TRPM8 Channels Research Articles

Cholinergic modulation shifts the response of CA1 pyramidal cells to depolarizing ramps via TRPM4 channels with potential implications for place field firing.

A synergistic combination of in vitro electrophysiology and multicompartmental modeling of rat CA1 pyramidal neurons identified TRPM4 channels as major drivers of cholinergic modulation of the firing rate during a triangular current ramp, which emulates the bump in synaptic input received while traversing the place field. In control, fewer spikes at lower frequencies are elicited on the down-ramp compared to the up-ramp due to long-term inactivation of the NaV channel. The cholinergic agonist carbachol (CCh) removes or even reverses this spike rate adaptation, causing more spikes to be elicited on the down-ramp than the up-ramp. CCh application during Schaffer collateral stimulation designed to simulate a ramp produces similar shifts in the center of mass of firing to later in the ramp. The non-specific TRP antagonist flufenamic acid and the TRPM4-specific blockers CBA and 9-phenanthrol, but not the TRPC-specific antagonist SKF96365, reverse the effect of CCh; this implicates the Ca2+-activated nonspecific cation current, ICAN, carried by TRPM4 channels. The cholinergic shift of the center of mass of firing is prevented by strong intracellular Ca2+ buffering but not by antagonists for IP3 and ryanodine receptors, ruling out a role for known mechanisms of release from intracellular Ca2+ stores. Pharmacology combined with modeling suggest that [Ca2+] in a nanodomain near the TRPM4 channel is elevated through an unknown source that requires both muscarinic receptor activation and depolarization-induced Ca2+ influx during the ramp. Activation of the regenerative inward TRPM4 current in the model qualitatively replicates and provides putative underlying mechanisms for the experimental observations.

Contribution of TRPC3-mediated Ca2+ entry to taste transduction.

The current concept of taste transduction implicates the TASR/PLCβ2/IP3R3/TRPM5 axis in mediating chemo-electrical coupling in taste cells of the type II. While generation of IP3 has been verified as an obligatory step, DAG appears to be a byproduct of PIP2 cleavage by PLCβ2. Here, we provide evidence that DAG-signaling could play a significant and not yet recognized role in taste transduction. In particular, we found that DAG-gated channels are functional in type II cells but not in type I and type III cells. The DAG-gated current presumably constitutes a fraction of the generator current triggered by taste stimulation in type II cells. Bitter stimuli and DAG analogs produced Ca2+ transients in type II cells, which were greatly decreased at low bath Ca2+, indicating their dependence on Ca2+ influx. Among DAG-gated channels, transcripts solely for TRPC3 were detected in the taste tissue, thus implicating this channel in mediating DAG-regulated Ca2+ entry. Release of the afferent neurotransmitter ATP from CV papillae was monitored online by using the luciferin/luciferase method and Ussing-like chamber. It was shown that ATP secretion initiated by bitter stimuli and DAG analogs strongly depended on mucosal Ca2+. Based on the overall findings, we speculate that in taste transduction, IP3-driven Ca2+ release is transient and mainly responsible for rapid activation of Ca2+-gated TRPM5 channels, thus forming the initial phase of receptor potential. DAG-regulated Ca2+ entry through apically situated TRPC3 channels extends the primary Ca2+ signal and preserves TRPM5 activity, providing a needful prolongation of the receptor potential.

TRPM2 Channels: A Potential Therapeutic Target in Melanoma?

The transient receptor potential, the melastatin (TRPM) subfamily, which consists of eight known members, appears to have significant importance in melanoma progression, treatment, and prognosis. As several members were originally cloned from cancerous tissue, initial studies aimed towards identifying TRPM involvement in cancer progression and tumorigenesis. For relevance in skin cancer, previous research has shown roles for several TRPM members in skin cancer progression, growth, and patient prognosis. One unique member, TRPM2, appears to have notable therapeutic potential in the treatment of melanoma. Previous and recent studies have demonstrated increased TRPM2 expression levels in melanoma, as well as important roles for TRPM2 in melanoma growth, proliferation, and survival. TRPM2 is thus an emerging target in the treatment of melanoma, where TRPM2 antagonism may offer an additional treatment option for melanoma patients in the future.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

