TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including Mg2+ homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM6S141L produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, Mg2+ and Ca2+ entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo.

m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.TRPM7 is one of only two ion channels to possess its own kinase domain (1). It is a member of the transient receptor potential ion channel family with the closest similarity to its bifunctional homologue TRPM6 as well as to melastatin (TRPM1), whose reduced expression has been used as a prognosis marker for metastasis in patients with localized melanoma (2-6). TRPM7 is also distinctive in its ion permeability, allowing Ca 2ϩ as well as Mg 2ϩ and other cations to compose its inward current (7,8). The channel kinase is a member of the recently discovered ␣-kinase family (9, 10). Annexin I has been identified as a substrate for the kinase, but the functional significance of annexin I phosphorylation by TRPM7 is not yet understood (11). Autophosphorylation of the channel does not alter channel activity (12). However, phospholipase C inactivates TRPM7 channel activity through hydrolysis of phosphatidylinositol 4,5-bisphosphate, which is presumably gating the channel (13,14). Magnesium ions block channel activity (8,(15)(16)(17), and, more recently, TRPM7 current has been shown to be potentiated by protons (18). Despite these recent advances in understanding TRPM7 channel regulation, the physiological role of this unique bifunctional protein still remains unclear.The passage of Mg 2ϩ by TRPM7 has linked it to the regulation of magnesium homeostasis in mammalian cells (19). Its capacity to carry calcium, in contrast, has been associated with calcium overload during anoxic cell death (20), calcium-dependent regulation of the cell cycle (21), and most recently, skeletogenesis and kidney stone formation in zebrafish (22). An early study by Nadler et al. (8) showed that overexpression of TRPM7 caused HEK-293 cells to detach and die, suggesting that the channel may have a role in controlling cell adhesion.Here we present evidence that TRPM7 is a potent regulator of m-calpain. Fourteen distinct members of the mammalian calpain famil...

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from −100 to −40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an ∼10-fold increase at pH 4.0 and 1–2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca2+ and Mg2+ for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca2+ and Mg2+ concentrations were increased. Third, the apparent affinity for Ca2+ and Mg2+ was significantly diminished at elevated external H+ concentrations. Fourth, the anomalous-mole fraction behavior of H+ permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca2+ and Mg2+ bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H+ concentrations, the affinity of TRPM7 for Ca2+ and Mg2+ is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg2+-inhibitable cation current (MIC) or Mg nucleotide–regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg 2؉ and Ca 2؉ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg 2؉ and Ca 2؉ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8-and 12-fold larger than TRPM7, respectively. permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca 2؉ and Mg 2؉ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.TRPM6 and TRPM7 belong to the TRP channel superfamily (1-5) and are distinguished from other known ion channels by virtue of having both ion channel and protein kinase activities (6 -11). In addition, TRPM6 and TRPM7 uniquely exhibit strong outward rectification, permeation to Ca 2ϩ , Mg 2ϩ , monovalent cations, and a wide array of trace metals (6 -8, 11, 12 (8,20,21,23), whereas TRPM7 is ubiquitously expressed, with highest expression in the kidney and heart (5, 6). In addition to these channels' regulation of Mg 2ϩ homeostasis, several studies have suggested multiple cellular and physiology functions for TRPM7, including anoxic neuronal death (24), cell adhesion and actomyosin contractility (25, 26), and skeletogenesis (27). Although the mechanisms by which TRPM6 and TRPM7 exert their physiological and/or pathological functions are not yet completely understood, it is clear that permeation of Ca 2ϩ and Mg 2ϩ contributes substantially to the known functions of these channels (7, 20 -22, 24, 25, 27). Moreover, a recent study demonstrated that the sensitivity of TRPM7 to external pH may contribute to controlling neurotransmitter release (28). Therefore, it is essential to understand the molecular mechanisms underlying the Ca 2ϩ and Mg 2ϩ permeability of TRPM6 and TRPM7, as well as their sensitivities to changes in pH.The aim of the present study was to identify the amino acid residues that determine Mg 2ϩ and Ca 2ϩ permeation of TRPM6 and TRPM7. We previously demonstrated that external protons significantly enhance TRPM6 and TRPM7 inward currents (11,19) by decreasing the divalent affinity to t...

BackgroundGestational diabetes mellitus (GDM) is a disease often manifests in mid to late pregnancy with symptoms including hyperglycemia, insulin resistance and fetal mal-development. The C57BL/KsJ-Lepdb/+ (db/+) mouse is a genetic GDM model that closely mimicked human GDM symptoms. Resveratrol (RV) is a naturally existing compound that has been reported to exhibit beneficial effects in treating type-2 diabetes.MethodsIn this study, we investigated the effect of RV on the pregnant db/+ GDM mouse model, and the underlying molecular mechanism.ResultsRV greatly improved glucose metabolism, insulin tolerance and reproductive outcome of the pregnant db/+ females. Moreover, we found that RV relieved GDM symptoms through enhancing AMPK activation, which in turn reduced production and activity of glucose-6-phosphatase in both pregnant db/+ females and their offspring.ConclusionsOur findings further supported the potential therapeutic effect of RV on not only diabetes, but also alleviating GDM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0114-0) contains supplementary material, which is available to authorized users.

In this study, we identified a novel circRNA, circ_0002483, and further investigated its functions in the progression and Taxol resistance of NSCLC. We found that circ_0002483 was expressed at low levels in NSCLC tissues and cell lines. Functional assays indicated that circ_0002483 overexpression significantly inhibited NSCLC cell proliferation and invasion in vitro and in vivo and enhanced the sensitivity of NSCLC cells to Taxol. Mechanistically, circ_0002483 was identified to sponge multiple miRNAs including miR-182-5p (also named miR-182), miR-520q-3p, miR-582-3p, miR-587, and miR-655. In addition, circ_0002483 was also demonstrated to regulate the expression of GRB2, FOXO1, and FOXO3, three target genes of miR-182-5p, by sponging miR-182-5p. Circ_0002483 was demonstrated to inhibit NSCLC progression in vitro and in vivo and enhanced the sensitivity of NSCLC cells to Taxol by sponging miR-182-5p to release the inhibition on GRB2, FOXO1, and FOXO3 mRNAs.

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The potential role of metformin in treating endometrial cancer remains to be explored. The current study investigated the role of metformin in 17β-estradiol-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells. We found that 17β-estradiol promoted proliferation and migration, attenuated apoptosis in both estrogen receptor (ER) positive and ER negative endometrial adenocarcinoma cells (Ishikawa and KLE cells, respectively). Metformin abolished 17β-estradiol-induced cell proliferation and reversed 17β-estradiol-induced EMT in Ishikawa cells. In addition, metformin increased the expression of βKlotho, a fibroblast growth factors (FGFs) coreceptor, and decreased ERK1/2 phosphorylation in both Ishikawa and KLE cells. Decreased expression of βKlotho was noted in human endometrial adenocarcinomas, and plasmid-driven expression of βKlotho in Ishikawa cells abolished 17β-estradiol-induced EMT via inhibiting ERK1/2 signaling. βKlotho expression and metformin show synergetic effects on the proliferation and the EMT in Ishikawa cells. Furthermore, we demonstrated that the anti-EMT effects of metformin could be partly abolished by introducing Compound C, a specific AMPKα signaling inhibitor. In conclusion, metformin abolishes 17β-estradiol-induced cell proliferation and EMT in endometrial adenocarcinoma cells by upregulating βKlotho expression, inhibiting ERK1/2 signaling, and activating AMPKα signaling. Our study provides novel mechanistic insight into the anti-tumor effects of metformin.