Assessment of TRPM7 functions by drug-like small molecules

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a plasma membrane ion channel linked to a cytosolic protein kinase domain. Genetic inactivation of this bi-functional protein revealed its crucial role in Ca²⁺ signalling, Mg²⁺ metabolism, immune responses, cell motility, proliferation and differentiation. Malfunctions of TRPM7 are associated with anoxic neuronal death, cardiac fibrosis, tumour progression and macrothrombocytopenia. Recently, several groups have identified small organic compounds acting as inhibitors or activators of the TRPM7 channel. In follow-up studies, the identified TRPM7 modulators were successfully used to uncover new cellular functions of TRPM7 in situ including a crucial role of TRPM7 in Ca²⁺ signaling and Ca²⁺ dependent cellular processes. Hence, TRPM7 has been defined as a promising drug target. Here, we summarize the progress in this quickly developing field.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

EGF enhances the migration of cancer cells by up-regulation of TRPM7

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca²⁺ from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200 μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.     

TRPM7 and its role in neurodegenerative diseases

Calcium (Ca²⁺) and magnesium (Mg²⁺) ions have been shown to play an important role in regulating various neuronal functions. In the present review we focus on the emerging role of transient potential melastatin-7 (TRPM7) channel in not only regulating Ca²⁺ and Mg²⁺ homeostasis necessary for biological functions, but also how alterations in TRPM7 function/expression could induce neurodegeneration. Although eight TRPM channels have been identified, the channel properties, mode of activation, and physiological responses of various TRPM channels are quite distinct. Among the known 8 TRPM channels only TRPM6 and TRPM7 channels are highly permeable to both Ca²⁺ and Mg²⁺; however here we will only focus on TRPM7 as unlike TRPM6, TRPM7 channels are abundantly expressed in neuronal cells. Importantly, the discrepancy in TRPM7 channel function and expression leads to various neuronal diseases such as Alzheimer disease (AD) and Parkinson disease (PD). Further, it is emerging as a key factor in anoxic neuronal death and in other neurodegenerative disorders. Thus, by understanding the precise involvement of the TRPM7 channels in different neurodegenerative diseases and by understanding the factors that regulate TRPM7 channels, we could uncover new strategies in the future that could evolve as new drug therapeutic targets for effective treatment of these neurodegenerative diseases.     

TRPM7 and its role in neurodegenerative diseases

Calcium (Ca²⁺) and magnesium (Mg²⁺) ions have been shown to play an important role in regulating various neuronal functions. In the present review we focus on the emerging role of transient potential melastatin-7 (TRPM7) channel in not only regulating Ca²⁺ and Mg²⁺ homeostasis necessary for biological functions, but also how alterations in TRPM7 function/expression could induce neurodegeneration. Although eight TRPM channels have been identified, the channel properties, mode of activation, and physiological responses of various TRPM channels are quite distinct. Among the known 8 TRPM channels only TRPM6 and TRPM7 channels are highly permeable to both Ca²⁺ and Mg²⁺; however here we will only focus on TRPM7 as unlike TRPM6, TRPM7 channels are abundantly expressed in neuronal cells. Importantly, the discrepancy in TRPM7 channel function and expression leads to various neuronal diseases such as Alzheimer disease (AD) and Parkinson disease (PD). Further, it is emerging as a key factor in anoxic neuronal death and in other neurodegenerative disorders. Thus, by understanding the precise involvement of the TRPM7 channels in different neurodegenerative diseases and by understanding the factors that regulate TRPM7 channels, we could uncover new strategies in the future that could evolve as new drug therapeutic targets for effective treatment of these neurodegenerative diseases.     

The ion channel TRPM7 regulates zinc-depletion-induced MDMX degradation

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn²⁺ concentration to promote tumorigenesis.     

A structural overview of the ion channels of the TRPM family

The TRPM (transient receptor potential melastatin) family belongs to the superfamily of TRP cation channels. The TRPM subfamily is composed of eight members that are involved in diverse biological functions such as temperature sensing, inflammation, insulin secretion, and redox sensing. Since the first cloning of TRPM1 in 1998, tremendous progress has been made uncovering the function, structure, and pharmacology of this family. Complete structures of TRPM2, TRPM4, and TRPM8, as well as a partial structure of TRPM7, have been determined by cryo-EM, providing insights into their channel assembly, ion permeation, gating mechanisms, and structural pharmacology. Here we summarize the current knowledge about channel structure, emphasizing general features and principles of the structure of TRPM channels discovered since 2017. We also discuss some of the key unresolved issues in the field, including the molecular mechanisms underlying voltage and temperature dependence, as well as the functions of the TRPM channels’ C-terminal domains.     

TRPM7 is involved in angiotensin II induced cardiac fibrosis development by mediating calcium and magnesium influx

Cardiac fibrosis is involved in a lot of cardiovascular pathological processes. Cardiac fibrosis can block conduction, cause hypoxia, strengthen myocardial stiffness, create electrical heterogeneity, and hamper systolic ejection, which is associated with the development of arrhythmia, heart failure and sudden cardiac death. Besides the initial stimulating factors, the cardiac fibroblasts (CFs) are the principal responsible cells in the fibrogenesis cascade of events. TRPM7, a member of the TRPM (Melastatin) subfamily, is a non-selective cation channel, which permeates both Ca²⁺ and Mg²⁺. Here we demonstrated TRPM7 expression in CFs, and 2-APB (TRPM7 inhibitor), inhibited Ang II-induced CTGF, α-SMA expression and CFs proliferation. Besides, knocking down TRPM7 by shRNA, we proved that TRPM7 mediated both calcium and magnesium changes in cardiac fibroblasts which contribute to fibrosis progress. This study suggested that TRPM7 should play a pivotal role in cardiac fibroblast functions associated to cardiac fibrosis development.     

Endotoxin-induced vascular endothelial cell migration is dependent on TLR4/NF-κB pathway,NAD(P)H oxidase activation,and transient receptor potential melastatin 7 calcium channel activity

Endothelial dysfunction is decisive and leads to the development of several inflammatory diseases. Endotoxemia-derived sepsis syndrome exhibits a broad inflammation-induced endothelial dysfunction. We reported previously that the endotoxin, lipopolysaccharide (LPS), induces the conversion of endothelial cells (ECs) into activated fibroblasts, showing a myofibroblast-like protein expression profile. Enhanced migration is a hallmark of myofibroblast function. However, the mechanism involved in LPS-induced EC migration is no totally understood. Some studies have shown that the transient receptor potential melastatin 7 (TRPM7) ion channel is involved in fibroblast and tumor cell migration through the regulation of calcium influx. Furthermore, LPS modulates TRPM7 expression. However, whether TRPM7 is involved in LPS-induced EC migration remains unknown.Here, we study the participation of LPS as an inducer of EC migration and study the mechanism underlying evaluating the participation of the TRPM7 ion channel.Our results demonstrate that LPS induced EC migration in a dose-dependent manner. Furthermore, this migratory process was mediated by the TLR-4/NF-κB pathway and the generation of ROS through the PKC-activated NAD(P)H oxidase. In addition, LPS increased the intracellular calcium level and the number of focal adhesion kinase (FAK)-positive focal adhesions in EC. Finally, we demonstrate that using TRPM7 blockers or suppressing TRPM7 expression through siRNA successfully inhibits the calcium influx and the LPS-induced EC migration.These results point out TRPM7 as a new target in the drug design for several inflammatory diseases that impair vascular endothelium function.     

TRPM7 regulates gastrulation during vertebrate embryogenesis

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca²⁺- and Mg²⁺-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg²⁺ supplementation or by expression of the Mg²⁺ transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg²⁺ in Rac-dependent polarized cell movements during vertebrate gastrulation.     

Inhibition of TRPM7 with waixenicin A reduces glioblastoma cellular functions

Glioblastoma (GBM) is the most common malignant primary brain tumour originating in the CNS. Median patient survival is <15 months with standard treatment which consists of surgery alongside radiation therapy and temozolomide chemotherapy. However, because of the aggressive nature of GBM, and the significant toxicity of these adjuvant therapies, long-term therapeutic effects are unsatisfactory. Thus, there is urgency to identify new drug targets for GBM. Recent evidence shows that the transient receptor potential melastatin 7 (TRPM7) cation channel is aberrantly upregulated in GBM and its inhibition leads to reduction of GBM cellular functions. This suggests that TRPM7 may be a potential drug target for GBM treatment. In this study, we assessed the effects of the specific TRPM7 antagonist waixenicin A on human GBM cell lines U87 or U251 both in vitro and in vivo. First, we demonstrated in vitro that application of waixenicin A reduced TRPM7 protein expression and inhibited the TRPM7-like currents in GBM cells. We also observed reduction of GBM cell viability, migration, and invasion. Using an intracranial xenograft GBM mouse model, we showed that with treatment of waixenicin A, there was increased cleaved caspase 3 activity, alongside reduction in Ki-67, cofilin, and Akt activity in vivo. Together, these data demonstrate higher GBM cell apoptosis, and lower proliferation, migration, invasion and survivability following treatment with waixenicin A.     

Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.     

Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca²⁺- and Mg²⁺-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca²⁺ overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H₂O₂) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H₂O₂ to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H₂O₂ under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H₂O₂ and NMM were dependent on free [Mg²⁺]_i; the inhibition was stronger when cells were perfused with higher free [Mg²⁺]_i solutions via pipette. In addition, TRPM7 current was not inhibited by H₂O₂ when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg²⁺]_i, which is sufficient for TRPM7 inhibition by H₂O₂ in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H₂O₂ just like the wild-type TRPM7 in a [Mg²⁺]_i- and [ATP]_i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg²⁺]_i elevation.     

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg²⁺ (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg²⁺ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg²⁺ showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression.     

The channel-kinase TRPM7 regulates phosphorylation of the translational factor eEF2 via eEF2-k

Protein translation is an essential but energetically expensive process, which is carefully regulated in accordance to the cellular nutritional and energy status. Eukaryotic elongation factor 2 (eEF2) is a central regulation point since it mediates ribosomal translocation and can be inhibited by phosphorylation at Thr56. TRPM7 is the unique fusion of an ion channel with a functional Ser/Thr-kinase. While TRPM7's channel function has been implicated in regulating vertebrate Mg²⁺ uptake required for cell growth, the function of its kinase domain remains unclear. Here, we show that under conditions where cell growth is limited by Mg²⁺ availability, TRPM7 via its kinase mediates enhanced Thr56 phosphorylation of eEF2. TRPM7-kinase does not appear to directly phosphorylate eEF2, but rather to influence the amount of eEF2's cognate kinase eEF2-k, involving its phosphorylation at Ser77. These findings suggest that TRPM7's structural duality ensures ideal positioning of its kinase in close proximity to channel-mediated Mg²⁺ uptake, allowing for the adjustment of protein translational rates to the availability of Mg²⁺.     

Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.     

The TRPM8 Protein Is a Testosterone Receptor: I. BIOCHEMICAL EVIDENCE FOR DIRECT TRPM8-TESTOSTERONE INTERACTIONS*

The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca²⁺ responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.     

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、...