Transient Receptor Potential Melastatin 3 Channels Research Articles

Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target.

Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells. This research aimed to: i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS; and ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients. Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years). NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol. This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients. Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC. Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX. Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients. This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX. The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca2+) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

Neurodevelopmental disorders caused by variants in TRPM3

Developmental and epileptic encephalopathies (DEE) are a broad and varied group of disorders that affect the brain and are characterized by epilepsy and comorbid intellectual disability (ID). These conditions have a broad spectrum of symptoms and can be caused by various underlying factors, including genetic mutations, infections, and other medical conditions. The exact cause of DEE remains largely unknown in the majority of cases. However, in around 25 % of patients, rare nonsynonymous coding variants in genes encoding ion channels, cell-surface receptors, and other neuronally expressed proteins are identified. This review focuses on a subgroup of DEE patients carrying variations in the gene encoding the Transient Receptor Potential Melastatin 3 (TRPM3) ion channel, where recent data indicate that gain-of-function of TRPM3 channel activity underlies a spectrum of dominant neurodevelopmental disorders.

Development of a high throughput-compatible fluorescence-based assay for TRPM3 channel activity and surface expression.

The Transient Receptor Potential Melastatin 3 (TRPM3) channel is a calcium-permeable non-selective cation channel that can be activated by heat and by a variety of chemical ligands, including neurosteroid pregnenolone sulfate (PS). The TRPM3 channel senses noxious heat in nociceptive neurons, and it is also expressed in the brain where its gating mechanisms and functions remain poorly understood. TRPM3 channel mutations in human patients were recently reported to be associated with intellectual disability and epilepsy, and to cause a gain of function phenotype by increasing the open probability of the channel. By using these mutations along with wild-type TRPM3 channels, expressed in tandem with a genetically encoded fluorescent calcium indicator, we developed and benchmarked a fluorescence-based assay to evaluate ion channel activity. We will apply this fluorescence-based assay to investigate genotype to phenotype relations in the TRPM3 channel at high-throughput. To distinguish between mutations affecting channel activity from those that impact surface expression in the plasma membrane, we are screening diverse engineered TRPM3 channel constructs that would enable specific fluorescence labelling of surface-expressed ion channels. This trafficking assay will further facilitate our effort to systematically identify residues in the transmembrane domain of the channel that are crucial for activation by different types of agonists.

Transient receptor potential melastatin 3 dysfunction in post COVID-19 condition and myalgic encephalomyelitis/chronic fatigue syndrome patients

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients.MethodsWhole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist.ResultsAs reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001).ConclusionThe results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

Phosphoinositide regulation of TRPM3 channels

Transient receptor potential melastatin 3 (TRPM3) is a calcium permeable non-selective ion channel in the transient receptor potential (TRP) superfamily. TRPM3 has been identified as a thermosensitive nociceptor channel which can be activated by noxious heat. TRPM3 knockout mice show impairment in avoiding noxious heat and have defects in developing inflammatory heat hyperalgesia. TRPM3 can also be activated by chemical agonists such as pregnenolone sulfate (PregS), CIM0216 and clotrimazole. Phosphatidylinositol 4,5-biphosphate (PIP2) is a minor component of the cell membrane, but its presence is required for the activation of many ion channels including TRPM3.

Migraine-relevant sex-dependent activation of mouse meningeal afferents by TRPM3 agonists

BackgroundMigraine is a common brain disorder that predominantly affects women. Migraine pain seems mediated by the activation of mechanosensitive channels in meningeal afferents. Given the role of transient receptor potential melastatin 3 (TRPM3) channels in mechanical activation, as well as hormonal regulation, these channels may play a role in the sex difference in migraine. Therefore, we investigated whether nociceptive firing induced by TRPM3 channel agonists in meningeal afferents was different between male and female mice. In addition, we assessed the relative contribution of mechanosensitive TRPM3 channels and that of mechanosensitive Piezo1 channels and transient receptor potential vanilloid 1 (TRPV1) channels to nociceptive firing relevant to migraine in both sexes.MethodsTen- to 13-week-old male and female wildtype (WT) C57BL/6 J mice were used. Nociceptive spikes were recorded directly from nerve terminals in the meninges in the hemiskull preparations.ResultsSelective agonists of TRPM3 channels profoundly activated peripheral trigeminal nerve fibres in mouse meninges. A sex difference was observed for nociceptive firing induced by either PregS or CIM0216, both agonists of TRPM3 channels, with the induced firing being particularly prominent for female mice. Application of Yoda1, an agonist of Piezo1 channels, or capsaicin activating TRPV1 channels, although also leading to increased nociceptive firing of meningeal fibres, did not reveal a sex difference. Cluster analyses of spike activities indicated a massive and long-lasting activation of TRPM3 channels with preferential induction of large-amplitude spikes in female mice. Additional spectral analysis revealed ​a dominant contribution of spiking activity in the α- and β-ranges following TRPM3 agonists in female mice.ConclusionsTogether, we revealed a specific mechanosensitive profile of nociceptive firing in females and suggest TRPM3 channels as a potential novel candidate for the generation of migraine pain, with particular relevance to females.

