CaT1 and the calcium release-activated calcium channel manifest distinct pore properties

The calcium release-activated calcium channel (CRAC) is a highly Ca(2+)-selective ion channel that is activated on depletion of inositol triphosphate (IP(3))-sensitive intracellular Ca(2+) stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na(+) and Cs(+); effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg(2+) inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg(2+)-gated channel not directly related to CRAC.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

Store-operated calcium entry is present in HL-1 cardiomyocytes and contributes to resting calcium

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.     

Acidic stress induced G1 cell cycle arrest and intrinsic apoptotic pathway in Jurkat T-lymphocytes

Background

Low extracellular pH (pH_e) is a common hallmark of tumor microenvironment, which will also affect pH sensitive T-lymphocytes in this environment. Due to the growing interest on T-cell mediated cancer therapies, acidic stress induced consequences on this lymphocyte deserves through investigations.

Changes in the calcium current among different transmural regions contributes to action potential heterogeneity in rat heart

To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca²⁺ current (I_Ca,L) by voltage and Ca²⁺-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak I_Ca,L was significantly greater in EPI than in other cells. The inactivation of I_Ca,L by Ca²⁺-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca²⁺ sensitive, with Ca²⁺ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I_Ca,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca²⁺ influx.     

Effect of copper deficiency on oxidative DNA damage in Jurkat T-lymphocytes

The micronutrient copper is a catalytic cofactor for copper, zinc superoxide dismutase and ceruloplasmin, which are two important antioxidant enzymes. As such, a lack of copper may promote oxidative stress and damage. The purpose of this study was to determine the effect of copper deficiency on oxidative damage to DNA in Jurkat T-lymphocytes. To induce copper deficiency, cells were incubated for 48 h with 5-20 microM 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Such treatment did not affect cell proliferation/viability, as assessed by measuring mitochondrial reduction of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben zen e disulfonate). Furthermore, the induction of copper deficiency did not promote oxidative DNA damage as evaluated by the comet assay. Comet scores were 15 +/- 0 and 16 +/- 1 for control and copper-deficient cells, respectively. However, the copper-deficient cells sustained greater oxidative DNA damage than the control cells (comet scores of 175 +/- 15 and 50 +/- 10, respectively) when both were oxidatively challenged with 50 microM hydrogen peroxide (H(2)O(2)). Supplemental copper but not zinc or iron prevented the potentiation of the H(2)O(2)-induced oxidative DNA damage caused by 2,3,2-tet. These data suggest that copper deficiency compromises the antioxidant defense system of cells, thereby increasing their susceptibility to oxidative DNA damage.     

Single-Channel Activities of the Human Epithelial Ca2+ Transport Proteins CaT1 and CaT2

The human epithelial channels, CaT1 and CaT2, were expressed in oocytes, and their single-channel characteristics were compared. In the presence of Na⁺ and K⁺ as charge carriers in the pipette solutions, channel activities were observed only when the the extracellular sides of the patches were exposed to nominally Ca²⁺- and Mg²⁺-free solutions. In patches of both CaT1- and CaT2-expressing oocytes, multiple channel openings were observed, but the current levels were higher in CaT2-expressing oocytes, particularly at more negative voltages. With K⁺ as a charge carrier in patches of CaT1-expressing oocytes, the channel activity was low at −10 to −60 mV, but increased dramatically at more negative potentials. This voltage dependence was observed in the presence of both Na⁺ and K⁺. The channel activity with Na⁺, however, was higher at all potentials. Differences between the voltage dependencies for the two cations were also observed in CaT2-expressing oocytes, but the channel activities were higher than those in CaT1-expressing oocytes, particularly in the presence of Na⁺. We also found that low concentrations of extracellular Mg²⁺ (5–50 μm) elicited a strong inhibitory action on the CaT channels. Activation of the CaT1 and CaT2 channels by hyperpolarization and other factors may promote increased Ca²⁺ entry that participates in stimulation of intestinal absorption and renal reabsorption and/or other Ca²⁺ transport mechanisms in epithelial cells. Received: 8 March 2001/Revised: 24 July 2001     

Structural conservation of the genes encoding CaT1, CaT2, and related cation channels

We report here the genomic structures of the genes encoding human calcium transport proteins CaT1 and CaT2, which belong to a recently identified class of highly selective calcium entry channels. The mRNA for CaT1 was expressed more abundantly than that for CaT2 in three major tissues involved in transcellular calcium transport, namely intestine, kidney, and placenta, as determined by quantitative PCR. The genes encoding CaT1 and CaT2, ECAC2 and ECAC1, respectively, are completely conserved in terms of exon size in the coding regions. They also share similar intron-exon structures with the genes encoding the closely related, nonselective cation channels VR1, VRL-1, OTRPC4 (also known as VR-OAC, Trp12, and VRL-2), and a hypothetical protein, VRL-3. We conclude that ECAC2 and ECAC1, which encode calcium selective channels, share a common ancestral gene with the genes encoding the related nonselective cation channels.     

