Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg2+, protons, and polyamines. Currents through these channels (ITRPM7) are robustly potentiated when the cell interior is exchanged with low Mg2+-containing buffers. ITRPM7 is also potentiated by phosphatidyl inositol bisphosphate (PI(4,5)P2) and suppressed by its hydrolysis. Here we characterized internal Mg2+- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg2+ and pH. Proton concentrations that were too low to inhibit ITRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP–expressing cells. At pH 6.5, protons inhibited ITRPM7 both in WT and VSP C363S–expressing cells but with a faster time course in the WT VSP–expressing cells. Inhibition by 150 μm Mg2+ was also significantly faster in the WT VSP–expressing cells. Cellular PI(4,5)P2 depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg2+, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PI(4,5)P2 depletion than the WT. We conclude that the internal Mg2+-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PI(4,5)P2–channel interactions.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg 2+ and divalent metal cations such as Ca 2+ , Zn 2+ , Cd 2+ , Mn 2+ and Ni 2+ . TRPM7 channels are inhibited by cytosolic Mg 2+ , rendering them largely inactive in intact cells. Dependence of channel activity on extracellular Mg 2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg 2+ concentrations varying between 400 nM and 1.4 mM for 1-2 days, obtaining an IC 50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg 2+ ] o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg 2+ depletion. 1.4 mM [Mg 2+ ] o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg 2+ depletion, but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni 2+ , Zn 2+ , Cd 2+ , Co 2+ , Mn 2+ , Sr 2+ and Ba 2+ requires TRPM7 channel activity. Mg 2+ loading modestly reduced cytotoxicity of Zn 2+ , Co 2+ , Ni 2+ and Mn 2+ but not of Cd 2+ . Channel blocker NS8593 reduced Co 2+ and Mn 2+ but not Cd 2+ or Zn 2+ cytotoxicity and interfered with Mg 2+ loading as evaluated by TRPM7 channel basal activity. Ba 2+ and Sr 2+ were neither detectably toxic, nor permeant through the plasma membrane. These results indicate that in Jurkat T cells entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg 2+ entry requires TRPM7 channels.

Summary Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4-substituted analogs were not as potent. However, to date the structure activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel “caged” 4- and 5- substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.

Dermal fibroblasts provide structural support by producing collagen and other structural/support proteins beneath the epidermis. Fibroblasts also produce insulin-like growth factor-1 (IGF-1), which binds to the IGF-1 receptors (IGF-1Rs) on keratinocytes to activate signaling pathways that regulate cell proliferation and cellular responses to genotoxic stressors like ultraviolet B radiation. Our group has determined that the lack of IGF-1 expression due to fibroblast senescence in the dermis of geriatric individuals is correlated with an increased incidence of skin cancer. The present studies tested the hypothesis that pro-energetics creatine monohydrate (Cr) and nicotinamide (NAM) can protect normal dermal human fibroblasts (DHF) against experimentally induced senescence. To that end, we used an experimental model of senescence in which primary DHF are treated with hydrogen peroxide (H2O2) in vitro, with senescence measured by staining for beta-galactosidase activity, p21 protein expression, and senescence associated secretory phenotype cytokine mRNA levels. We also determined the effect of H2O2 on IGF-1 mRNA and protein expression. Our studies indicate that pretreatment with Cr or NAM protects DHF from the H2O2-induced cell senescence. Treatment with pro-energetics post-H2O2 had no effect. Moreover, these agents also inhibited reactive oxygen species generation from H2O2 treatment. These studies suggest a potential strategy for protecting fibroblasts in geriatric skin from undergoing stress-induced senescence, which may maintain IGF-1 levels and therefore limit carcinogenesis in epidermal keratinocytes.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg 2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca 2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg 2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.