Cytoplasmic Mg2 Research Articles

Increased cytoplasmic Mg2+ level contributes to rice salicylic acid accumulation and broad-spectrum resistance.

Elevating Mg2+ levels in rice cytoplasm by genetic modification or exogenous supply increases salicylic acid accumulation and enhances broad-spectrum resistance to pathogens.

Chaperone Hsp70 helps Salmonella survive infection-relevant stress by reducing protein synthesis.

In all domains of life, Hsp70 chaperones preserve protein homeostasis by promoting protein folding and degradation and preventing protein aggregation. We now report that the Hsp70 from the bacterial pathogen Salmonella enterica serovar Typhimurium-termed DnaK-independently reduces protein synthesis in vitro and in S. Typhimurium facing cytoplasmic Mg2+ starvation, a condition encountered during infection. This reduction reflects a 3-fold increase in ribosome association with DnaK and a 30-fold decrease in ribosome association with trigger factor, the chaperone normally associated with translating ribosomes. Surprisingly, this reduction does not involve J-domain cochaperones, unlike previously known functions of DnaK. Removing the 74 C-terminal amino acids of the 638-residue long DnaK impeded DnaK association with ribosomes and reduction of protein synthesis, rendering S. Typhimurium defective in protein homeostasis during cytoplasmic Mg2+ starvation. DnaK-dependent reduction in protein synthesis is critical for survival against Mg2+ starvation because inhibiting protein synthesis in a dnaK-independent manner overcame the 10,000-fold loss in viability resulting from DnaK truncation. Our results indicate that DnaK protects bacteria from infection-relevant stresses by coordinating protein synthesis with protein folding capacity.

TRPM7 kinase inactivation results in macrophage alkalinization and increased phagocytic activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level.

Cellular Adaptations to Cytoplasmic Mg2+ Limitation.

Mg2+ is the most abundant divalent cation in living cells. It is essential for charge neutralization, macromolecule stabilization, and the assembly and activity of ribosomes and as a cofactor for enzymatic reactions. When experiencing low cytoplasmic Mg2+, bacteria adopt two main strategies: They increase the abundance and activity of Mg2+ importers and decrease the abundance of Mg2+-chelating ATP and rRNA. These changes reduce regulated proteolysis by ATP-dependent proteases and protein synthesis in a systemic fashion. In many bacterial species, the transcriptional regulator PhoP controls expression of proteins mediating these changes. The 5' leader region of some mRNAs responds to low cytoplasmic Mg2+ or to disruptions in translation of open reading frames in the leader regions by furthering expression of the associated coding regions, which specify proteins mediating survival when the cytoplasmic Mg2+ concentration is low. Microbial species often utilize similar adaptation strategies to cope with low cytoplasmic Mg2+ despite relying on different genes to do so.

Low Cytoplasmic Magnesium Increases the Specificity of the Lon and ClpAP Proteases.

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium, the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyltransferase Pat from acetyl coenzyme A (acetyl-CoA) bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg2+ promotes qad expression, which protects substrates of Lon and ClpSAP by increasing HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP also promotes qad transcription in Escherichia coli, which shares a similar PhoP box in the qad promoter region with S. Typhimurium, Salmonella bongori, and Enterobacter cloacae. Our findings identify cytoplasmic Mg2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. IMPORTANCE The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg2+, a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of preexisting proteins.

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis

Phosphorus (P) is an essential component of core biological molecules. In bacteria, P is acquired mainly as inorganic orthophosphate (Pi) and assimilated into adenosine triphosphate (ATP) in the cytoplasm. Although P is essential, excess cytosolic Pi hinders growth. We now report that bacteria limit Pi uptake to avoid disruption of Mg2+-dependent processes that result, in part, from Mg2+ chelation by ATP. We establish that the MgtC protein inhibits uptake of the ATP precursor Pi when Salmonella enterica serovar Typhimurium experiences cytoplasmic Mg2+ starvation. This response prevents ATP accumulation and overproduction of ribosomal RNA that together ultimately hinder bacterial growth and result in loss of viability. Even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake increases ATP synthesis, depletes free cytoplasmic Mg2+, inhibits protein synthesis, and hinders growth. Our results provide a framework to understand the molecular basis for Pi toxicity. Furthermore, they suggest a regulatory logic that governs P assimilation based on its intimate connection to cytoplasmic Mg2+ homeostasis.

Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

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 Mg2+ 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 Ca2+ 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 Mg2+ 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.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

Accumulation of inorganic phosphate (Pi ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established. We showed that Pi promoted Ca2+ release at concentrations below 10mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR . Pre-treatment of permeabilized myofibres with 2mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi . The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature. Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54±1.6 at 5mm, and then decreased monotonically, reaching 0.09±0.03 at [Pi ]=80mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

Depletion of plasma membrane–associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg2+, spermine, and pH

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.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

Modulation of Mg2+ influx and cytoplasmic free Mg2+ concentration in rat ventricular myocytes.

To examine whether TRPM7, a member of the melastatin family of transient receptor potential channels, is a physiological pathway for Mg2+ entry in mammalian cells, we studied the effect of TRPM7 regulators on cytoplasmic free Mg2+ concentration ([Mg2+]i) of rat ventricular myocytes. Acutely isolated single cells were AM-loaded with the fluorescent indicator furaptra, and [Mg2+]i was estimated at 25°C. After [Mg2+]i was lowered by soaking the cells with a high-K+ and Mg2+-Ca2+-free solution, [Mg2+]i was recovered by extracellular perfusion of Ca2+-free Tyrode's solution that contained 1mMMg2+. The initial rate of increase in [Mg2+]i was analyzed as the Mg2+ influx rate. The Mg2+ influx rate was increased by the TRPM7 activator, naltriben (2-50μM), in a concentration-dependent manner with a half maximal effective concentration (EC50) of 24μM. This EC50 value is similar to that reported for the activation of recombinant TRPM7 overexpressed in HEK293 cells. Naltriben (50μM) caused little change in basal [Mg2+]i (~ 0.9mM) in Ca2+-free Tyrode's solution, but significantly raised [Mg2+]i to 1.31 ± 0.03mM in 94min after the removal of extracellular Na+. Re-introduction of extracellular Na+ lowered [Mg2+]i back to the basal level even in the presence of naltriben. Application of 10μM NS8593, an inhibitor of TRPM7, significantly lowered [Mg2+]i to 0.72 ± 0.03mM in 50-60min independent of extracellular Na+. The results suggest that Mg2+ entry through TRPM7 significantly contributes to physiological Mg2+ homeostasis in mammalian heart cells.

Reduction in adaptor amounts establishes degradation hierarchy among protease substrates

ATP-dependent proteases control critical cellular processes, including development, physiology, and virulence. A given protease may recognize a substrate directly via an unfoldase domain or subunit or indirectly via an adaptor that delivers the substrate to the unfoldase. We now report that cells achieve differential stability among substrates of a given protease by modulating adaptor amounts. We establish that the regulatory protein PhoP represses transcription of the gene specifying the ClpAP protease adaptor ClpS when the bacteria Salmonella enterica and Escherichia coli experience low cytoplasmic Mg2+ The resulting decrease in ClpS amounts diminishes proteolysis of several ClpSAP-dependent substrates, including the putrescine aminotransferase Oat, which heightens the formation of antibiotic persisters, and the transcriptional regulators UvrY and PhoP, which alter the expression of genes controlled by these proteins. By contrast, the decrease in ClpS amounts did not impact the abundance of the ClpSAP substrate FtsA, reflecting that FtsA binds to ClpS more tightly than to UvrY and PhoP. Our findings show how physiological conditions that reduce adaptor amounts modify the abundance of selected substrates of a given protease.

