Transient Receptor Potential Melastatin 8 Channels Research Articles

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Expression of TRPM8 in sheep reproductive tissues and brain areas important for the expression of reproductive behavior

The Transient Receptor Potential Melastatin 8 (TRPM8) channel is a temperature-sensitive, calcium permeable ion channel and purported testosterone receptor. To determine how the hormone environment influences the expression of TRPM8 in gonadal tissue and areas of the brain important for reproduction, tissue from western white-faced cross-bred ewes, rams, and gonadectomized males (wethers; n = 6 per group) approximately 6 mo of age were collected. TRPM8 mRNA expression was greater (P = 0.01) in prostate of rams than wethers. Testes had greater (P = 0.004) expression of TRPM8 mRNA than the ovary. Differences in protein expression was similar with the testes having greater (P = 0.007) TRPM8 protein than the ovary. Protein expression did not differ (P = 0.6) in the prostate due to presence (ram) or absence (wether) of the testes. In the brain, TRPM8 varied in the amygdala with rams tending (P = 0.07) to express more mRNA which was reflected in greater (P = 0.04) number of neurons staining positive for TRPM8 in the central amygdala. Differences among ewes and wethers were not detected. This pattern was not observed (P ≥ 0.16) in the hypothalamus or olfactory bulb. To determine if TRPM8 was associated with the expression of ram sexual behavior, brains from rams categorized as high (n = 4) or low (n = 3) sexual activity were collected and blocked. Presence of TRPM8 channels was verified in the amygdala and hypothalamus of rams but was absent in the ventral tegmental area. Numbers of neurons staining positive for TRPM8 did not differ by expression of sexual behavior (P ≥ 0.2) in any area quantified. While expression of TRPM8 is more robust in tissues from intact males, expression of the channel does not appear to be important in the expression of sexual behavior.

Therapeutic potential of TRPM8 channels in cancer treatment.

Cancer is a multifactorial process associated with changes in signaling pathways leading to cell cycle variations and gene expression. The transient receptor potential melastatin 8 (TRPM8) channel is a non-selective cation channel expressed in neuronal and non-neuronal tissues, where it is involved in several processes, including thermosensation, differentiation, and migration. Cancer is a multifactorial process associated with changes in signaling pathways leading to variations in cell cycle and gene expression. Interestingly, it has been shown that TRPM8 channels also participate in physiological processes related to cancer, such as proliferation, survival, and invasion. For instance, TRPM8 channels have an important role in the diagnosis, prognosis, and treatment of prostate cancer. In addition, it has been reported that TRPM8 channels are involved in the progress of pancreatic, breast, bladder, colon, gastric, and skin cancers, glioblastoma, and neuroblastoma. In this review, we summarize the current knowledge on the role of TRPM8 channels in cancer progression. We also discuss the therapeutic potential of TRPM8 in carcinogenesis, which has been proposed as a molecular target for cancer therapy.

Suppressive effects of a transient receptor potential melastatin 8 (TRPM8) agonist on hyperthermia-induced febrile seizures in infant mice.

Background: Febrile seizures (FSs) are the most frequent type of seizures in infancy and childhood. Epileptiform discharges (EDs) on electroencephalogram at the time of first FS recurrence can increase the risk of epilepsy development. Therefore, inhibition of EDs is important. Recently, WS-3, a transient receptor potential melastatin 8 (TRPM8) agonist, reportedly suppressed penicillin G-induced cortical-focal EDs. However, the effects of TRPM8 agonists on FSs remain unknown. In this study, we aimed to clarify the effects of the TRPM8 agonist, and the absence of TRPM8 channels, on hyperthermia-induced FS by analyzing the fast ripple band. Methods: Hyperthermia (43°C for 30min) induced by a heating pad caused FSs in postnatal day 7 wild-type (WT) and TRPM8 knockout (TRPM8KO) mice. FSs were defined as EDs occurring during behavioral seizures involving hindlimb clonus and loss of the righting reflex. Mice were injected with 1% dimethyl sulfoxide or 1mM WS-3 20min before the onset of hyperthermia, and electroencephalograms; movies; and rectal, brain and heating pad temperatures were recorded. Results: In wild-type mice, WS-3 reduced the fast ripple amplitude in the first FS without changing rectal and brain temperature thresholds. In contrast, the anti-FS effect induced by the TRPM8 agonist was not observed in TRPM8KO mice and, compared with wild-type mice, TRPM8 deficiency lowered the rectal and brain temperature thresholds for FSs, exacerbated the fast ripple amplitude, and prolonged the duration of the initial FS induced by hyperthermia. Conclusion: Our findings suggest that TRPM8 agonists can be used to treat hyperthermia-induced FSs.