Kcnq1 contributes to an adrenergic-sensitive steady-state K+ current in mouse heart

It has been suggested that Kcne1 subunits are required for adrenergic regulation of Kcnq1 potassium channels. However, in adult mouse hearts, which do not express Kcne1, loss of Kcnq1 causes a Long QT phenotype during adrenergic challenge, raising the possibility that native Kcnq1 currents exist and are adrenergically regulated even in absence of Kcne1. Here, we used immunoblotting and immunohistochemical staining to show that Kcnq1 protein is present in adult mouse hearts. Voltage-clamp experiments demonstrated that Kcnq1 contributes to a steady-state outward current (I(SS)) in wild-type (Kcnq1(+/+)) ventricular myocytes during isoproterenol stimulation, resulting in a significant 7.1% increase in I(SS) density (0.43+/-0.16 pA/pF, p <0.05, n =15), an effect that was absent in Kcnq1-deficient (Kcnq1(-/-)) myocytes (-0.14+/-0.13 pA/pF, n =17). These results demonstrate for the first time that Kcnq1 protein is expressed in adult mouse hearts where it contributes to a beta-adrenergic-induced component of I(SS) that does not require co-assembly with Kcne1.     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.     

Apparent cytosolic calcium gradients in T-lymphocytes due to fura-2 accumulation in mitochondria

Fura-2 is the most common dye to measure cytosolic Ca2+ concentrations ([Ca2+]i). To facilitate simultaneous imaging of many cells while preserving their cytosolic environment, fura-2 is often loaded into the cytosol in its membrane-permeant ester form. It has been reported that small amounts of fura-2 accumulate in intracellular compartments, an effect that is usually neglected. We show that either focal or non-focal stimulation methods induce large [Ca2+]i gradients in T-lymphocytes during both, Ca2+ release and Ca2+ influx across the plasma membrane. Interfering with mitochondrial Ca2+ homeostasis and by labeling mitochondria with MitoTracker, we demonstrate that [Ca2+]i gradients co-localize with mitochondria and are attributable to mitochondrial fura-2 sequestration. Gradients could not be avoided by different loading protocols, compromising measurements of "real" [Ca2+]i gradients following T-cell stimulation. They were observed in human blood and lamina propria lymphocytes, Jurkat T-cells, mast cells, but not to the same extent in HEK-293 cells. Finally, we show that T-lymphocytes can be efficiently loaded with the membrane-impermeant fura-2 salt by electroporation and by osmotic lysis of pinocytic vesicles, which result in the loss of [Ca2+]i gradients. These methods are therefore suitable to study localized Ca2+ signals in large populations of T-cells while preserving their cytosolic integrity.     

CaT1 expression correlates with tumor grade in prostate cancer

Ca(2+) signaling is important for growth and survival of prostatic carcinoma (PCa) cells. Here we report that the gene for CaT1, a channel protein highly selective for Ca(2+), is expressed at high levels in human PCa and in the LNCaP PCa cell line. CaT1 mRNA levels were elevated in PCa specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlated with Gleason grade in a PCa series. CaT1 mRNA was suppressed by androgen and was induced by a specific androgen receptor antagonist in LNCaP cells, suggesting that the gene is negatively regulated by androgen. These findings are the first to implicate a Ca(2+) channel in PCa progression and suggest that CaT1 may be a novel target for therapy.     

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.     

Constitutively active calcium channels in plasma membrane of T-lymphocytes

Compensated influx and efflux of calcium ions maintain the constancy of Ca²⁺ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca²⁺ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca²⁺-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca²⁺ influx occurs through special constitutively active Ca²⁺ channels, permeable to Ni²⁺ and Mn²⁺. These channels differ from the receptor-activated SOC channels, from Ca²⁺ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca²⁺ influx rate in quiescent cells increases with a rise in temperature (Q₁₀ =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca²⁺-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca²⁺ channels, SOC channels, Ca²⁺-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca²⁺ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca²⁺ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca²⁺ channels in plasma membrane of non-excitable cells.     

Tunable calcium current through TRPV1 receptor channels

TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore.     

TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes

Stimulation of the T-cell receptor (TCR) activates Ca2+ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca2+ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca2+ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca2+ currents as well as TCR-dependent Ca2+ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca2+ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.     

Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent protein kinase.

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.