Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.     

Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels

To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.     

Store-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes

Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca(2+) entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) in neutrophil-like HL-60 cells. When extracellular Ca(2+) is initially removed, early addition of Ca(2+) after stimulation causes a complete restoration of Ca(2+) entry and H(2)O(2) production. Both Ca(2+) entry and H(2)O(2) production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca(2+) channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca(2+) influx and H(2)O(2) production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca(2+) entry and H(2)O(2) production observed in cells transfected by TRPC3 siRNA. In summary, Ca(2+) influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE.     

Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca(2+) signaling via a phosphatidylinositol 3,4, 5-trisphosphate PI(3,4,5)P(3)-sensitive Ca(2+) entry pathway. First, exogenous PI(3,4,5)P(3) at concentrations close to its physiological levels induces Ca(2+) influx in T cells, whereas PI(3,4)P(2), PI(4, 5)P(2), and PI(3)P have no effect on [Ca(2+)](i). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) entry. This PI(3,4,5)P(3)-stimulated Ca(2+) influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-gamma form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P(3)-induced Ca(2+) entry acts concertedly with Ins(1,4,5)P(3)-induced Ca(2+) release in initiating T cell Ca(2+) signaling. By using a biotinylated analog of PI(3,4,5)P(3) as the affinity probe, we have detected several putative PI(3,4,5)P(3)-binding proteins in T cell plasma membranes.     

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.     

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.     

Functional organization of TRPC-Ca2+ channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.     

Inhibition of store-operated calcium entry in human lymphocytes by radiation: protection by glutathione

The influence of gamma radiation on basal compared to activation-dependent Ca(2+) influx in human lymphocytes was investigated. A new quantitative fluorescence technique termed differential ratiometric fluorescence spectroscopy (DRFS) was employed. DRFS facilitated the real-time detection of changes in fluorescence in experimental and control cell samples simultaneously, enabling the resolution of acute moderate changes ( congruent with10-30%) in Ca(2+) (manganese) influx after exposure to ionizing radiation and other oxidant interventions. Exposure to radiation inhibited thapsigargin-stimulated store-operated Ca(2+) influx but not basal Ca(2+) influx in Jurkat T cells and human peripheral blood lymphocytes. The response of store-operated Ca(2+) influx to gamma radiation was dependent on dose between 5 and 40 Gy and was inhibited by preincubation with the Ca(2+) channel blocker Ni(2+), as determined with Jurkat T cells. Elevation of the intracellular concentration of glutathione significantly reduced the inhibition of Ca(2+) influx by gamma radiation. Similar to radiation, both the superoxide anion-generating xanthine/xanthine oxidase system and hydrogen peroxide inhibited thapsigargin-stimulated Ca(2+) influx in Jurkat T cells, and this inhibition was reversed in the presence of the antioxidant N-acetyl-l-cysteine. In conclusion, (1) ionizing radiation inhibited store-operated Ca(2+) entry in human lymphocytes, (2) the sensitivity of Ca(2+) influx to radiation was strictly dependent on depletion of Ca(2+) stores, and (3) glutathione protected against the inhibition of store-operated Ca(2+) entry by gamma radiation.     

The overexpression of presenilin2 and Alzheimer's-disease-linked presenilin2 variants influences TRPC6-enhanced Ca2+ entry into HEK293 cells

Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer's disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer's-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca(2+) signaling are unknown. The present study investigated the influence of two familial Alzheimer's-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca(2+) entry channel. We show that transient coexpression of Alzheimer's-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca(2+) entry. The inhibitory effect of presenilin2 and the Alzheimer's-disease-linked presenilin2 variants was not due to an increase in amyloid beta-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer's-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca(2+) entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca(2+) entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca(2+) entry into HEK293 cells.     

TRPC1 proteins confer PKC and phosphoinositol activation on native heteromeric TRPC1/C5 channels in vascular smooth muscle: comparative study of wild-type and TRPC1-/- mice

Ca(2+)-permeable cation channels consisting of canonical transient receptor potential 1 (TRPC1) proteins mediate Ca(2+) influx pathways in vascular smooth muscle cells (VSMCs), which regulate physiological and pathological functions. We investigated properties conferred by TRPC1 proteins to native single TRPC channels in acutely isolated mesenteric artery VSMCs from wild-type (WT) and TRPC1-deficient (TRPC1(-/-)) mice using patch-clamp techniques. In WT VSMCs, the intracellular Ca(2+) store-depleting agents cyclopiazonic acid (CPA) and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) both evoked channel currents, which had unitary conductances of ～2 pS. In TRPC1(-/-) VSMCs, CPA-induced channel currents had 3 subconductance states of 14, 32, and 53 pS. Passive depletion of intracellular Ca(2+) stores activated whole-cell cation currents in WT but not TRPC1(-/-) VSMCs. Differential blocking actions of anti-TRPC antibodies and coimmunoprecipitation studies revealed that CPA induced heteromeric TRPC1/C5 channels in WT VSMCs and TRPC5 channels in TRPC1(-/-) VSMCs. CPA-evoked TRPC1/C5 channel activity was prevented by the protein kinase C (PKC) inhibitor chelerythrine. In addition, the PKC activator phorbol 12,13-dibutyrate (PDBu), a PKC catalytic subunit, and phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) activated TRPC1/C5 channel activity, which was prevented by chelerythrine. In contrast, CPA-evoked TRPC5 channel activity was potentiated by chelerythrine, and inhibited by PDBu, PIP(2), and PIP(3). TRPC5 channels in TRPC1(-/-) VSMCs were activated by increasing intracellular Ca(2+) concentrations ([Ca(2+)](i)), whereas increasing [Ca(2+)](i) had no effect in WT VSMCs. We conclude that agents that deplete intracellular Ca(2+) stores activate native heteromeric TRPC1/C5 channels in VSMCs, and that TRPC1 subunits are important in determining unitary conductance and conferring channel activation by PKC, PIP(2), and PIP(3).     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) results from Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through Ca(2+)-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca(2+) entry (CCE) induced by Ca(2+) store depletion represents a major Ca(2+) influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca(2+)-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca(2+) currents and CCE generated by Ca(2+) influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca(2+) channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca(2+)](cyt).     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.     

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Non-stimulated, agonist-stimulated and store-operated Ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry

In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca(2+) reserves, molecular components of the store-operated Ca(2+) entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca(2+) influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca(2+) influx. The potential roles for specific Ca(2+) channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca(2+) influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca(2+) influx in a manner dependent on Ca(2+) influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca(2+) release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (time to peak [Ca(2+)](CYT) = 188.7 ± 34.6 s (TRPC1 siRNA) versus 124.0 ± 9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca(2+) influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca(2+) influx in non-stimulated cells.     

A pyrazole derivative,YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.     

A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.     

The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels

Na(+)/Ca(2+) exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca(2+) homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca(2+) influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC(50)=0.46 microM), TRPC6 (IC(50)=0.71 microM), and TRPC5 (IC(50)=1.38 microM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry was nearly completely suppressed by 10 microM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.