TRPV1 and TRPM8 antagonists reduce cystitis-induced bladder hypersensitivity via inhibition of different sensitised classes of bladder afferents in guinea pigs.

Interstitial cystitis (=painful bladder syndrome) is a chronic bladder syndrome characterised by pelvic and bladder pain, urinary frequency and urgency, and nocturia. Transient receptor potential (TRP) channels are an attractive target in reducing the pain associated with interstitial cystitis. The current study aims to determine the efficacy of combination of TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8) channel inhibition in reducing the pain associated with experimental cystitis in guinea pigs. A novel animal model of non-ulcerative interstitial cystitis has been developed using protamine sulfate/zymosan in female guinea pigs. Continuous voiding cystometry was performed in conscious guinea pigs. Ex vivo "close-to-target" single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder. Visceromotor responses in vivo were used to determine the effects of TRP channel antagonists on cystitis-induced bladder hypersensitivity. Protamine sulfate/zymosan treatment evoked mild inflammation in the bladder and increased micturition frequency in conscious animals. In cystitis, high threshold muscular afferents were sensitised via up-regulation of TRPV1 channels, high threshold muscular-mucosal afferents were sensitised via TRPM8 channels, and mucosal afferents by both. Visceromotor responses evoked by noxious bladder distension were significantly enhanced in cystitis and were returned to control levels upon administration of combination of low doses of TRPV1 and TRPM8 antagonists. The data demonstrate the therapeutic promises of combination of TRPV1 and TRPM8 antagonists for the treatment of bladder hypersensitivity in cystitis.

Structures of a mammalian TRPM8 in closed state

Transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable non-selective cation channel that acts as the primary cold sensor in humans. TRPM8 is also activated by ligands such as menthol, icilin, and phosphatidylinositol 4,5-bisphosphate (PIP2), and desensitized by Ca2+. Here we have determined electron cryo-microscopy structures of mouse TRPM8 in the absence of ligand, and in the presence of Ca2+ and icilin at 2.5–3.2 Å resolution. The ligand-free state TRPM8 structure represents the full-length structure of mammalian TRPM8 channels with a canonical S4-S5 linker and the clearly resolved selectivity filter and outer pore loop. TRPM8 has a short but wide selectivity filter which may account for its permeability to hydrated Ca2+. Ca2+ and icilin bind in the cytosolic-facing cavity of the voltage-sensing-like domain of TRPM8 but induce little conformational change. All the ligand-bound TRPM8 structures adopt the same closed conformation as the ligand-free structure. This study reveals the overall architecture of mouse TRPM8 and the structural basis for its ligand recognition.

Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice.

Migraine is a complex neurovascular disorder that is one of the leading causes of disability and a reduced quality of life. Even with such a high societal impact, our understanding of the cellular and molecular mechanisms that contribute to migraine headaches is limited. To address this complex disorder, several groups have performed genome-wide association studies to elucidate migraine susceptibility genes, with many identifying transient receptor potential melastatin 8 (TRPM8), a cold-sensitive cation channel expressed in peripheral afferents innervating the trigeminovascular system, and the principal mediator of cold and cold pain associated with injury and disease. Interestingly, these migraine-associated single-nucleotide polymorphisms reside in noncoding regions of TRPM8, with those correlated with reduced migraine risk exhibiting lower TRPM8 expression and decreased cold sensitivity. Nonetheless, as a role for TRPM8 in migraine has yet to be defined, we sought to address this gap in our knowledge using mouse genetics and TRPM8 antagonism to determine whether TRPM8 channels or neurons are required for migraine-like pain (mechanical allodynia and facial grimace) in inducible migraine models. Our results show that both evoked and spontaneous pain behaviors are dependent on both TRPM8 channels and neurons, as well as required in both acute and chronic migraine models. Moreover, inhibition of TRPM8 channels prevented acute but not established chronic migraine-like pain. These results are consistent with its association with migraine in genetic analyses and establish that TRPM8 channels are a component of the underlying mechanisms of migraine.

The TRPM8 channel as a potential therapeutic target for bladder hypersensitive disorders.