#4394TACROLIMUS BUT NOT VOCLOSPORIN INHIBITS KIDNEY TUBULAR CALCIUM AND MAGNESIUM REABSORPTION IN RATS AT CLINICALLY THERAPEUTIC DOSES

Abstract Background and Aims Clinically, the calcineurin inhibitor tacrolimus frequently causes hypercalciuria and hypomagnesemia by inhibiting kidney tubular calcium and magnesium reabsorption. Voclosporin, a novel calcineurin inhibitor approved in the USA and Europe for the treatment of adults with active lupus nephritis, has fewer side-effects including less hypomagnesemia. However, the differences between the kidney tubular effects of tacrolimus and voclosporin are unknown. To address this, we compared the effects of tacrolimus, voclosporin, and vehicle in rats. Method Tacrolimus (0.5 mg/kg) and voclosporin (0.5 mg/kg) were administrated by daily intraperitoneal injections in male Wistar rats and were compared against vehicle (Cremophor EL: 95% ethanol:saline (5:5:90, v:v:v)) for 28 days (n = 8–9/group). Dosages were determined based on pharmacokinetic studies in rats and aimed to achieve the area under the concentration-time curve observed in clinical studies. At day 18, blood and 24 h urine were collected to measure trough levels of the drugs and to analyze the fractional excretions of calcium and magnesium. At sacrifice, kidneys were harvested for immunoblotting of tubular proteins. Results Both tacrolimus and voclosporin reached clinically therapeutic doses with trough levels of 2.4 μg/L ± 0.6 μg/L and 25.8 μg/L ± 9.6 μg/L, respectively. Compared to vehicle, tacrolimus caused significantly higher fractional excretions of calcium (+348% ± 127%, P < 0.001) and magnesium (+60% ± 38%, P < 0.01) and also caused hypomagnesemia (plasma magnesium 0.65 mmol/L ± 0.04 mmol/L vs. 0.81 mmol/L ± 0.02 mmol/L, P < 0.001). Compared to vehicle, voclosporin only caused a slight, but non-statistically significant, increase in fractional calcium excretion (+ 44% ± 38%, P = 0.08) and did not cause higher fractional magnesium excretion (+5% ± 30%, P = 0.96) or hypomagnesemia (plasma magnesium 0.8 mmol/L ± 0.04 mmol/L, P = 0.9). Compared to vehicle, tacrolimus caused an 11-fold decrease in the protein abundance of the cytosolic calcium-binding protein calbindin-D28K and a 2-fold decrease in the abundance of the sodium-chloride cotransporter (NCC). In contrast, voclosporin did not decrease the protein abundances of calbindin-D28K and NCC. No differences were observed between vehicle, tacrolimus, and voclosporin in the calcium channel TRPV5 and magnesium channel TRPM6 (Figure 1). Conclusion In contrast to the calcineurin inhibitor tacrolimus, voclosporin does not inhibit the kidney tubular reabsorption of calcium and magnesium and therefore does not cause hypercalciuria or hypomagnesemia. A possible explanation for this difference is that tacrolimus but not voclosporin affects tubular transport in the distal convoluted tubule, which was further supported by the selective inhibition of calbindin-D28K and NCC by tacrolimus. Our data show that tubulotoxicity of tacrolimus is not apparent with voclosporin treatment, at clinically relevant doses.

Molecular determinants of TRPM8 function: key clues for a cool modulation.

Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

Topical borneol relieves nonhistaminergic pruritus via targeting TRPA1 and TRPM8 channels in peripheral nerve terminals of mice

Borneol has been used successfully for the treatment of itchy skin in traditional Chinese medicine. However, the antipruritic effect of borneol has rarely been studied, and the mechanism is unclear. Here, we showed that topical application of borneol on skin substantially suppressed pruritogen chloroquine- and compound 48/80-induced itching in mice. The potential targets of borneol, including transient receptor potential cation channel subfamily V member 3 (TRPV3), transient receptor potential cation channel subfamily A member 1 (TRPA1), transient receptor potential cation channel subfamily M member 8 (TRPM8), and gamma-aminobutyric acid type A (GABAA) receptor were pharmacologically inhibited or genetically knocked out one by one in mouse. Itching behavior studies demonstrated that the antipruritic effect of borneol is largely independent of TRPV3 and GABAA receptor, and TRPA1 and TRPM8 channels are responsible for a major portion of the effect of borneol on chloroquine-induced nonhistaminergic itching. Borneol activates TRPM8 and inhibits TRPA1 in sensory neurons of mice. Topical co-application of TRPA1 antagonist and TRPM8 agonist mimicked the effect of borneol on chloroquine-induced itching. Intrathecal injection of a group II metabotropic glutamate receptor antagonist partially attenuated the effect of borneol and completely abolished the effect of TRPM8 agonist on chloroquine-induced itching, suggesting that a spinal glutamatergic mechanism is involved. In contrast, the effect of borneol on compound 48/80-induced histaminergic itching occurs through TRPA1-and TRPM8-independent mechanisms. Our work demonstrates that borneol is an effective topical itch reliever, and TRPA1 inhibition and TRPM8 activation in peripheral nerve terminals account for its antipruritic effect.

Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells.

NOX4 activation has been implicated to have vasoprotective and blood pressure (BP)-lowering effects. Molecular mechanisms underlying this are unclear, but NOX4-induced regulation of the redox-sensitive Ca2+ channel TRPM2 and effects on endothelial nitric oxide synthase (eNOS)-nitric oxide signalling may be important. Wild-type and LinA3, renin-expressing hypertensive mice, were crossed with NOX4 knockout mice. Vascular function was measured by myography. Generation of superoxide (O2-) and hydrogen peroxide (H2O2) were assessed by lucigenin and amplex red, respectively, and Ca2+ influx by Cal-520 fluorescence in rat aortic endothelial cells (RAEC). BP was increased in NOX4KO, LinA3 and LinA3/NOX4KO mice. This was associated with endothelial dysfunction and vascular remodelling, with exaggerated effects in NOX4KO groups. The TRPM2 activator, ADPR, improved vascular relaxation in LinA3/NOX4KO mice, an effect recapitulated by H2O2. Inhibition of PARP and TRPM2 with olaparib and 2-APB, respectively, recapitulated endothelial dysfunction in NOX4KO. In endothelial cells, Ang II increased H2O2 generation and Ca2+ influx, effects reduced by TRPM2 siRNA, TRPM2 inhibitors (8-br-cADPR, 2-APB), olaparib and GKT137831 (NOX4 inhibitor). Ang II-induced eNOS activation was blocked by NOX4 and TRPM2 siRNA, GKT137831, PEG-catalase and 8-br-cADPR. Our findings indicate that NOX4-induced H2O2 production activates PARP/TRPM2, Ca2+ influx, eNOS activation and nitric oxide release in endothelial cells. NOX4 deficiency impairs Ca2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. We define a novel pathway linking endothelial NOX4/H2O2 to eNOS/nitric oxide through PARP/TRPM2/Ca2+. This vasoprotective pathway is perturbed when NOX4 is downregulated and may have significance in conditions associated with endothelial dysfunction, including hypertension.

(224) Icilin, a TRPM8 Agonist, Induces Relaxation and Reduces Sensitivity to Phenylephrine in Pudendal Artery from Control and Diabetic Mice

