New insights into the molecular mechanisms of store-operated Ca2+ signaling in T cells

The activation of Ca(2+) entry through store-operated channels by agonists that deplete Ca(2+) from the endoplasmic reticulum (ER) is an ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past 20 years. In T lymphocytes, store-operated Ca(2+)-release-activated Ca(2+) (CRAC) channels constitute the sole pathway for Ca(2+) entry following antigen-receptor engagement, and their function is essential for driving the program of gene expression that underlies T-cell activation by antigen. The first molecular components of this pathway have recently been identified: stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and Orai1, a pore-forming subunit of the CRAC channel. Recent work shows that CRAC channels are activated in a complex fashion that involves the co-clustering of STIM1 in junctional ER directly opposite Orai1 in the plasma membrane. These studies reveal an abundance of sites where Ca(2+) signaling might be controlled to modulate the activity of T cells during the immune response.     

Na+/Ca2+ exchanger activity modulates connective tissue growth factor mRNA expression in transforming growth factor beta1- and Des-Arg10-kallidin-stimulated myofibroblasts

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.     

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation

To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.     

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.     

Inhibition by calyculin A and okadaic acid of the Ca(2+) release-activated Ca(2+) entry pathway in rat basophilic leukemia cells: evidence for regulation by type 1/2A serine/threonine phosphatase activity

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.     

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.     

Disruption of the cortical actin cytoskeleton does not affect store operated Ca2+ channels in human T-cells

Lymphocyte signaling and activation leads to the influx of extracellular Ca(2+) via the activation of Ca(2+) release activated Ca(2+) (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca(2+) stores. One model to explain the coupling of store-emptying to CRAC activation is the secretion-like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store-operated Ca(2+) entry in T-cells.     

pH-dependent effect of mitochondria on calcium influx into Jurkat cells; a novel mechanism of cell protection against calcium entry during energy stress

Loss of the mitochondrial membrane potential results in a significant inhibition of calcium influx through calcium release-activated channels (CRAC) in Jurkat cells suspended in the medium of pH lower than 7.4. This effect disappears when the medium pH increases. Alkalinisation of the cytosol achieved by the addition of NH(4)Cl to the cells pretreated with thapsigargin, CCCP and CaCl(2), suspended in the medium of pH 7.2, does not affect CRAC activity, while alkalisation of the extracellular milieu by NaOH results in a strong stimulation of calcium entry. Thus, the mitochondrial effect on CRAC is exclusively related to the extracellular pH.Coupled mitochondria are able to take up Ca(2+) accumulated in the close proximity of CRAC. This protects these channels against feedback inhibition exerted by high [Ca(2+)](c). We conclude that CRAC may exist in two conformations: inhibitable and not inhibitable by cytosolic Ca(2+). Lower extracellular pH promotes the former one. This explains a much higher inhibitory effect of mitochondrial uncouplers on the calcium influx into the cells exposed to pH 7.2 than that observed in the cells suspended in the medium of pH 7.8. This phenomenon may provide an additional mechanism protecting cells against calcium overloading in transient episodes of energy stress.     

Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli

Intracellular Ca(2+) plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca(2+)-release-activated calcium (CRAC) channels are the primary Ca(2+) entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca(2+)-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca(2+) signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca(2+) permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.     

Functional consequences of activating store-operated CRAC channels

Store-operated CRAC channels, which are activated by the emptying of the endoplasmic reticulum Ca(2+) stores, are an important and widespread route for triggering rises in cytoplasmic Ca(2+). The cellular responses that are activated in response to Ca(2+) entry through CRAC channels are being dissected out, and recent evidence has established that CRAC channels can induce both short-term (safeguarding the Ca(2+) content of the endoplasmic reticulum, maintenance of cytoplasmic Ca(2+) oscillations, enzyme activation, secretion) and long-term (gene expression) changes in cells. CRAC channel activation is therefore capable of evoking a range of temporally distinct responses, highlighting the versatility of this ubiquitous Ca(2+) entry pathway.     

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Ca2+ modulation of Ca2+ release-activated Ca2+ channels is responsible for the inactivation of its monovalent cation current

The Ca(2+) release-activated Ca(2+) (CRAC) channel is the most well documented of the store-operated ion channels that are widely expressed and are involved in many important biological processes. However, the regulation of the CRAC channel by intracellular or extracellular messengers as well as its molecular identity is largely unknown. Specifically, in the absence of extracellular divalent cations it becomes permeable to monovalent cations with a larger conductance, however this monovalent cation current inactivates rapidly by an unknown mechanism. Here we found that Ca(2+) dissociation from a site on the extracellular side of the CRAC channel is responsible for the inactivation of its Na(+) current, and Ca(2+) occupancy of this site otherwise potentiates its Ca(2+) as well as Na(+) currents. This Ca(2+)-dependent potentiation is required for the normal functioning of CRAC channels.     

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion

Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked cytosolic free Ca2+ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+ entry.     

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

Potentiation of Fcepsilon receptor I-activated Ca(2+) current (I(CRAC)) by cholera toxin: possible mediation by ADP ribosylation factor

Antigen-evoked influx of extracellular Ca(2+) into mast cells may occur via store-operated Ca(2+) channels called calcium release-activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3), cholera toxin (CT) potentiates antigen-driven uptake of (45)Ca(2+) through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca(2)+ release-activated Ca(2+) current (I(CRAC)) elicited by suboptimal concentrations of antigen, without itself inducing I(CRAC), and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of I(CRAC) by thapsigargin, an inhibitor of organelle Ca(2+) pumps, or by intracellular dialysis with low Ca(2+) pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gsalpha. Nor was the potentiation of I(CRAC) due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked I(CRAC) to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked I(CRAC) without inhibiting ADP ribosylation of Gsalpha, but it did not affect I(CRAC) induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for Fcin receptor I-triggered Ca(2+) influx, and they suggest that ARF may modulate the induction of I(CRAC) by antigen.     

Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity

The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.     

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions

The activation of store-operated Ca(2+) entry by Ca(2+) store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca(2+) sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10-25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca(2+) influx through open Ca(2+) release-activated Ca(2+) (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca(2+) entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca(2+) influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca(2+) entry.