In the lower urinary tract, transient receptor potential (TRP) channels are primarily involved in physiological function, especially in cellular sensors responding to chemical and physical stimuli. Among TRP channels, TRP melastatin 8 (TRPM8) channels, responding to cold temperature and/or chemical agents, such as menthol or icilin, are mainly expressed in the nerve endings of the primary afferent neurons and in the cell bodies of dorsal root ganglia innervating the urinary bladder (via Aδ- and C-fibers); this suggests that TRPM8 channels primarily contribute to bladder sensory (afferent) function. Storage symptoms of overactive bladder, benign prostatic hyperplasia, and interstitial cystitis are commonly related to sensory function (bladder hypersensitivity); thus, TRPM8 channels may also contribute to the pathophysiology of bladder hypersensitivity. Indeed, it has been reported in a pharmacological investigation using rodents that TRPM8 channels contribute to the pathophysiological bladder afferent hypersensitivity of mechanosensitive C-fibers. Similar findings have also been reported in humans. Therefore, a TRPM8 antagonist would be a promising therapeutic target for bladder hypersensitive disorders, including urinary urgency or nociceptive pain. In this review article, the functional role of the TRPM8 channel in the lower urinary tract and the potential of its antagonist for the treatment of bladder disorders was described.

Spontaneous excitatory transmission enhancement produced by linalool and its isomer geraniol in rat spinal substantia gelatinosa neurons - involvement of transient receptor potential channels

BackgroundMany of plant-derived compounds inhibiting nerve conduction enhance glutamatergic spontaneous excitatory transmission by activating transient receptor potential (TRP) channels in spinal substantia gelatinosa (SG) neurons that play a crucial role in regulating nociceptive transmission. Although (±)-linalool and its isomer geraniol having antinociceptive effects inhibit nerve conduction, it has not been examined yet how they affect excitatory transmission in SG neurons. We examined the effects of the compounds on action potential-independent spontaneous excitatory transmission with a focus on an involvement of TRP channels. MethodsThe whole-cell patch-clamp technique was applied to SG neurons in adult rat spinal cord slices. Results(±)-Linalool increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a small increase in its amplitude. The (±)-linalool activity was sensitive to TRP vanilloid-1 (TRPV1) antagonist capsazepine and TRP ankyrin-1 (TRPA1) antagonist HC-030,031 while resistant to BCTC that is antagonist for cloned TRPV1 and TRP melastatin-8 (TRPM8) channels and for TRPM8 channels in the SG, indicating TRPV1 and TRPA1 activation. In 73% of the neurons tested, (±)-linalool produced an outward current at -70 mV. In SG neurons sensitive to (±)-linalool, geraniol produced a quantitatively similar effect to (±)-linalool. Geraniol-induced sEPSC frequency increase was sensitive to BCTC but resistant to capsazepine and HC-030,031, indicating TRPM8 activation. Outward currents produced by (±)-linalool and geraniol were unaffected by all of the TRP antagonists. ConclusionIn SG neurons, (±)-linalool and geraniol presynaptically enhanced spontaneous excitatory transmission by activating different types of TRP channel while hyperpolarizing membranes in a manner independent of TRP channels. Such a TRP channel modulation and hyperpolarization could contribute to the antinociceptive effects of (±)-linalool and geraniol.

Rational Design of a Modality-Specific Inhibitor of TRPM8 Channel against Oxaliplatin-Induced Cold Allodynia.

Platinum‐based compounds in chemotherapy such as oxaliplatin often induce peripheral neuropathy and neuropathic pain such as cold allodynia in patients. Transient Receptor Potential Melastatin 8 (TRPM8) ion channel is a nociceptor critically involved in such pathological processes. Direct blockade of TRPM8 exhibits significant analgesic effects but also incurs severe side effects such as hypothermia. To selectively target TRPM8 channels against cold allodynia, a cyclic peptide DeC‐1.2 is de novo designed with the optimized hot‐spot centric approach. DeC‐1.2 modality specifically inhibited the ligand activation of TRPM8 but not the cold activation as measured in single‐channel patch clamp recordings. It is further demonstrated that DeC‐1.2 abolishes cold allodynia in oxaliplatin treated mice without altering body temperature, indicating DeC‐1.2 has the potential for further development as a novel analgesic against oxaliplatin‐induced neuropathic pain.

Suppressive Effects of Transient Receptor Potential Melastatin 8 Agonist on Epileptiform Discharges and Epileptic Seizures.

