarly, the percentage of neurons responding to PSS from mice with colitis was increased compared to healthy mice; however, this did not reach statistical significance (75% vs 70%, p=0.351, Fischer’s Exact Test). Furthermore, CIM-0216 (20µM)-induced change in the basal firing of lumbar splanchnic nerves was significantly increased in mice with colitis (1.23±0.24Hz) compared to healthy mice (0.60±0.14Hz, p<0.05, unpaired t-test). Conclusion(s) TRPM3 activation excited DRG neurons and lumbar splanchnic nerves and these excitatory effects were augmented in mice with colitis. Disclosure of Interest None Declared

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends onthe membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PI(4,5)P2 and pregnenolone sulfate. Similarly, mutating residues in the PI(4,5)P2 binding site in TRPM8 resulted in increased sensitivity to PI(4,5)P2 depletion and reduced sensitivity to menthol. Mutations of most PI(4,5)P2-interacting residues in TRPM3 also increased sensitivity to inhibition byGβγ, indicating allosteric interaction between Gβγ and PI(4,5)P2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

Hypoxia Augments TRPM3‐mediated Calcium Influx in Vagal Sensory Neurons

Exposure to low systemic oxygen (hypoxia, Hx) elevates blood pressure, heart rate and respiration via reflex sensory and central nervous system activation. We have shown transient receptor potential (TRP) vanilloid channels contribute to afferent‐evoked activation of the brainstem nucleus tractus solitarii (nTS) following chronic Hx. Short sustained Hx exposure (10% O2, 24hr) in rats also enhances nTS activity, although the mechanisms are not completely understood. Recent evidence from others shows TRP melastatin 3 (TRPM3) channels contribute to afferent activity and nTS glutamate signaling, but their role in afferent signaling in Hx is unknown. We hypothesize that TRPM3 increases afferent neuronal activity in nTS‐projecting nodose ganglia (NG) neurons, and its contribution is elevated following Hx. Rats were exposed to either in vivo normal room air (normoxia) or 24‐hr of 10% O2 (Hx). A subset of NG neurons from normoxic rats were exposed to in vitro incubation for 24‐hr in 21% or 1% O2, 5% CO2. Intracellular calcium (Ca2+) of dissociated NG neurons was monitored via Fura‐2 fluorescence imaging. TRPM3 was activated via 1‐min bath application of Pregnenolone sulfate (Preg, 30 and 100 µM) or CIM0216 (5 ‐ 20 µM). The increase in cellular fluorescence to Preg or CIM0216 was normalized to that of its prior baseline fluorescence (ΔF/F%). NG neurons were also exposed to vehicle (Veh) alone and their Ca2+ ΔF/F% responses were used as the threshold for TRPM3 responders under comparable timeframes. RNAScope identified TRPM3 transcripts in the normoxic NG. Veh alone did not alter Ca2+ from their initial baseline (ΔF/F%, less than 102.5% increase across all preparations). Activation of TRPM3 via Preg (30 and 100 µM) elevated Ca2+ in 51% and 55% of NG neurons by a similar magnitude [120.4 ± 3.8% and 116.6 ± 3.0% (mean ± SEM), respectively]. CIM0216 also augmented Ca2+ with the greatest response occurring during 20 µM (~56% of NG neurons, 115.6 ± 3.1% elevation). Preg responses were significantly reduced by pre‐application of the TRPM3 antagonist ononetin (20 µM; Preg, 125.9 ± 7.7% vs. ononetin + Preg, 101.7 ± 0.6%), confirming drug specificity. Elevated Ca2+ by 30 µM Preg was also eliminated by bathing cells in 0 mM Ca2+ (reduced to 100.9 ± 0.4%) suggesting Ca2+ elevation occurs via opening of membrane‐bound TRPM3. In NG neurons isolated from Hx‐exposed rats, the Preg‐induced elevation of Ca2+ was significantly greater than their paired normoxic‐exposed rats (134.1 ± 4.2% vs 118.1 ± 3.9%), although the number of responders were comparable (40% vs 43%). To determine if the increase in TRPM3 Ca2+ responses to in vivo Hx was due to the reduction of oxygen or the reflex‐induced change in neuronal activity, cells were isolated then incubated in either 21% or 1% O2 for 24 hrs. Following 24 hr in vitro Hx, 30 µM Preg enhanced Ca2+ more than in 24 hr normoxic controls (115.3 ± 2.2% vs 108.1 ± 0.1%). The proportion of Preg responders was similar between groups (58% vs 61%). Altogether, these results suggest a Hx specific increase in TRPM3 function, driven by the influx of extracellular calcium.

