Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes

TRPM6 and TRPM7 are distinct from all other ion channels in that they are composed of linked channel and protein kinase domains. Recent studies demonstrate that these 'chanzymes' are essential for Mg(2+) homeostasis, which is critical for human health and cell viability.     

Molecular determinants of Mg2+ and Ca2+ permeability and pH sensitivity in TRPM6 and TRPM7

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ 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. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ 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 Ca2+ and Mg2+ 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.     

Mrs2p forms a high conductance Mg2+ selective channel in mitochondria

Members of the CorA-Mrs2-Alr1 superfamily of Mg²⁺ transporters are ubiquitous among pro- and eukaryotes. The crystal structure of a bacterial CorA protein has recently been solved, but the mode of ion transport of this protein family remained obscure. Using single channel patch clamping we unequivocally show here that the mitochondrial Mrs2 protein forms a Mg²⁺-selective channel of high conductance (155 pS). It has an open probability of ∼60% in the absence of Mg²⁺ at the matrix site, which decreases to ∼20% in its presence. With a lower conductance (∼45 pS) the Mrs2 channel is also permeable for Ni²⁺, whereas no permeability has been observed for either Ca²⁺, Mn²⁺, or Co²⁺. Mutational changes in key domains of Mrs2p are shown either to abolish its Mg²⁺ transport or to change its characteristics toward more open and partly deregulated states. We conclude that Mrs2p forms a high conductance Mg²⁺ selective channel that controls Mg²⁺ influx into mitochondria by an intrinsic negative feedback mechanism.     

Characterization of Na+/Mg2+ antiport by simultaneous 28Mg2+ influx

During net Mg2+ efflux from Mg2+-preloaded chicken erythrocytes, which occurs via Na+/Mg2+ antiport, 28Mg2+ is taken up intracellularly. Km of 28Mg2+ influx amounted to 1 mM. In Na+-free medium Vmax of 28Mg2+ influx was increased and Km was reduced to 0.2 mM. 28Mg2+ influx was noncompetitively inhibited by amiloride as was found for Na+/Mg2+ antiport. The results indicate that, extracellularly, Mg2+ can compete with Na+ for common binding sites of the Na+/Mg2+ antiporter, resulting in 28Mg2+-24Mg2+ exchange. The rate of Mg2+ exchange depends on extracellular Na+ and on the rate of net Mg2+ efflux.     

Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel

Modulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. Here, we report that Mg2+-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in direct binding of Mg2+ ions. Moreover, mutation of the only positively charged residue in the other intracellular linker between S4 and S5 also results in a dramatic reduction of Mg2+-dependent modulation of Eag activation kinetics. Collectively, the two mutations in eag eliminate or even paradoxically reverse the effect of Mg2+ on channel activation and inactivation kinetics. Together, these results suggest an important role of the intracellular linker regions in gating processes of Eag channels.     

The Mg2+ transporter MagT1 partially rescues cell growth and Mg2+ uptake in cells lacking the channel-kinase TRPM7

Magnesium (Mg(2+)) transport across membranes plays an essential role in cellular growth and survival. TRPM7 is the unique fusion of a Mg(2+) permeable pore with an active cytosolic kinase domain, and is considered a master regulator of cellular Mg(2+) homeostasis. We previously found that the genetic deletion of TRPM7 in DT40 B cells results in Mg(2+) deficiency and severe growth impairment, which can be rescued by supplementation with excess extracellular Mg(2+). Here, we show that gene expression of the Mg(2+) selective transporter MagT1 is upregulated in TRPM7(-/-) cells. Furthermore, overexpression of MagT1 in TRPM7(-/-) cells augments their capacity to uptake Mg(2+), and improves their growth behavior in the absence of excess Mg(2+).     

TRPM2 Ca2+ channel regulates energy balance and glucose metabolism

TRPM2 Ca(2+)-permeable cation channel is widely expressed and activated by markers of cellular stress. Since inflammation and stress play a major role in insulin resistance, we examined the role of TRPM2 Ca(2+) channel in glucose metabolism. A 2-h hyperinsulinemic euglycemic clamp was performed in TRPM2-deficient (KO) and wild-type mice to assess insulin sensitivity. To examine the effects of diet-induced obesity, mice were fed a high-fat diet for 4-10 mo, and metabolic cage and clamp studies were conducted in conscious mice. TRPM2-KO mice were more insulin sensitive partly because of increased glucose metabolism in peripheral organs. After 4 mo of high-fat feeding, TRPM2-KO mice were resistant to diet-induced obesity, and this was associated with increased energy expenditure and elevated expressions of PGC-1α, PGC-1β, PPARα, ERRα, TFAM, and MCAD in white adipose tissue. Hyperinsulinemic euglycemic clamps showed that TRPM2-KO mice were more insulin sensitive, with increased Akt and GSK-3β phosphorylation in heart. Obesity-mediated inflammation in adipose tissue and liver was attenuated in TRPM2-KO mice. Overall, TRPM2 deletion protected mice from developing diet-induced obesity and insulin resistance. Our findings identify a novel role of TRPM2 Ca(2+) channel in the regulation of energy expenditure, inflammation, and insulin resistance.     

The channel kinases TRPM6 and TRPM7 are functionally nonredundant

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg(2+) deficiency, and cell death within 24-48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg(2+)-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5-10 mm Mg(2+) concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each other's biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg(2+) homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.     

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.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

TRPM7: a unique channel involved in magnesium homeostasis

TRPM7 is a ubiquitously expressed cation channel with a fused alpha kinase domain. It is highly permeable to magnesium and calcium, and is negatively gated by intracellular Mg(2+) and Mg-ATP. Substrates for the TRPM7 kinase domain include annexinA1 and myosin IIA heavy chain, and there is evidence to suggest a functional interaction between the channel and kinase domains. Alterations in the expression and activity of TRPM7 have profound effects on cell proliferation and differentiation. Genetic deletion of TRPM7 in model systems demonstrates that this channel is critical for cellular growth and embryonic development. Here, we provide a brief overview of the activity of TRPM7 and the associated regulatory mechanisms. We will then discuss the biological functions of TRPM7, emphasizing its role in development and the potential pathophysiological significance of TRPM7 in neurological and cardiovascular disease.     

Voltage dependence of the Ca2+-activated cation channel TRPM4

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.     

Functional characterization of homo- and heteromeric channel kinases TRPM6 and TRPM7

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 TRPM6(S141L) 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.     

Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+

It is known that extracellular Mg²⁺ and Ca²⁺ can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg²⁺ and Ca²⁺ on the Cs+ currents, decreasing their binding affinities: their IC50 values increased 510- and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs+ currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC50 values were increased 5.5- and 3.9-fold, respectively, for Mg2+ and 10.5- and 6.7-fold, respectively, for Ca2+. D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg2+ (1.7-fold) and Ca2+ (3.9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg²⁺ and Ca²⁺. In the E1052A and D1059A mutants, inward Mg²⁺ and Ca²⁺ currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg²⁺ and Ca²⁺, (2) Asp-1054 is an essential determinant of Mg²⁺ and Ca²⁺ conductivity, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.     

Characterization of furosemide-sensitive Mg2+ influx in Yoshida ascites tumor cells

Partially Mg2+-depleted Yoshida ascites tumor cells took up Mg2+ after reincubation in Mg2+- and HCO3(-)-containing media. Mg2+ influx was insensitive to ouabain, amiloride and disulfonic stilbenes, but was noncompetitively inhibited by furosemide (Ki = 0.4 mM) and bumetanide. Mg2+ influx obeyed Michaelis-Menten kinetics with respect to Mg2+ concentration (Km = 1.1 mM) and was sigmoidal with respect to HCO3- concentration. Electroneutral Mg2+, HCO3- cotransport was supposed to be the mechanism of Mg2+ influx.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.     

Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels

TRPM7 channel kinase is a protein highly expressed in cells of hematopoietic lineage, such as lymphocytes. Studies performed in native and heterologous expression systems have shown that TRPM7 forms nonselective cation channels functional in the plasma membrane and activated on depletion of cellular Mg(2+). In addition to internal Mg(2+), cytosolic pH and the phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] are potent physiological regulators of this channel: protons inhibit, while PI(4,5)P(2) is required for TRPM7 channel activity. These channels are also inhibited from inside by other metal cations and polyamines. While the regulation of TRPM7 channels by internal metal ions, acidic pH, and PI(4,5)P(2) is voltage independent, extracellular metal cations and polyamines block voltage dependently at micromolar concentrations and appear to occupy a distinct blocking site. In the present study we investigated intracellular Mg(2+) and pH dependence of native TRPM7 currents using whole cell patch-clamp electrophysiology in human Jurkat T lymphocytes and HEK293 cells. Our main findings are 1) Mg(2+) inhibition involves not one but two separate sites of high (～10 μM) and low (～165 μM) affinity; and 2) while sharing certain characteristics with Mg(2+) inhibition, protons most likely inhibit through one inhibitory site, corresponding to the low-affinity Mg(2+) site, with an estimated IC(50) of pH 6.3. Additionally, we present data on amplitude distribution of preactivated TRPM7 currents in Jurkat T lymphocytes in the absence of prior Mg(2+) or proton depletion.     

Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.