Epilepsy is a relatively common condition, but more than 30% of patients have refractory epilepsy that is inadequately controlled by or is resistant to multiple drug treatments. Thus, new antiepileptic drugs based on newly identified mechanisms are required. A previous report revealed the suppressive effects of transient receptor potential melastatin 8 (TRPM8) activation on penicillin G-induced epileptiform discharges (EDs). However, it is unclear whether TRPM8 agonists suppress epileptic seizures or affect EDs or epileptic seizures in TRPM8 knockout (TRPM8KO) mice. We investigated the effects of TRPM8 agonist and lack of TRPM8 channels on EDs and epileptic seizures. Mice were injected with TRPM8 agonist 90 min after or 30 min before epilepsy-inducer injection, and electrocorticograms (ECoGs) were recorded under anesthesia, while behavior was monitored when awake. TRPM8 agonist suppressed EDs and epileptic seizures in wildtype (WT) mice, but not in TRPM8KO mice. In addition, TRPM8KO mice had a shorter firing latency of EDs, and EDs and epileptic seizures were deteriorated by the epilepsy inducer compared with those in WT mice, with the EDs being more easily propagated to the contralateral side. These findings suggest that TRPM8 activation in epileptic regions has anti-epileptic effects.

Efficacy of the combination of KPR-5714, a novel transient receptor potential melastatin 8 (TRPM8) antagonist, and β3-adrenoceptor agonist or anticholinergic agent on bladder dysfunction in rats with bladder overactivity

Transient receptor potential melastatin 8 (TRPM8) channels may contribute to the pathophysiological bladder afferent hyperactivity, thus a TRPM8 antagonist would be a promising therapeutic target for the bladder hypersensitive disorders including urinary urgency in overactive bladder (OAB). We aimed to investigate a pharmacological effect of KPR-5714, a novel selective TRPM8 antagonist, on TRPM8 channels, M3 receptors and β3-adrenoceptors using the transfected cells of each gene at first. Then, combination effects of KPR-5714 and mirabegron, a β3-adrenoceptor agonist, or tolterodine tartrate, an anticholinergic agent, were studied on rhythmic bladder contractions (RBCs) in normal rats and bladder function in frequent-voiding rats. In vitro measurements showed that KPR-5714 acts on neither β3-adrenoceptor nor M3 receptor. In normal rats, KPR-5714 and mirabegron significantly reduced the frequency of RBCs, and a combined administration showed an additive effect. In rats with cerebral infarction, KPR-5714 and mirabegron significantly reduced the voiding frequency, and a combined administration showed an additive effect. In rats exposed to cold temperature, KPR-5714 and tolterodine tartrate significantly reduced the voiding frequency accompanied by the increased mean voided volume, and a combined administration showed additive effects. The present study demonstrated that the combined administration of KPR-5714 and mirabegron or tolterodine tartrate showed the additive effects on bladder dysfunction in different animal models, suggesting that the combination therapy of TRPM8 antagonist and β3-adrenoceptor agonist or anticholinergic agent can be the potential treatment option for obtaining additive effects in comparison with monotherapy for OAB.

PSII-23 Aflatoxin B1 influences expression of TRPM8 in SKOV, ovarian epithelial cancer cells

Abstract Aflatoxins are mold products of Aspergillus flavus. They are common food contaminants that affect dietary staples. Aflatoxins are most prevalent in emerging countries and negatively impact the health and reproduction of several animals. Aflatoxin B1 (AFB1) affects fertility in many species by decreasing the number of gametes. The mechanism for aflatoxin’s effect on gamete production has not been fully elucidated. In aflatoxin treated rats, steroidogenesis is affected by the competitive binding of aflatoxin to the StAR protein. The transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel highly expressed in male reproductive tracts, and sheep ovaries. TRPM8 binds testosterone and is responsive to steroids. The objective of these experiments is to determine if AFB1 influenced the expression of TRPM8 channels in an ovarian cell line. Human epithelial ovarian cells, SKOV, were seeded into chamber slides and treated in triplicate with media containing ethanol, 5µg/ml AFB1, 10µg/ml AFB1, or 20µg/ml AFB1 for 24 hours. The cells were stained for TRPM8 using immunocytochemistry and in a replicate experiment, analyzed for mRNA using Real Time RT-PCR protocols. Images were captured at 200x magnification and analyzed. Mean gray scale intensity was used to determine staining intensity as a reflection of TRPM8 expression. SKOV cells treated with AFB1 had increased staining (P ≤ 0.001) compared to ethanol treated cells. Relative TRPM8 mRNA did not differ with AFB1 treatments (P = 0.2). Inconsistency between mRNA and protein expression may be due to the timing of mRNA synthesis and translation of the protein. Based on these results, expression of TRPM8 in SKOV cells increased in response to AFB1. Although it is not certain if this effect is restricted to these transformed cells, the possibility remains that changes in TRPM8 expression may contribute to the negative reproductive consequences of AFB1 exposure in humans and livestock species.