Disease-Related TRPM3 Mutations Render the Channel Overactive via Different Mechanisms

The Transient Receptor Potential Melastatin 3 (TRPM3) ion channelis a calcium permeable non-selective ion channel and it belongs to the Transient Receptor Potential (TRP) superfamily. TRPM3 has been identified as a thermosensitive nociceptor channel which can be activated by noxious heat. TRPM3 knockout mice show impairment in avoiding noxious heat and have defects in developing inflammatory heat hyperalgesia. TRPM3 can also be activated by chemical agonists such as pregnenolone sulfate (PregS), CIM0216 and clotrimazole.The presence of de novo substitutions of TRPM3 has been reported in human pediatric patients with intellectual disability and epilepsy recently. Either the Val 990 (992) Met or the Pro 1090 (1092) Gln was found in those patients, however the functional effects of those mutations were not reported. We find that both mutations make the channel overactive increasing basal activity, as well as increasing sensitivity to agonist and heat. The TRPM3-Val990Met mutant shows higher basal activity and it makes the channel more sensitive towards different agonists such as PregS and CIM0216 than the Pro1090Gln mutant. The Val to Mel substitution is located in the S4-S5 loop which could help to understand the increased sensitivity of mutated TRPM3 towards different agonists since the S4-S5 loop is the hot spots for ligand binding in many TRP channels. In contrast, the extracellular S6 segment mutation, Pro1090Gln, increases the sensitivity of TRPM3 towards heat activation to a larger extent than the Val990Met mutation. Both mutated TRPM3 channels can be inhibited by primidone, which is a clinically approved anti-epileptic drug, suggesting the potential treatment of this type of disease.

TRPM3 Channels Play Roles in Heat Hypersensitivity and Spontaneous Pain after Nerve Injury.

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel in primary sensory neurons of the dorsal root ganglia (DRGs). Pharmacological and genetic studies implicated TRPM3 in various pain modalities, but TRPM3 inhibitors were not validated in TRPM3-/- mice. Here we tested two inhibitors of TRPM3 in male and female wild-type and TRPM3-/- mice in nerve injury-induced neuropathic pain. We found that intraperitoneal injection of either isosakuranetin or primidone reduced heat hypersensitivity induced by chronic constriction injury (CCI) of the sciatic nerve in wild-type, but not in TRPM3-/- mice. Primidone was also effective when injected locally in the hindpaw or intrathecally. Consistently, intrathecal injection of the TRPM3 agonist CIM0216 reduced paw withdrawal latency to radiant heat in wild-type, but not in TRPM3-/- mice. Intraperitoneal injection of 2 mg/kg, but not 0.5 mg/kg isosakuranetin, inhibited cold and mechanical hypersensitivity in CCI, both in wild-type and TRPM3-/- mice, indicating a dose-dependent off-target effect. Primidone had no effect on cold sensitivity, and only a marginal effect on mechanical hypersensitivity. Genetic deletion or inhibitors of TRPM3 reduced the increase in the levels of the early genes c-Fos and pERK in the spinal cord and DRGs in CCI mice, suggesting spontaneous activity of the channel. Intraperitoneal isosakuranetin also inhibited spontaneous pain related behavior in CCI in the conditioned place preference assay, and this effect was eliminated in TRPM3-/- mice. Overall, our data indicate a role of TRPM3 in heat hypersensitivity and in spontaneous pain after nerve injury.SIGNIFICANCE STATEMENT Neuropathic pain is a major unsolved medical problem. The heat-activated TRPM3 ion channel is a potential target for novel pain medications, but the pain modalities in which it plays a role are not clear. Here we used a combination of genetic and pharmacological tools to assess the role of this channel in spontaneous pain, heat, cold, and mechanical hypersensitivity in a nerve injury model of neuropathic pain in mice. Our findings indicate a role for TRPM3 in heat hyperalgesia, and spontaneous pain, but not in cold and mechanical hypersensitivity. We also find that not only TRPM3 located in the peripheral nerve termini, but also TRPM3 in the spinal cord or proximal segments of DRG neurons are important for heat hypersensitivity.

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.

The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.

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.

Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients

BackgroundChronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME) is a debilitating disorder that is accompanied by reduced cytotoxic activity in natural killer (NK) cells. NK cells are an essential innate immune cell, responsible for recognising and inducing apoptosis of tumour and virus infected cells. Calcium is an essential component in mediating this cellular function. Transient Receptor Potential Melastatin 3 (TRPM3) cation channels have an important regulatory role in mediating calcium influx to help maintain cellular homeostasis. Several single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in patients with CFS/ME and have been proposed to correlate with illness presentation. Moreover, a significant reduction in both TRPM3 surface expression and intracellular calcium mobilisation in NK cells has been found in CFS/ME patients compared with healthy controls. Despite the functional importance of TRPM3, little is known about the ion channel function in NK cells and the epiphenomenon of CFS/ME. The objective of the present study was to characterise the TRPM3 ion channel function in NK cells from CFS/ME patients in comparison with healthy controls using whole cell patch-clamp techniques.MethodsNK cells were isolated from 12 age- and sex-matched healthy controls and CFS patients. Whole cell electrophysiology recording has been used to assess TRPM3 ion channel activity after modulation with pregnenolone sulfate and ononetin.ResultsWe report a significant reduction in amplitude of TRPM3 current after pregnenolone sulfate stimulation in isolated NK cells from CFS/ME patients compared with healthy controls. In addition, we found pregnenolone sulfate-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients.ConclusionsTRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.

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.

Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits.

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.

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.

Inhibition of TRPM3 Ion Channels by G-Protein Beta-Gamma Subunits

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+ permeable cation channel activated by heat, and chemical activators such as pregnenolone sulphate and CIM0216. Here we show that activation of receptors coupled to heterotrimeric Gi proteins inhibits TRPM3 currents in a mammalian expression system. This inhibition was alleviated by co-expression of proteins that act as sinks for the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, but not a constitutively active mutant of Gαo inhibited pregnenolone sulphate-induced TRPM3 currents. TRPM3 was also inhibited by purified Gβγ proteins applied to excised inside out patches, indicating a direct effect. Baclofen and somatostatin, 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 Gi-coupled receptors regulate TRPM3 channels via direct inhibition by Gβγ.

Activation of gene transcription via CIM0216, a synthetic ligand of transient receptor potential melastatin-3 (TRPM3) channels

ABSTRACTSeveral compounds have been proposed to stimulate TRPM3 Ca2+ channels. We recently showed that stimulation of TRPM3 channels with pregnenolone sulfate activated the transcription factor AP-1, while other proposed TRPM3 ligands (nifedipine, D-erythro-sphingosine) exhibited either no or TRPM3-independent effects on gene transcription. Here, we have analyzed the transcriptional activity of CIM0216, a synthetic TRPM3 ligand proposed to have a higher potency and affinity for TRPM3 than pregnenolone sulfate. The results show that CIM0216 treatment of HEK293 cells expressing TRPM3 channels activated AP-1 and stimulated the transcriptional activation potential of c-Jun and c-Fos, 2 basic region leucine zipper transcription factors that constitute AP-1. CIM0216-induced gene transcription was attenuated by knock-down of TRPM3 or treatment with mefenamic acid, a TRPM3 inhibitor. CIM0216 was similarly or less capable in activating TRPM3-mediated gene transcription, suggesting that pregnenolone sulfate is still the ligand of choice for changing the gene expression pattern via TRPM3.

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.

CREB, AP-1, ternary complex factors and MAP kinases connect transient receptor potential melastatin-3 (TRPM3) channel stimulation with increased c-Fos expression.

The rise in intracellular Ca(2+) stimulates the expression of the transcription factor c-Fos. Depending on the mode of entry of Ca(2+) into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca(2+) influx via activation of transient receptor potential melastatin-3 (TRPM3) channels with enhanced c-Fos expression. Transcription of c-Fos promoter/reporter genes that were integrated into the chromatin via lentiviral gene transfer was analysed in HEK293 cells overexpressing TRPM3. The transcriptional activation potential of c-Fos was measured using a GAL4-c-Fos fusion protein. The signalling pathway connecting TRPM3 stimulation with enhanced c-Fos expression requires the activation of MAP kinases. On the transcriptional level, three Ca(2+) -responsive elements, the cAMP-response element and the binding sites for the serum response factor (SRF) and AP-1, are essential for the TRPM3-mediated stimulation of the c-Fos promoter. Ternary complex factors are additionally involved in connecting TRPM3 stimulation with the up-regulation of c-Fos expression. Stimulation of TRPM3 channels also increases the transcriptional activation potential of c-Fos. Signalling molecules involved in connecting TRPM3 with the c-Fos gene are MAP kinases and the transcription factors CREB, SRF, AP-1 and ternary complex factors. As c-Fos constitutes, together with other basic region leucine zipper transcription factors, the AP-1 transcription factor complex, the results of this study explain TRPM3-induced activation of AP-1 and connects TRPM3 with the biological functions regulated by AP-1.

Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel.

Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5'-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily.