TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.

The store-operated, calcium release-activated calcium current I(CRAC) is activated by the depletion of inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. The significantly different dose-response relationships of IP(3)-mediated Ca(2+) release and CRAC channel activation indicate that I(CRAC) is activated by a functionally, and possibly physically, distinct sub-compartment of the endoplasmic reticulum (ER), the so-called CRAC store. Vertebrate genomes contain three IP(3) receptor (IP(3)R) genes and most cells express at least two subtypes, but the functional relevance of various IP(3)R subtypes with respect to store-operated Ca(2+) entry is completely unknown. We here demonstrate in avian B cells (chicken DT40) that IP(3)R type II and type III participate in IP(3)-induced activation of I(CRAC), but IP(3)R type I does not. This suggests that the expression pattern of IP(3)R contributes to the formation of specialized CRAC stores in B cells.

Deviations from physiological pH (∼pH 7.2) as well as altered Ca(2+) signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca(2+) influx is the store-operated Ca(2+) entry (SOCE) or Ca(2+) release-activated Ca(2+) current (ICRAC), which is activated upon depletion of intracellular Ca(2+) stores. We here show that extracellular and intracellular changes in pH regulate both endogenous ICRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous ICRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP3-induced ICRAC. In the absence of IP3, external alkalization did not elicit endogenous ICRAC but was able to activate heterologous ICRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP3-induced activation of endogenous and heterologous ICRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of ICRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates ICRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate ICRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.

2-Aminoethoxydiphenyl borate (2-APB) has emerged as a useful pharmacological tool in the study of store-operated Ca(2+) entry (SOCE). It has been shown to potentiate store-operated Ca(2+) release-activated Ca(2+) (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2-APB. We now present a thorough pharmacological profile of 2-APB and report that it can activate CRACM3 channels in a store-independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2-APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2-APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2-APB-induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs(+) or K(+). These results suggest that 2-APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store-independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store-operated and STIM1-dependent activation. The inhibition of CRACM1 channels by high concentrations of 2-APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store-dependent multimerization of STIM1. Finally, we demonstrate that single-point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2-APB to gate CRACM1 in a STIM1-independent manner, suggesting that 2-APB facilitates CRAC channels by altering the pore architecture.

TRPM7 (transient-receptor-potential melastatin 7) is an ion channel with alpha-kinase function. TRPM7 is divalent-selective and regulated by a range of receptor-stimulated second messenger pathways, intracellular Mg-nucleotides, divalent and polyvalent cations and pH. TRPM7 is ubiquitously found in mammalian cells, including kidney, the responsible organ for osmolyte regulation, posing the question whether the channel is osmosensitive. Recent reports investigated the sensitivity of native TRPM7-like currents to cell swelling with contradictory results. Here, we assess the sensitivity of TRPM7 to both hypo- and hyperosmotic conditions and explored the involvement of the channel's kinase domain. We find that hypotonicity facilitates TRPM7 at elevated intracellular magnesium and Mg.ATP (3-4 mm), but has no effect in the absence of these solutes. Hypertonic conditions, in contrast, inhibit TRPM7 with an IC(50) of 430 mosmol l(-1). This inhibitory effect is maintained in the complete absence of intra- and extracellular divalent ions, although shifted to higher osmolarities (IC(50) = 510 mosmol l(-1)). TRPM7 senses osmotic gradients rather than ionic strength and this is independent of cAMP or not affected by cytochalasin D treatment. Furthermore, the kinase-domain deletion mutant of TRPM7 shows a similar behaviour to osmolarity as the wild-type protein, both in the presence and absence of divalent ions. This indicates that at least part of the osmosensitivity resides in the channel domain. Physiologically, TRPM7 channels do not seem to play an active role in regulatory volume changes, but rather those volume changes modulate TRPM7 activity through changes in the cytosolic concentrations of free Mg, Mg-nucleotides and a further unidentified factor. We conclude that TRPM7 senses osmotically induced changes primarily through molecular crowding of solutes that affect channel activity.

Microglia are the main immunocompetent cells of the mammalian central nervous system (CNS). Activation of cultured microglial cells and subsequent release of nitric oxide and cytokines critically depends on intracellular calcium levels. Since microglia undergo dramatic morphological, biochemical and electrophysiological changes in response to pathological events in the CNS, we investigated temporal changes in expression levels of ion channels involved in cellular calcium homeostasis in mouse cortical microglial cells in culture. Specifically, we assessed the inward and delayed outward rectifier potassium currents (I IRK and I DRK), calcium (Ca2+) release-activated Ca2+ currents (I CRAC) and Ca2+-activated TRPM4-like currents (I CAN) in non-activated microglia and cells that were activated by exposure to lipopolysaccharide (LPS) between 3 and 48 h. Unstimulated microglial cells, subcultured from an astrocyte coculture, typically exhibited a ramified, rod-shaped morphology. During the first 3 days of culture cell size and shape were maintained, but the percentage of cells showing prominent I IRK went up and those expressing I DRK went down. Cells retaining I DRK exhibited smaller amplitudes, whereas those of I IRK and I CRAC were not affected. However, after 24 h of exposure to 1 microg ml(-1) LPS, most cells showed an amoeboid ('fried egg'-shaped) morphology with a 62% increase in cell capacitance. At that point in time, only 14% of the cells revealed I IRK and 3% had I DRK exclusively, whereas the majority of cells expressed both currents. The amplitudes of I CRAC and I IRK progressively decreased after stimulation, whereas I DRK transiently reached a maximum after 6 h of LPS exposure and then returned to pre-stimulation expression levels. Cultured microglia also revealed TRPM4-like, Ca2+-activated non-selective currents (I CAN) with an EC50 of 1.2 microm [Ca2+]i. The expression levels of this current did not change significantly during and after 24 h of LPS exposure. We propose that LPS-induced down-regulation of I IRK and I CRAC will reduce the cell's capacity to produce significant calcium influx upon receptor activation and result in decreased sensitivity to exogenous stimulation. In this scenario, I CAN expression would remain constant, although its activity would automatically be reduced due to the diminished calcium influx capacity of the cell.

TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel's regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only.

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) >> Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.