Nociceptive afferent phenotyping reveals that transient receptor potential ankyrin 1 promotes cold pain through neurogenic inflammation upstream of the neurotrophic factor receptor GFRα3 and the menthol receptor transient receptor potential melastatin 8.

The proper detection and behavioral response to painfully cold temperatures is critical for avoiding potentially harmful tissue damage. Cold allodynia and hyperalgesia, pain associated with innocuous cooling and exaggerated pain with noxious cold, respectively, are common in patients with chronic pain. In peripheral somatosensory afferents, the ion channels transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) are candidate receptors for innocuous and noxious cold temperatures, respectively. However, the role of TRPA1 as a cold sensor has remained controversial, and recent evidence suggests that TRPM8 channels and afferents mediate the detection of both pleasant and painful cold. To determine the role of TRPA1 afferents in cold-induced mouse behaviors in vivo, we used functional phenotyping by targeted nerve conduction block with the cell-impermeant lidocaine derivative QX-314. Surprisingly, we find that injection of QX-314 with TRPA1 agonists reduces cold-induced behaviors in mice, but does so in a TRPM8-dependent manner. Moreover, this effect is sexually dimorphic and requires the glial cell line-derived neurotrophic factor receptor GFRα3, as does cold hypersensitivity produced by the activation of TRPA1 channels. Taken together, these results suggest that under conditions of neurogenic inflammation, TRPA1 works upstream of GFRα3 and TRPM8 to produce cold hypersensitivity, providing novel insights into the role of TRPA1 channels in cold pain.

Acute stress induces visceral hypersensitivity via glucocorticoid receptor-mediated membrane insertion of TRPM8: Involvement of a non-receptor tyrosine kinase Pyk2.

Psychological stress is an important factor for the development and recurrence of irritable bowel syndrome (IBS). The mechanisms underlying stress-induced visceral hypersensitivity (VH), a key pathophysiological component in IBS, are still incompletely understood. We aimed to test whether transient receptor potential melastatin 8 (TRPM8) participates in acute stress-induced VH. Rats were subjected to 1-hour water avoidance stress (WAS). Visceral sensitivity was measured with visceromotor response to colorectal distension. Western blot and immunofluorescence were applied to evaluate the expression of GR and TRPM8 and activation of PKA, Akt, and PKC pathways. WAS-caused VH depended on glucocorticoid receptors (GRs) and TRPM8 channels. In a dorsal root ganglion (DRG)-derived cell line, corticosterone rapidly (within 30minutes) induced membrane expression of TRPM8. This effect was inhibited by GR antagonism and was mimicked by membrane-impermeable corticosterone. PKA, PI3K/Akt, and PKC pathways, which lied downstream of GR and acted in parallel to promote membrane expression of TRPM8, contributed to WAS-induced VH. The non-receptor tyrosine kinase Pyk2, which may serve as a convergence point for PKA, PI3K/Akt, and PKC pathways, facilitated membrane insertion of TRPM8 via tyrosine-phosphorylating TRPM8 in L6-S2 DRGs and participated in WAS-induced VH. Collectively, acute stress-induced VH could involve membrane-bound GR-dependent enhancement of TRPM8 function in nociceptive DRG neurons. Mechanistically, Pyk2 could act as a key mediator that coordinates multiple protein kinase signaling and triggers phosphorylation and membrane insertion of TRPM8.

Evidence for the involvement of peripheral cold-sensitive TRPM8 channels in human cutaneous hygrosensation.

