Fluorescence-Based Measurements of the CRAC Channel Activity in Cell Populations.

Cytosolic Ca2+ plays an important role in cellular biology, and since its identification as a second messenger, a number of techniques and methods to analyze the changes in cytosolic Ca2+ concentration ([Ca2+]c) induced by physiological agonists have been developed. Changes in [Ca2+]c might be determined in single cells or in cell populations. Measurement in single cells allows to determine changes in [Ca2+]c at a subcellular level but often results in heterogeneous responses among cells. Determination of intracellular Ca2+ mobilization at the cell population level reduces this heterogeneity and allows [Ca2+]c measurements in small cells that load little amounts of indicator. Here, we describe the measurement of agonist-evoked changes in [Ca2+]c associated with Ca2+ influx in cell populations.

Homer proteins mediate the interaction between STIM1 and Cav1.2 channels.

STIM1 is a ubiquitous Ca2+ sensor of the intracellular, agonist-sensitive, Ca2+ stores that communicates the filling state of the Ca2+ compartments to plasma membrane store-operated Ca2+ (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca2+ influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, ß2 and α2δ-1 exhibited a significant Ca2+ entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca2+ stores, resulted in Ca2+ influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 α1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 α1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1.

STIM1 regulates TRPC6 heteromultimerization and subcellular location.

STIM1 (stromal interaction molecule 1) regulates store-operated channels in the plasma membrane, but the regulation of TRPC (transient receptor potential canonical) heteromultimerization and location by STIM1 is poorly understood. STIM1 is a single transmembrane protein that communicates the filling state of the endoplasmic reticulum to store-operated channels. STIM1 has been reported to regulate the activity of all of the TRPC family members, except TRPC7. TRPC6 has been predominantly associated to second messenger-activated Ca2+ entry pathways. In the present paper we report that STIM1 regulates the expression of TRPC6 in the plasma membrane and evokes translocation of this channel to the endoplasmic reticulum. Attenuation of TRPC6 expression in the plasma membrane resulted in a reduction in the association of this channel with TRPC1 and TRPC3. We have found that expression of TRPC6 in the endoplasmic reticulum results in an increase in the passive Ca2+ efflux and basal cytosolic Ca2+ concentration, but not in the ability of cells to accumulate Ca2+ into the endoplasmic reticulum. We propose a novel mechanism for the regulation of TRPC6 channel location and function by STIM1, probably as a mechanism to modulate second messenger-operated Ca2+ entry while potentiating store-operated Ca2+ influx.

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets.

Cytosolic-free Ca(2+) plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca(2+) concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca(2+) channel in Ca(2+) homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca(2+) homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca(2+)]c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca(2+) accumulation in wild-type and TRPC6-deficient mice; however, passive Ca(2+) leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1-TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn(2+) entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca(2+)]c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca(2+)]c, controlling the passive Ca(2+) leak rate from agonist-sensitive intracellular Ca(2+) stores in resting platelets.

Development and characterization of multilayer films of polyaniline, titanium dioxide and CTAB for potential antimicrobial applications.

Composites prepared from polyaniline (PANI) and the ceramic technology of titanium dioxide (TiO2) have been proposed, however, the interaction of these materials with greater control of molecular arrangement becomes attractive in order to achieve properties not previously described or yet the optimization of those already reported. Therefore, in this study, thin hybrid films made of polyaniline (PANI), a conductive polymer, and the technological ceramic, titanium dioxide (TiO2), were prepared by the layer-by-layer (LbL) self-assembly technique. The films were characterized by cyclic voltammetry (CV), UV-VIS spectroscopy and atomic force microscopy (AFM). Aiming to improve the dispersion of the ceramic in the polymer matrix, the commercial surfactant, cetyl trimethylammonium bromide (CTAB), was used in the formation of the films. The best condition of deposition was found showing synergic interactions between the conjugated materials. The antibacterial activity of the PANI(TiO2)/CTAB films was studied and the obtained results suggest their use as antimicrobial coatings.

Cytoskeletal and scaffolding proteins as structural and functional determinants of TRP channels.

