Agonist of TRPM8 channel,menthol, facilitates the initiation of thermoregulatory responses to external cooling

The effects of activation of the cold and menthol sensitive TRPM8 ion channel on different thermoregulatory parameters (total oxygen consumption, carbon dioxide release, respiratory coefficient, vasoconstriction response of skin blood vessels and shivering) were studied in anaesthetized rats subjected to two types of external cooling—rapid and slow.     

Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol

The Ca²⁺-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel has been recently identified in detrusor smooth muscle (DSM) of the urinary bladder. Two recent publications by our research group provide evidence in support of the novel hypothesis that TRPM4 channels enhance DSM excitability and contractility. This is a critical question as prior studies have primarily targeted hyperpolarizing currents facilitated by K⁺ channels, but the depolarizing component in DSM cells is not well understood. For the first time, we utilized the selective TRPM4 channel inhibitor, 9-phenanthrol, to investigate TRPM4 channel functional effects in DSM at both cellular and tissue levels in rodents. Our new data presented here showed that in rat DSM cells, 9-phenanthrol attenuates spontaneous inward currents in the presence of the muscarinic receptor agonist, carbachol, thus reducing DSM cell excitability. In support of our original hypothesis, we found that TRPM4 channel mRNA levels are much higher in DSM vs. vascular smooth muscle and that inhibition of TRPM4 channels can potentially attenuate DSM excitability. Thus, we postulate the novel concept that selective pharmacological inhibition of TRPM4 channels can limit both excitability and contractility of DSM.     

瞬时受体电位M8离子通道功能及其翻译后修饰调节机制

瞬时受体电位M8(transient receptor potential melastatin 8, TRPM8)又称冷及薄荷醇感受器,位于细胞膜或细胞器膜上,是瞬时受体电位(transient receptor potential, TRP)通道超家族中的一员。TRPM8通道分布广泛,是一个非选择性阳离子通道,可作为冷热传感器和冷痛传感器进行信号传导,参与众多生物过程的调节,在维持细胞内外稳态、控制离子进出细胞方面具有重要作用。研究发现,蛋白质翻译后修饰(post-translational modification, PTM)通过调控TRPM8通道的功能,进而影响多种疾病的发生和发展。因此,探究TRPM8的翻译后修饰的过程,对深入了解TRPM8的功能及调控机制是十分必要的。目前,已报道的TRPM8翻译后修饰包括磷酸化、泛素化和糖基化等,它们能够调控蛋白质的相互作用和改变TRPM8离子通道的活性,从而调控细胞增殖、迁移和凋亡。值得注意的是,TRPM8的表达与前列腺癌、膀胱癌和乳腺癌等多种癌症密切相关。本文将从TRPM8离子通道的结构出发,系统地阐述TRPM8蛋白翻译后修饰和激动剂、...     

Comparative modeling of the quaternary structure for the human TRPM8 channel and analysis of its binding features

The aim of this study was to generate a reliable model for the homotetrameric structure of the human TRPM8 cation channel, a temperature sensor involved in innocuous cold perceptions. The described model was generated using a fragmental strategy and its interaction capacities were explored by docking a representative set of ligands. The analysis of the quaternary structure suggests that the N-terminus possesses a solenoidal topology which could be involved in tetramerization due to its electrostatic characteristics. Again, the tetramer model unveils a precise fitting between the segments of neighbouring monomers affording attractive suggestions for the multifaceted mechanism of channel gating. Docking results are in convincing agreement with mutational analyses and confirm that S4 and S4-S5 linker play a key role in channel activation. Overall, the proposed model could find fertile applications to further investigate the gating mechanism and to design truly selective ligands able to clarify the pathophysiological roles of the TRPM8 channel.     

Exploring the activation mechanism of TRPM8 channel by targeted MD simulations

The TRPM8 cation channel belongs to the superfamily of transient receptor potential (TRP) channels. It is involved in non-painful cool sensation and triggered by diverse chemical and physical stimuli whose precise activation mechanism is still unknown. The study presents a set of targeted molecular dynamics (MD) simulations involving selected complexes of the TRPM8 channel whose homology model was recently generated by some of us. More in detail, the MD simulations concerned the TRPM8 alone and in complex with agonists and antagonists. These simulations were focused on voltage sensor module and designed to validate the ligand induced activation mechanism as hypothesized in our previous study. The obtained results are in encouraging agreement with the proposed mechanism and allow a clear discrimination between agonists and antagonists. In addition, the MD runs confirm that the agonist binding triggers a set of concatenate conformational shifts which induce the approaching of the S3 segment toward the S4 segment and culminate in an extension of the latter. By introducing suitable constraints, the reported MD simulations were rendered as fast as possible in order to achieve a truly productive compromise between reliability and computational costs. The obtained results emphasize that suitably targeted MD runs can be fast enough to be systematically applied to predict the bioactivity of large datasets providing it as an useful tool in rational ligand design process.     

The influence of TRPM8 ion channel activation on immune response at different temperature conditions

In experiments on rats it was shown that it is possible to modulate the immune response in a whole organism by activating cold-sensitive TRPM8 ion channel by its agonist menthol. The most pronounced changes in the conditions without external temperature stimulation were related to immune parameters for the spleen cells and immunoglobulin level in blood: the activation of TRPM8 ion channel by menthol enhances antigen binding and inhibits antibody formation in spleen, significantly reduces the level of IgG in blood. Activation of TRPM8 ion channel changes the effect of subsequent temperature exposure—cooling or heating. Preliminary application of menthol eliminates the inhibitory effect of deep cooling on immune response. Stimulation of the antigen binding in spleen at deep heating is inversed to suppression in case of heating on the background of TRPM8 activation by menthol. On the contrary, suppression of antibody formation caused by deep heating is eliminated if heating is carried out on the background of TRPM8 stimulation.     

