Cleavage of TRPM7 releases the kinase domain from the ion channel and regulates its participation in Fas-induced apoptosis

Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.     

Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1

TRPM7/ChaK1 is a unique channel/kinase that contains a TRPM channel domain with 6 transmembrane segments fused to a novel serine-threonine kinase domain at its C terminus. The goal of this study was to investigate a possible role of kinase activity and autophosphorylation in regulation of channel activity of TRPM7/ChaK1. Residues essential for kinase activity were identified by site-directed mutagenesis. Two major sites of autophosphorylation were identified in vitro by mass spectrometry at Ser(1511) and Ser(1567), and these sites were found to be phosphorylated in intact cells. TRPM7/ChaK1 is a cation-selective channel that exhibits strong outward rectification and inhibition by millimolar levels of internal [Mg(2+)]. Mutation of the two autophosphorylation sites or of a key catalytic site that abolished kinase activity did not alter channel activity measured by whole-cell recording or Ca(2+) influx. Inhibition by internal Mg(2+) was also unaffected in the autophosphorylation site or "kinase-dead" mutants. Moreover, kinase activity was enhanced by Mg(2+), was decreased by Zn(2+), and was unaffected by Ca(2+). In contrast, channel activity was inhibited by all three of these divalent cations. However, deletion of much of C-terminal kinase domain resulted in expression of an apparently inactive channel. We conclude that neither current activity nor regulation by internal Mg(2+) is affected by kinase activity or autophosphorylation but that the kinase domain may play a structural role in channel assembly or subcellular localization.     

The TRPM7 channel is inactivated by PIP(2) hydrolysis

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel.     

TRPM7 channel is regulated by magnesium nucleotides via its kinase domain

TRPM7 is a Ca(2+)- and Mg(2+)-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg(2+), the functional roles of intracellular levels of Mg.ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg.ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg(2+). Mg.ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg(2+) and Ca(2+) overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP > or = GTP > or = UTP > ITP approximately free Mg(2+) alone. These nucleotides also enhanced TRPM7 inhibition by free Mg(2+), suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg(2+)-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg.NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg(2+) binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg(2+) binding site extrinsic to that domain.     

Identification of TG100-115 as a new and potent TRPM7 kinase inhibitor,which suppresses breast cancer cell migration and invasion

BackgroundTransient receptor potential melastatin 7 (TRPM7) regulates breast cancer cell proliferation, migration, invasion and metastasis in its ion channel- and kinase domain-dependent manner. The pharmacological effects of TRPM7 ion channel inhibitors on breast cancer cells have been studied, but little is known about the effects of TRPM7 kinase domain inhibitors due to lack of potent TRPM7 kinase inhibitors.MethodsScreening was performed by using TRPM7 kinase assay. Effects of TG100-115 on breast cancer cell proliferation, migration, invasion, myosin IIA phosphorylation, and TRPM7 ion channel activity were assessed by using MTT, wound healing, transwell assay, Western blotting, and patch clamping, respectively.ResultsWe found that CREB peptide is a potent substrate for the TR-FRET based TRPM7 kinase assay. Using this method, we discovered a new and potent TRPM7 kinase inhibitor, TG100-115. TG100-115 inhibited TRPM7 kinase activity in an ATP competitive fashion with over 70-fold stronger activity than that of rottlerin, known as a TRPM7 kinase inhibitor. TG100-115 has little effect on proliferation of MDA-MB-231 cells, but significantly decreases cell migration and invasion. Moreover, TG100-115 inhibits TRPM7 kinase regulated phosphorylation of the myosin IIA heavy chain and phosphorylation of focal adhesion kinase. TG100-115 also suppressed TRPM7 ion channel activity.ConclusionsTG100-115 can be used as a potent TRPM7 kinase inhibitor and a potent inhibitor of breast cancer cell migration.General significanceTG100-115 could be a useful tool for studying the pharmacological effects of TRPM7 kinase activity aimed at providing insight into new therapeutic approaches to the treatment of breast cancer.     

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

The activity of the TRPM7 channel is negatively regulated by intracellular Mg²⁺. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg²⁺. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H₂O₂). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H₂O₂-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg²⁺ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg²⁺ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg²⁺-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.     

Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel

Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called alpha-kinases that display no sequence homology to conventional protein kinases. It is an unusual bifunctional protein that contains an alpha-kinase domain fused to an ion channel. The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis. However, little is known about its protein kinase activity. To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria. ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues. The kinase is specific for ATP and cannot use GTP as a substrate. ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin. Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity. The kinase requires Mg(2+) (optimum at 4-10 mM) or Mn(2+) (optimum at 3-5 mM), with activity in the presence of Mn(2+) being 2 orders of magnitude higher than in the presence of Mg(2+). Zn(2+) and Co(2+) inhibited ChaK1-cat kinase activity. Ca(2+) at concentrations up to 1 mM did not affect kinase activity. Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg(2+) can directly modulate TRPM7/ChaK1 kinase activity in vivo.     

Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7's kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.     