In contrast to other species, humans are believed to lack hygroreceptors for sensing skin wetness. Yet, the molecular basis of human hygrosensation is currently unknown, and it remains unclear whether we possess a receptor-mediated sensing mechanism for skin wetness. The aim of this study was to assess the role of the cutaneous cold-sensitive transient receptor potential melastatin-8 (TRPM8) channel as a molecular mediator of human hygrosensation. To this end, we exploited both the thermal and chemical activation of TRPM8-expressing cutaneous Aδ cold thermoreceptors, and we assessed wetness sensing in healthy young men in response to 1) dry skin cooling in the TRPM8 range of thermosensitivity and 2) application of the TRPM8 agonist menthol. Our results indicate that 1) independently of contact with moisture, a cold-dry stimulus in the TRPM8 range of activation induced wetness perceptions across 12 different body regions and those wetness perceptions varied across the body following regional differences in cold sensitivity; and 2) independently of skin cooling, menthol-induced stimulation of TRPM8 triggered wetness perceptions that were greater than those induced by physical dry cooling and by contact with an aqueous cream containing actual moisture. For the first time, we show that the cutaneous cold-sensing TRPM8 channel plays the dual role of cold and wetness sensor in human skin and that this ion channel is a peripheral mediator of human skin wetness perception.

Characterization of New TRPM8 Modulators in Pain Perception.

Background: Transient Receptor Potential Melastatin-8 (TRPM8) is a non-selective cation channel activated by cold temperature and by cooling agents. Several studies have proved that this channel is involved in pain perception. Although some studies indicate that TRPM8 inhibition is necessary to reduce acute and chronic pain, it is also reported that TRPM8 activation produces analgesia. These conflicting results could be explained by extracellular Ca2+-dependent desensitization that is induced by an excessive activation. Likely, this effect is due to phosphatidylinositol 4,5-bisphosphate (PIP2) depletion that leads to modification of TRPM8 channel activity, shifting voltage dependence towards more positive potentials. This phenomenon needs further evaluation and confirmation that would allow us to understand better the role of this channel and to develop new therapeutic strategies for controlling pain. Experimental approach: To understand the role of TRPM8 in pain perception, we tested two specific TRPM8-modulating compounds, an antagonist (IGM-18) and an agonist (IGM-5), in either acute or chronic animal pain models using male Sprague-Dawley rats or CD1 mice, after systemic or topical routes of administration. Results: IGM-18 and IGM-5 were fully characterized in vivo. The wet-dog shake test and the body temperature measurements highlighted the antagonist activity of IGM-18 on TRPM8 channels. Moreover, IGM-18 exerted an analgesic effect on formalin-induced orofacial pain and chronic constriction injury-induced neuropathic pain, demonstrating the involvement of TRPM8 channels in these two pain models. Finally, the results were consistent with TRPM8 downregulation by agonist IGM-5, due to its excessive activation. Conclusions: TRPM8 channels are strongly involved in pain modulation, and their selective antagonist is able to reduce both acute and chronic pain.

Pharmacological inhibition of TRPM8-induced gene transcription

Transient receptor potential melastatin-8 (TRPM8) channels are activated by cold temperature, menthol and icilin, leading to cold sensation. TRPM8 activation is connected with various diseases, indicating that a specific pharmacological antagonist, allowing nongenetic channel suppression, would be a valuable tool for therapy and basic research. Here, we assessed the biological activity and specificity of various TRPM8 inhibitors following stimulation of TRPM8 channels with either icilin or menthol. Recently, we showed that icilin strikingly upregulates the transcriptional activity of AP-1. By measuring AP-1 activity, we assessed which compound interrupted the TRPM8-induced intracellular signaling cascade from the plasma membrane to the nucleus. We tested the specificity of various TRPM8 inhibitors by analyzing AP-1 activation following stimulation of TRPM3 and TRPV1 channels, L-type voltage-gated Ca2+ channels, and Gαq-coupled receptors, either in the presence or absence of a particular TRPM8 inhibitor. The results show that the TRPM8 inhibitors BCTC, RQ-00203078, TC-1 2014, 2-APB, and clotrimazole blocked TRPM8-mediated activation of AP-1. However, only the compound RQ-00203078 showed TRPM8-specificity, while the other compounds function as broad-spectrum Ca2+ channel inhibitors. In addition, we show that progesterone interfered with TRPM8-induced activation of AP-1, as previously shown for TRPM3 and TRPC6 channels. TRPM8-induced transcriptional activation of AP-1 was additionally blocked by the compound PD98059, indicating that extracellular signal-regulated protein kinase-1/2 is essential to couple TRPM8 stimulation with transcriptional activation of AP-1. Moreover, an influx of Ca2+-ions is essential to induce the intracellular signaling cascade leading to the activation of AP-1.

