Expression of renal distal tubule transporters TRPM6 and NCC in a rat model of cyclosporine nephrotoxicity and effect of EGF treatment

Renal magnesium (Mg(2+)) and sodium (Na(+)) loss are well-known side effects of cyclosporine (CsA) treatment in humans, but the underlying mechanisms still remain unclear. Recently, it was shown that epidermal growth factor (EGF) stimulates Mg(2+) reabsorption in the distal convoluted tubule (DCT) via TRPM6 (Thébault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ. J Am Soc Nephrol 20: 78-85, 2009). In the DCT, the final adjustment of renal sodium excretion is regulated by the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is activated by the renin-angiotensin-aldosterone system (RAAS). The aim of this study was to gain more insight into the molecular mechanisms of CsA-induced hypomagnesemia and hyponatremia. Therefore, the renal expression of TRPM6, TRPM7, EGF, EGF receptor, claudin-16, claudin-19, and the NCC, and the effect of the RAAS on NCC expression, were analyzed in vivo in a rat model of CsA nephrotoxicity. Also, the effect of EGF administration on these parameters was studied. CsA significantly decreased the renal expression of TRPM6, TRPM7, NCC, and EGF, but not that of claudin-16 and claudin-19. Serum aldosterone was significantly lower in CsA-treated rats. In control rats treated with EGF, an increased renal expression of TRPM6 together with a decreased fractional excretion of Mg(2+) (FE Mg(2+)) was demonstrated. EGF did not show this beneficial effect on TRPM6 and FE Mg(2+) in CsA-treated rats. These data suggest that CsA treatment affects Mg(2+) homeostasis via the downregulation of TRPM6 in the DCT. Furthermore, CsA downregulates the NCC in the DCT, associated with an inactivation of the RAAS, resulting in renal sodium loss.     

Methionine Sulfoxide Reductase B1 (MsrB1) Recovers TRPM6 Channel Activity during Oxidative Stress

Mg²⁺ is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg²⁺ homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg²⁺ channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg²⁺ transport. Remarkably, TRPM6 combines a Mg²⁺-permeable channel with an α-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 α-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg²⁺-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H₂O₂) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H₂O₂ (TRPM6, 67 ± 5% of control; TRPM6 + MsrB1, 81 ± 5% of control). Cell surface biotinylation assays showed that H₂O₂ treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met¹⁷⁵⁵ to Ala in TRPM6 reduced the inhibitory effect of H₂O₂ on TRPM6 channel activity (TRPM6 M1755A: 84 ± 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met¹⁷⁵⁵ and could, thereby, function as a modulator of TRPM6 during oxidative stress.     

Upregulation of transient receptor potential melastatin 6 channel expression by rosiglitazone and all-trans-retinoic acid in erlotinib-treated renal tubular epithelial cells

Anti-epidermal growth factor receptor (EGFR) drugs including erlotinib cause a side effect of hypomagnesemia. In lung adenocarcinoma A549 cells, anticancer agents such as cisplatin and doxorubicin dose-dependently increased toxicity, but the effects were significantly suppressed by culturing the cells in low Mg²⁺-containing media. To obtain the maximum effect in cancer chemotherapy, it should be necessary to prevent the reduction of body Mg ²⁺ content. Anti-EGFR drugs inhibit EGF-induced elevation of transient receptor potential melastatin 6 (TRPM6) Mg ²⁺ channel in renal tubular epithelial NRK-52E cells. Here, we found that rosiglitazone, an antidiabetic drug, and all- trans-retinoic acid (ATRA), a vitamin A derivative, increase the messenger RNA (mRNA) level of TRPM6 in the presence of erlotinib. The rosiglitazone- and ATRA-induced elevation of mRNA level, Mg ²⁺ influx, and promoter activity of TRPM6 were inhibited by GW-9662, a potent antagonist of peroxisome proliferator-activated receptor (PPAR)γ, and LE135, a retinoic acid receptor (RAR) antagonist, respectively. Rosiglitazone increased the phosphorylation and nuclear localization levels of PPARγ, which were inhibited by GW-9662. In contrast, RAR was mainly distributed in the nuclei under control conditions, which was unchanged by ATRA and LE135. The promoter activity of TRPM6 was inhibited by a mutation in the peroxisome proliferator hormone response element (PPRE). A chromatin immunoprecipitation assay revealed that PPARγ and RAR bind to the PPRE, which was blocked by GW-9662 and LE135, respectively. These results suggest that rosiglitazone and ATRA reverse the reduction in Mg ²⁺ reabsorption caused by anti-EGFR drugs.     