Abstract Introduction Diabetes mellitus (DM) is a chronic metabolic disease with epidemiological relevance worldwide. One of the most common and most underestimated complications of diabetes is erectile dysfunction (ED). It is estimated that approximately 35-90% of diabetic men present ED as an early complication. Previous studies have shown that TRPM8 activation triggers relaxation of pudendal artery with increased sensitivity in the hypertensive rats. However, despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the vasculature responsible for sexual function in diabetic condition still needs to be elucidated. Objective In this way, the aim of this study was to investigate TRPM8 channels in arteries vital to erection of the penis. We hypothesized that TRPM8 activation would induce relaxation and reduce sensitivity to alpha-adrenergic stimulation in pudendal arteries from diabetic mice. Methods Male control and db/db mice were euthanized, and the internal pudendal arteries were mounted on DMT wire myographs for the measurement of isometric force. Results Cumulative administration of icilin (10-8 to 10-4M), a TRPM8 agonist, induced endothelium-dependent and endothelium-independent relaxation in pre-contracted rings with phenylephrine (Phe,1μM). Relaxant effect induced by icilin was similar between pudendal arteries from db/db (Emax: 56.31 +/− 1.90%, n=5) and control (Emax:46.57 +/− 4.45%, n=5) mice. Diabetes increased the contractility of the pudendal artery in response to phenylephrine in concentration of 3x10-6M (99.40 +/− 4.65%, n=4) compared to control (85.44 +/− 2.36%, n=4). Interestingly, pre-incubation with icilin (10-4M) decreased the potency of the contraction induced by phenylephrine (10-9 to 3x10-5M) compared to untreated rings in both diabetic (pD2: 6.39 +/− 0.07 vs 6.67 +/− 0.05, n=4) and control (pD2: 6.43 +/− 0.09 vs 6.82 +/− 0.05, n=4) animals. In the pudendal artery, diabetes decreased the sensitivity to acetylcholine compared to control (pD2: 7.17 +/− 0.06 vs 7.42 +/− 0.03, n=4). However, the relaxation induced by acetylcholine (10-9 to 3x10-5M) was not changed by pre-incubation with icilin. Conclusions Icilin is able to promote relaxation with the same magnitude in pudendal arteries from diabetic and control mice. Increased responses to phenylephrine in pudendal artery may contribute to the development of ED. In addition, pre-incubation with icilin reduced sensitivity to phenylephrine, suggesting that the icilin could be a helpful treatment for ED. Disclosure No

Downregulation of TRPM7, TRPM8, and TRPV1 channels modulate apoptotic parameters and neurodegenerative markers: Focus on neuronal differentiation and Parkinson's disease model

The transient receptor potential channel (TRP) channels are expressed in neuronal tissues and involved in neurological diseases such as pain, epilepsy, neuronal apoptosis, and neurodegenerative diseases. Formerly, we have investigated how neuronal differentiation changes TRP channels expression profile and how Parkinson's disease model is related with this expression levels. We have found that transient receptor potential channel melastatin subtype 7 (TRPM7), transient receptor potential channel melastatin subtype 8 and transient receptor potential channel vanilloid subtype 1 (TRPV1) channels have pivotal effects on differentiation and 1-Methyl-4-phenylpyridinium (MPP+ )-induced Parkinson's disease model in SH-SY5Y cells. In this study, we have investigated that downregulation of the TRP channels to evaluate how differentiation status changes to Parkinson's disease pathological hallmarks. We have also performed to other analyses to elucidate these TRP channels' function in MPP+ -induced neurotoxicity related apoptosis, cell viability, caspase 3 and 9 enzyme activities, intracellular reactive oxygen species production, mitochondrial depolarization levels, Ca2+ signaling, Alpha-synuclein and Dopamine levels, mono amino oxidase A and B enzymatic activities, both in differentiated and undifferentiated neuronal cells. Herein we have concluded that especially TRPM7 and TRPV1 channels have distinct role in Parkinson's disease pathology via their activity changings in pathological state, and downregulation of these channels or specific antagonists can be useful for the possible treatment strategy for Parkinson's disease and related markers.

Melatonin promotes orofacial antinociception in adult zebrafish by modulating TRP channels

Melatonin is an endogenous hormone, known as the sleep hormone, which has already demonstrated its antinociceptive effect. This study aimed to evaluate the participation of TRP's channels in the orofacial antinociceptive effect of melatonin (MT) in adult zebrafish. Initially, the open field test was performed to evaluate the effect of MT on the locomotor activity of adult zebrafish. Then, the animals were pre-treated with MT (0.1, 0.3 or 1 mg/mL; gavage) and acute orofacial nociception was induced by the application of capsaicin (TRPV1 agonist), cinnamaldehyde (TRPA1 agonist) or menthol (TRPM8 agonist) applied into the animal's lip. Naive groups were included. MT, per se, did not alter the locomotor activity of the animals. MT reduced the nociceptive behavior induced by the three agonists; however, the most significant effect was obtained with the lowest concentration tested (0.1 mg/mL) in the capsaicin test. The orofacial antinociceptive effect of melatonin was prevented by capsazepine (TRPV1 antagonist), but not by HC-030031 (TRPA1 antagonist). The molecular docking study indicated interaction between MT and the TRPV1, TRPA1 and TRPM8 channels and, in line with the in vivo results, there was greater affinity between MT and the TRPV1 channel. The results confirm the pharmacological relevance of melatonin as an inhibitor of orofacial nociception and this effect seems to be related to the modulation of TRP's channels.