Phosphorylation of annexin I by TRPM7 channel-kinase

TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death. TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs at a conserved serine residue (Ser5) located within the N-terminal amphipathic alpha-helix of annexin 1. The N-terminal region plays a crucial role in interaction of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.     

Identification of the phosphorylation sites on intact TRPM7 channels from mammalian cells

Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca(2+) and Mg(2+) in mammalian cells. Although studies have shown that the kinase domain of TRPM7 is required for channel activation and can phosphorylate other target proteins, a systematic analysis of intact TRPM7 channel phosphorylation sites expressed in mammalian cells is lacking. We applied mass spectrometric proteomic techniques to identify and characterize the key phosphorylation sites in TRPM7 channels. We identified 14 phosphorylation sites in the cytoplasmic domain of TRPM7, eight of which have not been previously reported. The identification of phosphorylation sites using antibody-based immunopurification and mass spectrometry is an effective approach for defining the phosphorylation status of TRPM7 channels. The present results show that TRPM7 channels are phosphorylated at multiple sites, which serves as a mechanism to modulate the dynamic functions of TRPM7 channels in mammalian cells.     

Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7

TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical alpha-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32+/-4 mol/mol), which strongly increases the rate of substrate phosphorylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.     

The TRPM6 Kinase Domain Determines the Mg·ATP Sensitivity of TRPM7/M6 Heteromeric Ion Channels

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg²⁺) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg²⁺ and therefore unlikely to be active at physiological levels of [Mg²⁺]_i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.     

Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein''s expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE''s downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel capable of attenuating TRPM7''s function during cell stress, making them effective tools for the biophysical characterization and suppression of TRPM7 channel conductance in vivo.     

TRPM7 reflected in Cryo-EMirror

TRPM7 is an atypical type of ion channel because its pore-forming moiety is covalently linked to a protein kinase domain. The channel-kinase TRPM7 controls a wide range of biological processes such as mineral homeostasis, immune responses, cell motility, proliferation and differentiation. Earlier this year, Duan J & co-workers [1] published three TRPM7 structures resolved by cryo-electron microscopy (cryo-EM). This study tremendously advances our mechanistic understanding of TRPM7 channel function and forms the basis for informed structure-function assessment of this extraordinary protein.     

The role of transient receptor potential channel blockers in human gastric cancer cell viability

Transient receptor potential cation channel, subfamily M, receptor 7 (TRPM7) is a ubiquitous divalent-selective ion channel with its own kinase domain. Human gastric cancer cells express the TRPM7 channel, and the presence of this channel is essential for cell survival. Recent studies have suggested that 5-lipoxygenase (5-LOX) inhibitors are potent blockers of the TRPM7 channels. The aim of this study was to show the effects of 5-LOX inhibitors on the growth and survival of gastric cancer cells. Among 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886) were potent blockers of TRPM7-like currents in gastric cancer cells and also induced cell death. However, zileuton was ineffective in suppressing TRPM7-like current activity and inducing cell death. Moreover, a specific transient receptor potential cation channel, subfamily C, member 3 (TRPC3) inhibitor, a pyrazole compound (Pyr3), and a specific melastatin TRP (TRPM4) inhibitor, 9-phenanthrol, did not affect TRPM7-like currents or induce cell death. We conclude that TRPM7 has an important role in the growth and survival of gastric cancer cells and a likely potential target for the pharmacological treatment of gastric cancer.     

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.     

Role of the alpha-kinase domain in transient receptor potential melastatin 6 channel and regulation by intracellular ATP

Transient receptor potential melastatin 6 (TRPM6) plays an essential role in epithelial Mg(2+) transport. TRPM6 and its closest homologue, TRPM7, both combine a cation channel with an alpha-kinase domain. However, the role of this alpha-kinase domain in TRPM6 channel activity remains elusive. The aim of this study was to investigate the regulation of TRPM6 channel activity by intracellular ATP and the involvement of its alpha-kinase domain. We demonstrated that intracellular Na- and Mg-ATP decreased the TRPM6 current in HEK293 cells heterogeneously expressing the channel, whereas Na-CTP or Na-GTP had no effect on channel activity. Whole cell recordings in TRPM6-expressing HEK293 cells showed that deletion of the alpha-kinase domain prevented the inhibitory effect of intracellular ATP without abrogating channel activity. Mutation of the conserved putative ATP-binding motif GXG(A)XXG (G1955D) in the alpha-kinase domain of TRPM6 inhibited the ATP action, whereas this effect remained preserved in the TRPM6 phosphotransferase-deficient mutant K1804R. Mutation of the TRPM6 autophosphorylation site, Thr(1851), into either an alanine or an aspartate, resulted in functional channels that could still be inhibited by ATP. In conclusion, intracellular ATP regulates TRPM6 channel activity via its alpha-kinase domain independently of alpha-kinase activity.     

Waixenicin A inhibits cell proliferation through magnesium-dependent block of transient receptor potential melastatin 7 (TRPM7) channels

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).     

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.     

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain

We demonstrated a role for the Mg^2 + transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg^2 + and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg^2 +]_i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg^2 + responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg^2 + influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg^2 +-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.