TRPM7 Current Inactivation: Evidence for Inside-Out Signaling

TRPM7 channels conduct metal cations such as Na+, K+, Ca2+ and Mg2+. In the presence of external Ca2+/Mg2+ TRPM7 has a steeply outwardly rectifying current-voltage (I-V) relation. In the absence of Ca2+/Mg2+ the IV becomes semi-linear. This has been explained by the removal of pore blockade by divalent cations (e.g. Ca2+o/Mg2+o). TRPM7 channels are inhibited by cytoplasmic Mg2+ in a voltage-independent manner, primarily by a reversible reduction in the overall number of conducting channels. Here, we have examined the consequences of external Ca2+ removal and reintroduction on TRPM7 current kinetics. In whole-cell patch clamp with low internal Mg2+, we rapidly and repeatedly exchanged 2 mM Ca2+ with divalent cation free (DVF) Na+ or Cs+ containing solutions to compare the time dependent changes in current. This maneuver resulted in a declining current in DVF following Ca2+ exposure, which we call inactivation. This was followed by a slowly rising current when Ca2+o was reintroduced. The decay of monovalent current occurred over 1-4 minutes and was monoexponential in most cases. Internal Mg2+ and spermine, partially inhibiting TRPM7, revealed more pronounced decay/potentiation cycles than seen with Mg2+-free internal solutions. Inactivation was observed with both Na+ and Cs+ as charge carriers and extracellular Mg2+ could substitute for the potentiating Ca2+ effect. Current decay in DVF was distinct from TRPM7 rundown, which we previously documented in patch-clamp recordings. Inactivation persisted in perforated-patch recordings, which allow the maintenance of physiological Mg2+ concentrations and prevent rundown. We have also characterized TRPM7 point mutations that abolish Ca2+ potentiation/inactivation. TRPM7 current behavior in response to switches between divalent-containing and divalent-free solutions resembled the Ca2+ potentiation/depotentiation described for calcium release activated calcium (CRAC) channels. We conclude that TRPM7 response to extracellular ions depends on intracellular Mg2+/spermine concentrations, presenting a novel case of inside-out signaling.

Protein synthesis controls phosphate homeostasis.

Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium, this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg2+), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg2+ promotes an uptake in Mg2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature.

Mg Isotope Fractionation during Uptake by a Rock-Inhabiting, Model Microcolonial Fungus Knufia petricola at Acidic and Neutral pH.

The model rock-inhabiting microcolonial fungus Knufia petricola fractionates stable Mg isotopes in a time- and pH-dependent manner. During growth, the increase of 26Mg/24Mg in the fungal cells relative to the growth media amounted to 0.65 ± 0.14‰ at pH 6 and 1.11 ± 0.35‰ at pH 3. We suggest a constant equilibrium fractionation factor during incorporation of Mg into ribosomes and ATP as a cause of enrichment of 26Mg in the cells. We suggest too that the proton gradient across the cell wall and cytoplasmic membrane controls Mg2+ transport into the fungal cell. As the strength of this gradient is a function of extracellular solution pH, the pH-dependence on Mg isotope fractionation is thus due to differences in fungal cell mass fluxes. Through a mass balance model we show that Mg uptake into the fungal cell is not associated with a unique Mg isotope fractionation factor. This Mg isotope fractionation dependence on pH might also be observed in any organism with cells that follow similar Mg uptake and metabolic pathways and serves to reveal Mg cycling in ecosystems.

Ryanodine receptor modification and regulation by intracellular Ca2 + and Mg2 + in healthy and failing human hearts

RationaleHeart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel – the ryanodine receptor (RyR2) – whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2+. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. ObjectiveTo assess Ca2+ and Mg2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. Methods and resultsThe function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2+. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2+. ConclusionDespite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

Similarities and Differences in Gating of the Two-Pore Channels TPC1 and TPC2

Endolysosomal Ca2+ homeostasis is implicated in several diseases and controls many endolysosomal functions. A key to understanding endolysosomal Ca2+ signaling to understand the function of the newly discovered endolysosomal PI(3,5)P2-regulated two-pore channels (TPCs) and their potential activation by NAADP. Our recent work concluded that the lysosomal TPC2 function as a NAADP and PI(3,5)P2-activated channel regulated by cytoplasmic Mg2+. The properties and gating of the mostly endosomal TPC1 are not known. Recording whole-organelle currents of enlarged endosomes expressing GFP-TPC1, we discovered that TPC1 is potently activated by PI(3,5)P2 and NAADP. Moreover, PI(3,5)P2 facilitates TPC1 activation by NAADP. Unlike, TPC2, the activity of TPC1 showed poor inhibition by Mg2+. Most notably, the concentration dependence of activation of TPC1 and TPC2 by NAADP are remarkably different. While activation of TPC2 by NAADP followed normal saturation dependence with no sign of inhibition by high NAADP concentration, activation of TPC1 by NAADP followed a bell shaped dependency, resembling the NAADP-mediated Ca2+ release in intact cells. These findings provide the first direct evidence for gating of TPC1 by NAADP and indicate that endosomal, rather than lysosomal, Ca2+ release is the key trigger Ca2+ pool that sensitizes the IP3-mediated Ca2+ release from the ER. The interaction between TPC1-expressing endosomes and the ER are being examined.