Differential mRNA Expression and Glucocorticoid-Mediated Regulation of TRPM6 and TRPM7 in the Heart and Kidney throughout Murine Pregnancy and Development

The transient receptor potential (TRP) channels TRPM6 and TRPM7 are critically involved in maintaining whole body and cellular Mg²⁺ homeostasis and ensuring the normal function of organs such as the heart and kidney. However, we do not know how the expression of TRPM6 and TPRM7 in these organs changes throughout fetal development and adult life, and whether this expression can be hormonally regulated. This study determined the ontogeny of TRPM6 and TRPM7 mRNA expression from mid-gestation through to adulthood in the mouse. In a second series of experiments, we examined how maternal administration of the glucocorticoids corticosterone and dexamethasone between embryonic days 12.5–15 affected TRPM6 and TRPM7 channel mRNA expression in the mother and fetus. Whilst renal TRPM7 expression was relatively constant throughout development, renal TRPM6 expression was markedly upregulated after birth. In contrast, cardiac TRPM7 expression was 2–4 fold higher in the fetus than in the adult. Surprisingly, TRPM6 expression was detected in the fetal heart (qPCR and in situ hybridization). Glucocorticoid administration during gestation increased fetal cardiac expression of both channels without affecting renal expression. In contrast, in the dam renal TRPM6 and TRPM7 expression was increased by glucocorticoids with no change in the cardiac channel expression. These data suggest that TRPM6 and TRPM7 channels are important in organogenesis, and that elevated maternal glucocorticoid levels can alter the expression of these channels. This suggests that perturbations in hormonal regulatory systems during pregnancy may adversely impact upon normal fetal development, at least in part by altering expression of TRPM channels.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

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.     

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Modulation of TRPM6 and Na+/Mg2+ exchange in mammary epithelial cells in response to variations of magnesium availability

Mammary epithelial cells (HC11) chronically adapted to grow in a low‐magnesium (0.05 mM vs. 0.5 mM) or in a high‐magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non‐physiological magnesium availability. Magnesium influx was higher in low‐magnesium cells compared to control or high‐magnesium cells, whereas magnesium efflux was higher in high‐magnesium cells compared to control and low‐magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na⁺/Mg²⁺ exchange. Using a monoclonal antibody detecting a ～70 kDa protein associated with Na⁺/Mg²⁺ exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high‐magnesium cells > control cells > low‐magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low‐magnesium cells re‐added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na⁺/Mg²⁺ exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney. J. Cell. Physiol. 222: 374–381, 2010. © 2009 Wiley‐Liss, Inc.     

Extracellular Mg regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation

Claudin-16 is involved in the paracellular reabsorption of Mg²⁺ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg²⁺ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg²⁺ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg²⁺ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg²⁺ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg²⁺ deprivation, and recovered by re-addition of Mg²⁺. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg²⁺ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg²⁺ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg²⁺ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg²⁺ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.     

An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells

Magnesium (Mg²⁺) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate the effects of Mg²⁺ enrichment, and its influence on the immunomodulatory capacity of MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different concentrations of Mg²⁺ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg²⁺ on MSC immunomodulatory properties, cell proliferation rates, expression of NFκB and STAT-3, production of IL-1β, IL-6, TGF-β, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg²⁺, in the way that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg²⁺. Mg²⁺, especially at 5 mM, raised proliferation rates of MSCs, and modulated immune responses by decreasing levels of IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-α. In addition, MSCs cultured in 5 mM Mg²⁺ expressed lower levels of pNFκB/NFκB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg²⁺ did not affect this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg²⁺ modulated the production profile of cytokines, especially of IL-1β and IL-6 in macrophages. In conclusion, Mg²⁺ is able to modulate some immunoregulatory properties of MSCs.     

Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity

Magnesium (Mg²⁺) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg²⁺ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg²⁺ (re)absorption in kidney and intestine. The apical entry of Mg²⁺ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.     

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.     

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.     

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters

Magnesium ions (Mg²⁺) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg²⁺ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg²⁺ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg²⁺ channels and transporters. In this review article, we provide an overview of the families of Mg²⁺ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.     

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.     

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.