膀胱尿路上皮癌组织TRPM7、TRPM8表达与临床病理特征和预后的关系分析

摘要 目的：探究膀胱尿路上皮癌（BUC）组织瞬时受体电位阳离子通道亚家族M成员7（TRPM7）、瞬时受体电位阳离子通道亚家族M成员8（TRPM8）表达与临床病理特征和预后的关系。方法：选取127例BUC患者并取术中癌组织及癌旁组织。比较癌组织及癌旁组织中TRPM7、TRPM8表达情况,分析癌组织中TRPM7、TRPM8表达与临床病理特征的关系。随访3年,分析不同TRPM7、TRPM8表达患者的预后及预后的影响因素。结果：癌组织TRPM7、TRPM8阳性表达率显著高于癌旁组织（P<0.05）。TRPM7、TRPM8阳性表达患者病理分期T2~T4期、淋巴结转移占比显著高于TRPM7、TRPM8阴性表达患者（P<0.05）。TRPM7、TRPM8阳性表达患者3年生存率显著低于TRPM7、TRPM8阴性表达患者（P<0.05）。淋巴结转移、TRPM7阳性表达、TRPM8阳性表达是BUC患者预后不良的独立危险因素（P<0.05）。结论：BUC组织中TRPM7和TRPM8的阳性表达与病理分期和淋巴结转移密切相关,且TRPM7、TRPM8阳性表达是BUC患者预后不良的独立危险因素。     

Stimulation of TRPC5 cationic channels by low micromolar concentrations of lead ions (Pb)

Lead toxicity is long-recognised but continues to be a major public health problem. Its effects are wide-ranging and include induction of hyper-anxiety states. In general it is thought to act by interfering with Ca²⁺ signalling but specific targets are not clearly identified. Transient receptor potential canonical 5 (TRPC5) is a Ca²⁺-permeable ion channel that is linked positively to innate fear responses and unusual amongst ion channels in being stimulated by trivalent lanthanides, which include gadolinium. Here we show investigation of the effect of lead, which is a divalent ion (Pb²⁺). Intracellular Ca²⁺ and whole-cell patch-clamp recordings were performed on HEK 293 cells conditionally over-expressing TRPC5 or other TRP channels. Extracellular application of Pb²⁺ stimulated TRPC5 at concentrations greater than 1 μM. Control cells without TRPC5 showed little or no response to Pb²⁺ and expression of other TRP channels (TRPM2 or TRPM3) revealed partial inhibition by 10 μM Pb²⁺. The stimulatory effect on TRPC5 depended on an extracellular residue (E543) near the ion pore: similar to gadolinium action, E543Q TRPC5 was resistant to Pb²⁺ but showed normal stimulation by the receptor agonist sphingosine-1-phosphate. The study shows that Pb²⁺ is a relatively potent stimulator of the TRPC5 channel, generating the hypothesis that a function of the channel is to sense metal ion poisoning.     

Ethanol inhibits cold-menthol receptor TRPM8 by modulating its interaction with membrane phosphatidylinositol 4,5-bisphosphate

Ethanol has opposite effects on two members of the transient receptor potential (TRP) family of ion channels: it inhibits the cold-menthol receptor TRPM8, whereas it potentiates the activity of the heat- and capsaicin-gated vanilloid receptor TRPV1. Both thermosensitive cation channels are critically regulated by the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)). The effects of this phospholipid on TRPM8 and TRPV1 are also functionally opposite: PIP(2) is necessary for the activation of TRPM8 but it constitutively inhibits TRPV1. This parallel led us to investigate the possible role of PIP(2) in the ethanol-induced modulation of rat TRPM8, heterologously expressed in HEK293T cells. In this study, we characterize the effects of ethanol (0.1-10%) on whole-cell currents produced by menthol and by low temperature (< 17 degrees C). We show that the inclusion of PIP(2) in the intracellular solution results in a strong reduction in the ethanol-induced inhibition of menthol-evoked responses. Conversely, intracellular dialysis with anti-PIP(2) antibody or with the PIP(2) scavenger, poly L-lysine, enhanced the ethanol-induced inhibition of TRPM8. A 20 min pre-incubation with wortmannin caused a modest decrease in inhibition produced by 1% ethanol, indicating that the ethanol-induced inhibition is not mediated by lipid kinases. These findings suggest that ethanol inhibits TRPM8 by weakening the PIP(2)-TRPM8 channel interaction; a similar mechanism may contribute to the ethanol-mediated modulation of some other PIP(2)-sensitive TRP channels.     

Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

As the function of transient receptor potential melastatin member 8 (TRPM8) in osteosarcoma is still unknown, we aim to investigate the possible effects and potential mechanisms of TRPM8 on cell proliferation, metastasis and chemosensitivity in osteosarcoma cells. We find that TRPM8 is aberrantly over-expressed in human osteosarcoma tissues and cell lines. Knockdown of TRPM8 by siRNA in osteosarcoma cells leads to the impaired regulation of intracellular Ca²⁺ concentration and then the Akt-GSK-3β pathway and the phosphorylation of p44/p42 and FAK are suppressed. Knockdown of TRPM8 not only negatively influences the cell proliferation and metastasis but also enhances epirubicin-induced cell apoptosis. Such results reveal that TRPM8 is worthy further investigation for its potential as a clinical biomarker and therapeutic target in osteosarcoma.