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

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-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 Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/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 Gαi/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 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 Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, 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.

Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 μM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.

Ca2+-dependent regulation and binding of calmodulin to multiple sites of Transient Receptor Potential Melastatin 3 (TRPM3) ion channels

TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.

G-Protein Beta-Gamma Subunits Inhibit the Heat-Sensitive TRPM3 Ion Channels

Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated non-selective, Ca2+ permeable cation channel also stimulated by chemical agents such as pregnenolone sulphate and CIM0216. Here we show that activation of Gi-coupled cell surface receptors inhibits TRPM3 currents in a mammalian expression system, which was alleviated by co-expression of proteins that bind βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, Gβ3γ2, Gβ4γ2, but not Gβ5γ2 or constitutively active mutants of Gαo or Gαi, inhibited pregnenolone sulphate-induced TRPM3 currents. Purified Gβγ proteins applied to excised inside out patches also inhibited TRPM3 activity, indicating a direct effect. Baclofen, somatostatin, and DAMGO, agonists of Gi coupled receptors, inhibited Ca2+ signals induced by pregnenolone sulphate and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data show that Gβγ inhibits TRPM3 channels upon Gi-coupled receptor activation.

Activation of TRPM3 in Perivascular Sensory Nerves Induces Dilation of Mouse Resistance Arteries

The vascular tone is determined by a complex interplay of vasodilator and vasoconstrictor stimuli that modulate the contractile state of vascular smooth muscle cells (VSMCs). Activation of the cation channel Transient Receptor Potential Melastatin 3 (TRPM3) has been shown to induce contraction of VSMCs in aorta. However, the contribution of this channel to the vascular tone in resistance arteries remains unknown. Real-time qPCR and immunochemistry experiments showed Trpm3 expression in mesenteric arteries isolated from wild type (WT) C57BL/6J mice. Myography experiments carried out in intact pressurized mesenteric arteries from WT mice showed that the TRPM3 agonist PS induces vasodilation at concentrations above ∼5 µM, with a concentration-dependency featuring two distinct increasing phases. In contrast, PS only induced vasodilation above 10 µM following a single Hill-type behavior in preparations from Trpm3 knockout (KO) mice. Recordings in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096 recapitulated the results of Trpm3 KO preparations, indicating that the TRPM3-mediated effect of PS entails CGRP release from perivascular nerve endings. The effect of 10 µM PS was inhibited to about 50% by the combination of potassium channel blockers (500 nM paxilline, 10 µM correolide and 50 nM stromatoxin). Electrophysiological recordings in freshly isolated mesenteric VSMCs, revealed that basal currents are not sensitive to PS (10 µM and 30 µM) and that PS has no effect on potassium currents. Our data indicates that activation of TRPM3 channels in perivascular sensory nerves induces CGRP release, which leads to activation of potassium channels in smooth muscle cells, resulting in dilation of mesenteric arteries. These findings reveal a potential role of TRPM3 in vascular tone regulation, and support the recent notion that this channel may play roles in neurogenic inflammation.

Localization of an Alternative Ion Permeation Pathway in TRPM3

Transient receptor potential (TRP) channels are cationic channels that are involved in a broad array of functions, including homeostasis, motility and sensory functions such as taste transduction, nociception, and temperature sensing. In general, the activation of these channels leads to the opening of their central channel pore resulting in a cationic influx and a subsequent depolarization of the cell. TRP melastatin 3 (TRPM3) is a member of the melastatin family of TRP channels and was shown to be expressed in sensory neurons where it acts as noxious heat sensor and nociceptor. Activation of TRPM3 results typically in an outward-rectifying current-voltage (I-V) curve due to the opening of the central pore of the channel. Recently, it was shown that certain agonists can induce the opening of an alternative ion permeation pathway next to the central pore, resulting in outward- and inward-rectifying currents. Here we use mutagenesis studies to localize the alternative pore of TRPM3. Our data suggest the location of the alternative pore in the voltage-sensing domain (VSD) of the channel and more specifically in proximity to transmembrane segment 4 (S4). We show three residues (W982, D988 and G991) that seem to be involved in the shaping of the alternative ion permeation pathway. The results obtained in this study are in line with the location of other non-canonical pores described in mutated voltage-gated K+-, Na+- and Ca2+-channels and propose the potential occurrence of non-canonical pores in other ion channels that are lacking the typical gating charges found in voltage-gated channels.