Transient receptor potential (TRP) channels are six transmembrane-spanning proteins, with variable selectivity for cations, that play a relevant role in intracellular Ca(2+) homeostasis. There is a large body of evidence that shows association of TRP channels with the actin cytoskeleton or even the microtubules and demonstrating the functional importance of this interaction for TRP channel function. Conversely, cation currents through TRP channels have also been found to modulate cytoskeleton rearrangements. The interplay between TRP channels and the cytoskeleton has been demonstrated to be essential for full activation of a variety of cellular functions. Furthermore, TRP channels have been reported to take part of macromolecular complexes including different signal transduction proteins. Scaffolding proteins play a relevant role in the association of TRP proteins with other signaling molecules into specific microdomains. Especially relevant are the roles of the Homer family members for the regulation of TRPC channel gating in mammals and INAD in the modulation of Drosophila TRP channels. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

The canonical transient receptor potential 6 (TRPC6) channel is sensitive to extracellular pH in mouse platelets.

The canonical transient receptor potential-6 (TRPC6) is a receptor-activated non-selective Ca(2+) channel regulated by a variety of modulators such as diacylglycerol, Ca(2+)/calmodulin or phosphorylation. The present study is aimed to investigate whether different situations, such as acidic pH, exposure to reactive oxygen species (ROS) or hypoxic-like conditions modulate TRPC6 channel function. Here we show normal aggregation and Ca(2+) mobilization stimulated by thrombin in TRPC6 KO platelets; however, OAG (1-oleoyl-2-acetyl-sn-glycerol)-evoked Ca(2+) entry was attenuated in the absence of TRPC6. Exposure of mouse platelets to acidic pH resulted in abolishment of thrombin-evoked aggregation and attenuated platelet aggregation induced by thapsigargin (TG) or OAG. Both OAG-induced Ca(2+) entry and platelet aggregation were greatly attenuated in cells expressing TRPC6 channels. Exposure of platelets to H2O2 or deferoxamine did not clearly alter thrombin, TG or OAG-induced platelet aggregation. Our results indicate that TRPC6 is sensitive to acidic pH but not to exposure to ROS or hypoxic-like conditions, which might be involved in the pathogenesis of the altered platelet responsiveness to DAG-generating agonists in disorders associated to acidic pH.

Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca(2+) entry by regulation of STIM1-Orai1 association.

TRPA1 is a non-selective Ca(2+) permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca(2+)-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca(2+) entry without altering the release of Ca(2+) from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca(2+) entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca(2+) entry by the modulation of STIM1/Orai1 interaction.

Pharmacology of TRP channels in the vasculature.

The TRP family of cation-permeable channels owes its name to a Drosophila TRP mutant with impaired vision due to transient rather than sustained receptor potential. Mammalian TRP channels can be grouped into 6 subfamilies, including TRPC, TRPM, TRPV, TRPA, TRPP and TRPML and a number of TRP family members have been identified in the vasculature. TRP channels play an important functional role in the vasculature as mediators of cation influx across the plasma membrane, thus contributing to a large number of processes such as vascular smooth muscle contraction and vascular pressure or the responses to oxidative stress, mechanical stimuli, heat and hypoxia-induced vascular remodelling. TRP channelopaties are involved in the pathogenesis of different disorders including hypertension and cardiomyopathy. A number of identified natural compounds and synthetic agents have been reported to modulate TRP function, and are the base for therapeutical strategies.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.

AIMS: Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS: We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSION: Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

The membrane potential modulates thrombin-stimulated Ca²âº mobilization and platelet aggregation.

G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca(2+) concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca(2+) release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF4(-) and m-3M3FBS, respectively, leads to Ca(2+) release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca(2+) entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca(2+) influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca(2+) entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca(2+)-ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca(2+). These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets.

The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium.

STIM1 acts as an endoplasmic reticulum Ca(2+) sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca(2+) binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca(2+) concentrations in store-operated Ca(2+) entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca(2+) in a concentration-dependent manner. Expression of the Ca(2+)-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca(2+). Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn(2+) entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca(2+), which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca(2+) mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca(2+) concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.

Two-pore channel 2 (TPC2) modulates store-operated Ca(2+) entry.

Two-pore channels (TCPS) are NAADP-sensitive receptor channels that conduct Ca(2+) efflux from the intracellular stores. Discharge of the internal Ca(2+) pools results in the activation of store-operated Ca(2+) entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca(2+) into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca(2+) entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, assoicates STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca(2+) stores, but not in resting cells. These results provide strong evidence for modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells.