Structural mechanisms of TRPM7 activation and inhibition

The transient receptor potential channel TRPM7 is a master regulator of the organismal balance of divalent cations that plays an essential role in embryonic development, immune responses, cell mobility, proliferation, and differentiation. TRPM7 is implicated in neuronal and cardiovascular disorders, tumor progression and has emerged as a new drug target. Here we use cryo-EM, functional analysis, and molecular dynamics simulations to uncover two distinct structural mechanisms of TRPM7 activation by a gain-of-function mutation and by the agonist naltriben, which show different conformational dynamics and domain involvement. We identify a binding site for highly potent and selective inhibitors and show that they act by stabilizing the TRPM7 closed state. The discovered structural mechanisms provide foundations for understanding the molecular basis of TRPM7 channelopathies and drug development.

TRPM8 channel activation induces relaxation in pudendal artery and corpus cavernosum: Can TRPM8 channel be a target to improve diabetic erectile dysfunction?

Erectile dysfunction (ED) is one of the most common and underestimated complications of diabetes. Previous studies have shown that TRPM8 activation triggers relaxation of internal pudendal arteries (IPA) with increased sensitivity in hypertensive rats. Despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the IPA and corpus cavernosum (CC) in diabetes still needs to be elucidated. We hypothesized that preserved TRPM8 channel function in diabetic mice could be a therapeutic target for the treatment of ED. Male lean and db/db mice were euthanized, and the IPA were mounted on DMT wire myographs and CC were mounted on AVS organ bath for the measurement of isometric force. Concentration-response curves to icilin or menthol (TRPM8 agonists) were obtained. The CC TRPM8 channels expression was evaluated by western blot. Second harmonic generation was used to evaluate collagen deposition in IPA. Data are expressed as mean ± S.E.M of 3-5 mice. Non-linear regression curve was performed, and the maximum response (Emax) was analyzed using Student’s t test (p<0.05). Similar TRPM8 channel expression was observed in the CC from lean and db/db mice. Menthol-induced relaxation of the CC was reduced in diabetic mice (Emax: 105.00 ± 5.00%) compared to lean mice (Emax: 139.44 ± 15.91%) . Relaxant effect induced by icilin and menthol was similar between IPA from db/db (Emax icilin: 95.56 ± 0.86% and Emax menthol: 93.88 ± 2.33%) and lean (Emax icilin: 97.05 ± 0.87% and Emax menthol: 101.10 ± 1.75%) mice. Interestingly, pre-incubation with icilin (10-4M) for 30 minutes decreased the potency of phenylephrine-induced contraction compared to untreated IPA rings in both db/db and lean mice. In the IPA, diabetes decreased the sensitivity to acetylcholine compared to lean. However, the relaxation induced by acetylcholine was not changed by pre-incubation with icilin. Diabetic mice (db/db) IPA tended to have a decrease in arterial collagen content compared to lean mice. Although menthol-induced relaxation of the CC was reduced in diabetic mice, icilin and menthol were able to promote similar relaxation in IPA from diabetic and lean mice. In this way, TRPM8 function was preserved in IPA from diabetic mice. Icilin reduced sensitivity to phenylephrine in IPA from both mice. These data suggest that activation of TRPM8 could be a therapeutic target for the treatment of ED. SMSNA, NIH (RO1DK132948) and FAPESB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPM8 channels do not regulate endothelium-dependent and -independent vascular reactivity in mice