Effects of Inactivation of TRPM7 Kinase Activity on its Channel Activity in Mice

Transient receptor potential (TRP) family channels are involved in sensory pathways and are activated by various environmental stimuli. Among the members of this family, TRPM7 is a unique fusion of an ion channel and a C-terminus kinase domain that is ubiquitously expressed. TRPM7 is a key membrane protein governing cellular Mg2+ homeostasis in mammals since its channel pore is permeable to Mg2+ ions and can act as a Mg2+ influx pathway. Moreover, TRPM7 channel activity is inhibited by intracellular Mg2+. Mechanistic links between its kinase activity and channel function have remained uncertain, partly due to embryonic lethality of TRPM7 gene deletion in mice. In this study, we generated kinase inactive knock-in mutant mice by mutagenesis of a key lysine residue involved in Mg2+-ATP binding. K1646R mutant mice were normal in development and general locomotor activity. In peritoneal macrophages isolated from adult animals the basal activity of TRPM7 channels prior to cytoplasmic Mg2+ depletion was significantly potentiated, while maximal current densities measured after Mg2+ depletion were unchanged in the absence of detectable kinase function. The inhibition of TRPM7 channel currents by 300 uM intracellular Mg2+ or spermine was similar in WT and K1646R macrophages. Serum total Ca2+ and Mg2+ levels were not significantly altered in kinase-dead mutant mice either. Our findings suggest that 1) abolishing TRPM7 kinase activity does not impair its channel activity, but rather, potentiates basal current magnitudes; 2) kinase activity is not essential for regulation of mammalian Mg2+ homeostasis.

Physiological Pathway of Magnesium Influx in Rat Ventricular Myocytes

Cytoplasmic free Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution, [Mg2+]i decreased from ∼ 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg2+]i was recovered by perfusion with Ca2+-free Tyrode’s solution containing 1 mM Mg2+. The time course of the [Mg2+]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg2+ influx rate. The Mg2+ influx rate was inversely related to [Mg2+]i, being higher at low [Mg2+]i. The Mg2+ influx rate was augmented by the high extracellular Mg2+ concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K+. Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg2+ influx rate with half inhibitory concentrations (IC50) of, respectively, 17 μM, 2.0 μM, and 22 μM. We also studied Ni2+ influx by fluorescence quenching of intracellular furaptra by Ni2+. The Ni2+ influx was activated by lowering intra- and extracellular Mg2+ concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg2+ influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg2+ influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ∼ 50% by 100 μM 2-APB or 10 μM NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg2+ influx in rat ventricular myocytes.

Reduced cellular Mg2+ content enhances hexose 6-phosphate dehydrogenase activity and expression in HepG2 and HL-60 cells

We have reported that Mg2+ dynamically regulates glucose 6-phosphate entry into the endoplasmic reticulum and its hydrolysis by the glucose 6-phosphatase in liver cells. In the present study, we report that by modulating glucose 6-phosphate entry into the endoplasmic reticulum of HepG2 cells, Mg2+ also regulates the oxidation of this substrate via hexose 6-phosphate dehydrogenase (H6PD). This regulatory effect is dynamic as glucose 6-phosphate entry and oxidation can be rapidly down-regulated by the addition of exogenous Mg2+. In addition, HepG2 cells growing in low Mg2+ show a marked increase in hexose 6-phosphate dehydrogenase mRNA and protein expression. Metabolically, these effects on hexose 6-phosphate dehydrogenase are important as this enzyme increases intra-reticular NADPH production, which favors fatty acid and cholesterol synthesis. Similar effects of Mg2+ were observed in HL-60 cells.These and previously published results suggest that in an hepatocyte culture model changes in cytoplasmic Mg2+ content regulates glucose 6-phosphate utilization via glucose 6 phosphatase and hexose-6 phosphate dehydrogenase in alternative to glycolysis and glycogen synthesis. This alternative regulation might be of relevance in the transition from fed to fasted state.