Capacitative and non-capacitative signaling complexes in human platelets.

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1.

Store-operated Ca(2+) entry (SOCE) is a major pathway for Ca(2+) influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and Ca(2+) permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca(2+) permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca(2+) channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.

Acidic NAADP-releasable Ca(2+) compartments in the megakaryoblastic cell line MEG01.

BACKGROUND: A novel family of intracellular Ca(2+)-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca(2+) mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca(2+) release from the acidic compartments. OBJECTIVES: The present study is aimed to investigate NAADP-mediated Ca(2+) release from intracellular stores in the megakaryoblastic cell line MEG01. METHODS: Changes in cytosolic and intraluminal free Ca(2+) concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. RESULTS: Treatment of MEG01 cells with the H(+)/K(+) ionophore nigericin or the V-type H(+)-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca(2+) stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca(2+) from acidic lysosomal-like Ca(2+) stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca(2+) efflux from the acidic lysosomal-like Ca(2+) stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca(2+)-induced Ca(2+) release (CICR) from the complementary Ca(2+) compartment. CONCLUSION: Our results show for the first time NAADP-evoked Ca(2+) release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

Lipid rafts are essential for the regulation of SOCE by plasma membrane resident STIM1 in human platelets.

STIM1 is a transmembrane protein essential for the activation of store-operated Ca²+ entry (SOCE), a major Ca²+ influx mechanism. STIM1 is either located in the endoplasmic reticulum, communicating the Ca²+ concentration in the stores to plasma membrane channels or in the plasma membrane, where it might sense the extracellular Ca²+ concentration. Plasma membrane-located STIM1 has been reported to mediate the SOCE sensitivity to extracellular Ca²+ through its interaction with Orai1. Here we show that plasma membrane lipid raft domains are essential for the regulation of SOCE by extracellular Ca²+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn²+ entry, which was inhibited by increasing concentrations of extracellular Ca²+. Platelet treatment with methyl-ß-cyclodextrin, which removes cholesterol and disrupts the lipid raft domains, impaired the inactivation of Ca²+ entry induced by extracellular Ca²+. Methyl-ß-cyclodextrin also abolished translocation of STIM1 to the plasma membrane stimulated by treatment with TG and prevented TG-evoked co-immunoprecipitation between plasma membrane-located STIM1 and the Ca²+ permeable channel Orai1. These findings suggest that lipid raft domains are essential for the inactivation of SOCE by extracellular Ca²+ mediated by the interaction between plasma membrane-located STIM1 and Orai1.

Role of oxidant scavengers in the prevention of Ca²+ homeostasis disorders.

A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca²+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca²+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca²+ mobilization induced by ROS.

Lipid rafts modulate the activation but not the maintenance of store-operated Ca(2+) entry.

Different studies have reported that proteins involved in Ca(2+) entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca(2+) entry by facilitating STIM1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of STIM1 with TRPC1. Here we report that treatment of HEK293 cells with thapsigargin (TG) results in the activation of Ca(2+) entry with two components, an early, La(3+)-sensitive, component and a late component that shows both La(3+)-sensitive and -insensitive constituents. Preincubation with methyl-beta-cyclodextrin (MbetaCD) prevented TG-induced activation of Ca(2+) entry but, in contrast, enhanced this process after its activation. Addition of MbetaCD after store depletion did not modify the La(3+)-sensitive store-operated divalent cation entry but increased La(3+)-insensitive non-capacitative Ca(2+) entry. Cell stimulation with TG results in a transient increase in Orai1 co-immunoprecipitation with STIM1, TRPC1 and TRPC6. TG-induced association of these proteins was significantly attenuated by preincubation for 30 min with MbetaCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MbetaCD was added after store depletion with TG. Addition of MbetaCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca(2+) entry. TRPC6 expression silencing indicates that MbetaCD-enhanced non-capacitative Ca(2+) entry was mediated by TRPC6. In conclusion, lipid raft domains are necessary for the activation but not the maintenance of SOCE probably due to the support of the formation of Ca(2+) signalling complexes involving Orai1, TRPCs and STIM1.

Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.

BACKGROUND/AIMS: The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. METHODS: Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. RESULTS: Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells. CONCLUSIONS: NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.