Foremost among the ion channels that control vascular tone is the transient receptor potential (TRP) cation channels. The TRPM subfamily of the TRP channels includes eight members, expressed in various excitable and non-excitable cells and involved in diverse functions, such as sensing membrane potential, cold, taste, redox, osmolarity, and cell survival. Previous analysis of TRPM gene expression showed the highest expression of TRPM8 in intact blood vessels compared with other isoforms (TRPM1-7). Studies have also revealed that activating TRPM8 by menthol and icilin modulates endothelium-dependent and independent vascular reactivity. However, the non-selectivity of pharmacological TRPM8 modulators constitutes a significant limitation in the prior studies aimed at delineating TRPM8-dependent vasoregulation. In this study, we demonstrate that acetylcholine (ACH)- and sodium nitroprusside (SNP)-induced reduction in arterial pressure was unaltered in mice lacking TRPM8. We generated endothelial-specific knockout (KO) of TRPM8 (ecTRPM8-/-) by breeding TRPM8 floxed (TRPM8fl/fl) with Cdh5-Cre (VE-Cadherin-Cre) mice. Unlike global TRPM8 KO, ecTRPM8-/- mice were not resistant to cold temperatures. Phenylephrine-induced contraction of renal and mesenteric arteries isolated from TRPM8fl/fl and ecTRPM8-/- mice were similar. Also, ACH- and SNP-induced relaxation of phenylephrine-precontracted renal and mesenteric arteries from TRPM8fl/fl and ecTRPM8-/- mice were comparable. These findings suggest that TRPM8 channels do not regulate endothelium-dependent and -independent vasorelaxation in mice. Disclosure of Funding: NHLBI: R01 HL151735-01 and R01HL151735-S This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription.

Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes and ion channels. Here, we used genetic and pharmacological tools to study the functional role of calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3 and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Pathophysiological Roles of Transient Receptor Potential (Trp) Channels and Zinc Toxicity in Brain Disease.

Maintaining the correct ionic gradient from extracellular to intracellular space via several membrane-bound transporters is critical for maintaining overall cellular homeostasis. One of these transporters is the transient receptor potential (TRP) channel family that consists of six putative transmembrane segments systemically expressed in mammalian tissues. Upon the activation of TRP channels by brain disease, several cations are translocated through TRP channels. Brain disease, especially ischemic stroke, epilepsy, and traumatic brain injury, triggers the dysregulation of ionic gradients and promotes the excessive release of neuro-transmitters and zinc. The divalent metal cation zinc is highly distributed in the brain and is specifically located in the pre-synaptic vesicles as free ions, usually existing in cytoplasm bound with metallothionein. Although adequate zinc is essential for regulating diverse physiological functions, the brain-disease-induced excessive release and translocation of zinc causes cell damage, including oxidative stress, apoptotic cascades, and disturbances in energy metabolism. Therefore, the regulation of zinc homeostasis following brain disease is critical for the prevention of brain damage. In this review, we summarize recent experimental research findings regarding how TRP channels (mainly TRPC and TRPM) and zinc are regulated in animal brain-disease models of global cerebral ischemia, epilepsy, and traumatic brain injury. The blockade of zinc translocation via the inhibition of TRPC and TRPM channels using known channel antagonists, was shown to be neuroprotective in brain disease. The regulation of both zinc and TRP channels may serve as targets for treating and preventing neuronal death.

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism.

Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage

Stroke prognosis is negatively associated with an elevation of serum bilirubin, but how bilirubin worsens outcomes remains mysterious. We report that post-, but not pre-, stroke bilirubin levels among inpatients scale with infarct volume. In mouse models, bilirubin increases neuronal excitability and ischemic infarct, whereas ischemic insults induce the release of endogenous bilirubin, all of which are attenuated by knockout of the TRPM2 channel or its antagonist A23. Independent of canonical TRPM2 intracellular agonists, bilirubin and its metabolic derivatives gate the channel opening, whereas A23 antagonizes it by binding to the same cavity. Knocking in a loss of binding point mutation for bilirubin, TRPM2-D1066A, effectively antagonizes ischemic neurotoxicity in mice. These findings suggest a vicious cycle of stroke injury in which initial ischemic insults trigger the release of endogenous bilirubin from injured cells, which potentially acts as a volume neurotransmitter to activate TRPM2 channels, aggravating Ca2+-dependent brain injury.