Phosphoinositide-interacting regulator of TRP (PIRT) has opposing effects on human and mouse TRPM8 ion channels

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensitive ion channel with diverse physiological roles. TRPM8 activity is modulated by many mechanisms, including an interaction with the small membrane protein phosphoinositide-interacting regulator of TRP (PIRT). Here, using comparative electrophysiology experiments, we identified species-dependent differences between the human and mouse TRPM8-PIRT complexes. We found that human PIRT attenuated human TPRM8 conductance, unlike mouse PIRT, which enhanced mouse TRPM8 conductance. Quantitative Western blot analysis demonstrates that this effect does not arise from decreased trafficking of TRPM8 to the plasma membrane. Chimeric human/mouse TRPM8 channels were generated to probe the molecular basis of the PIRT modulation, and the effect was recapitulated in a pore domain chimera, demonstrating the importance of this region for PIRT-mediated regulation of TRPM8. Moreover, recombinantly expressed and purified human TRPM8 S1-S4 domain (comprising transmembrane helices S1-S4, also known as the sensing domain, ligand-sensing domain, or voltage sensing-like domain) and full-length human PIRT were used to investigate binding between the proteins. NMR experiments, supported by a pulldown assay, indicated that PIRT binds directly and specifically to the TRPM8 S1-S4 domain. Binding became saturated as the S1-S4:PIRT mole ratio approached 1. Our results have uncovered species-specific TRPM8 modulation by PIRT. They provide evidence for a direct interaction between PIRT and the TRPM8 S1-S4 domain with a 1:1 binding stoichiometry, suggesting that a functional tetrameric TRPM8 channel has four PIRT-binding sites.

Transient Receptor Potential Melastatin 8 (TRPM8) Induced Relaxation of Pulmonary Artery is Mediated Through Inhibition of Store‐Operated Ca2+ Entry in Normoxic and Chronic Hypoxic Rats

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Members of multiple transient receptor potential subfamilies (TRPC, TRPV, and TRPM) have been identified in pulmonary vascular tissue. Our previous studies showed that the expression and functions of the store‐operated TRPC1, receptor‐operated TRPC6, and the mechanosensitive TRPV4 channels are augmented in PASMCs during PH, and their upregulation contribute to PH development. In contrast, TRPM8 is down‐regulated in PASMCs of rats PH models, and activation of TRPM8 with agonists causes relaxation of pulmonary arteries (PAs)(Liu et al., Cell Physiol Biochem 2013, 31:892–904). However, the mechanism of TRPM8‐induced PA relaxation has not been determined. In this study, we examined the possible interactions of TRPM8 activation and store‐operated Ca2+ entry (SOCE) in PAs and PASMCs of normoxic and chronic hypoxic pulmonary hypertensive (CHPH) rats. We found that TRPM8 expression was down‐regulated in PAs and the TRPM8‐mediated cation entry was reduced significantly in the PASMCs of rats after 3 weeks of hypoxia (10% O2) exposure. Activation of TRPM8 with icilin (0.1–100 μM) caused concentration‐dependent relaxation of cyclopiazonic acid (CPA) and ET‐1 pre‐contracted endothelium‐denuded PAs in the presence of nifedipine. The vasorelaxant effect of icilin was abolished in the presence of the SOCE antagonist Gd3+ at submicromolar concentration. Icilin suppressed the CPA‐induced cation entry in PASMCs determined by the Mn2+ quenching technique and by Ca2+ transient measurement. The inhibitory effect of icilin on SOCE‐induced PA relaxation and Ca2+ entry were abolished by the TRPM8 antagonist AMTB, indicating that the effect was mediated specifically by TRPM8 channels. Moreover, TRPM8‐mediated inhibitory effects on CPA‐induced contraction in PAs and SOCE in PASMCs were significantly augmented in CHPH rats, probably related to the enhanced SOCE caused by PH. These results demonstrate for the first time that TRPM8 activation can cause relaxation of PA through inhibition of SOCE. Since SOCE plays many important roles in pulmonary vascular functions and is crucial for the vascular remodeling and the enhanced vasoconstriction in PH, the downregulation of TRPM8 expression and activity in PASMCs during PH may minimized the TRPM8‐dependent inhibition of SOCE, and allow unimpeded SOCE activity for PH development.Support or Funding InformationSupported by grants NSFC31571179, NSFC31371165, NSF of Fujian Province 2015J01313, and Fujian Province Hundred Experts Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.