TRPM3 Exhibits Slight Temperature Sensitivity in the Planar Lipid Bilayer System and Requires the Presence of PIP2

Transient Receptor Potential Melastatin 3 (TRPM3) channel is critical for various physiological processes. In sensory nervous system TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, while in pancreatic β-cells the channel was linked to glucose-induced insulin release. In cells, TRPM3 is activated by heat and chemical agonists, including neurosteroid, pregnenolone sulfate (PS), and L-type Ca2+ channel blocker, nifedipine. To define the physical interactions and nuances of TRPM3 channel activity with its modulators, we succeeded to incorporate purified TRPM3 protein into the artificial membrane system - planar lipid bilayers. We found that channel opening by PS required the presence of phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole. In the presence of 5 μM PIP2 the channel exhibited EC50 for PS of ∼1.3 μM, which is lower than that observed in cell recordings (∼23 μM), due to higher agonist's sensitivity of the channel in bilayers. Unlike PS, the presence of nifedipine alone sufficed to induce TRPM3 activity (EC50 = ∼1.7 μM), in which case channel demonstrated distinct gating behavior. Similarly to PS, EC50 of TRPM3 to nifedipine in cell recordings was much higher (∼32 μM), than that observed in the bilayers.We also performed an extensive thermodynamic analysis on TRPM3 temperature dependence. Intriguingly, we found that TRPM3 exhibits slight temperature sensitivity in comparison to highly temperature-sensitive TRPM8 or TRPV1. TRPM3 remained closed upon heat-induced stimuli, but exhibited openings in the presence of PIP2 (Q10 = 5.3), although only with low open probability profile (Po = 0.048 ± 0.016).Together our results resolved the details on TRPM3 channel function in isolated system, which confirmed its direct gating by PS and PIP2, but indicated lack of strong intrinsic temperature sensitivity, common to other thermo-sensitive TRP channels.

Regulation of the TRPM3 Channel in Planar Lipid Bilayers

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective cation channel that can be activated with nifedipine and pregnenolone sulfate (PS). The role of TRPM3 in nociceptive neurons has been linked to the heat sensation and development of heat hyperalgesia during inflammation. However, the functional characterization of TRPM3 and its specific agonists remain poorly understood. To investigate biophysical and pharmacological properties of TRPM3 we aimed to incorporate the purified channel in planar lipid bilayers. TRPM3 was purified from HEK-293 cells using immunoprecipitation technique. The single channel activity was then examined under various conditions. We investigated TRPM3 activity with different agonists. Application of nifedipine (1-10 µM) resulted in dose-dependent increase of open probability of the channel. These results suggest that nifedipine can alone activate TRPM3. Unlike nifedipine, addition to the bilayers of PS, did not induce the channel openings alone and required co-application of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or clotrimazole. TRPM3 demonstrated outward rectification upon activation with PS/PI(4,5)P2, but linear current-voltage relationship with PS/clotrimazole. In both conditions, TRPM3 tended to inactivate at higher voltages (100-120 mV) and with increased concentrations of PS. Interestingly, in the absence of any agonists TRPM3 exhibited basal channel activity that sustained for several minutes. Previously TRPM3 was proposed to serve as a temperature sensor. We tested temperature sensitivity of TRPM3 and found that in the framework of lipid bilayers the channel did not exhibit any temperature-induced activation in the absence of agonists. In the presence of nifedipine TRPM3 demonstrated weak temperature dependence with Q10of 1.5 at +100 mV and 1.7 at −100 mV. These results indicate that TRPM3 is unlikely to serve as a temperature sensor alone and may involve some alternative molecular mechanisms for temperature sensation.

Signal transduction via TRPM3 channels in pancreatic β-cells

Transient receptor potential melastatin 3 (TRPM3) channels are non-selective cation channels that are expressed in insulinoma cells and pancreatic β-cells. Stimulation of TRPM3 with the neurosteroid pregnenolone sulfate induces an intracellular signaling cascade, involving a rise in intracellular Ca(2)(+) concentration, activation of the protein kinases Raf and ERK, and a change in the gene expression pattern of the cells. In particular, biosynthesis of insulin is altered following activation of TRPM3 by pregnenolone sulfate. Moreover, a direct effect of TRPM3 stimulation on insulin secretion has been reported. The fact that stimulation of TRPM3 induces a signaling cascade that is very similar to the signaling cascade induced by glucose in β-cells suggests that TRPM3 may influence main functions of pancreatic β-cells. The view that TRPM3 represents an ionotropic steroid receptor of pancreatic β-cells linking insulin release with steroid hormone signaling is discussed.

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01–10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

TRPM3 Is a Nociceptor Channel Involved in the Detection of Noxious Heat

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its invivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.