Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Local cytosolic Ca2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca2+ entry

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.     

Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death

The goal of the current study was to determine the roles of ATP content, endoplasmic reticulum (ER) Ca(2+) stores, cytosolic free Ca(2+) (Ca(2+)(f)) and calpain activity in the signaling of rabbit renal proximal tubular (RPT) cell death (oncosis). Increasing concentrations (0.3-10 microM) of the mitochondrial inhibitor antimycin A produced rapid ATP depletion that correlated to a rapid and sustained increase in Ca(2+)(f), but not phospholipase C activation. The ER Ca(2+)-ATPase inhibitors thapsigargin (5 microM) or cyclopiazonic acid (100 microM) alone produced similar but transient increases in Ca(2+)(f). Pretreatment with thapsigargin prevented antimycin A-induced increases in Ca(2+)(f) and antimycin A pretreatment prevented thapsigargin-induced increases in Ca(2+)(f). Calpain activity increased in conjunction with ER Ca(2+) release. Pretreatment, but not post-treatment, with thapsigargin or cyclopiazonic acid prevented antimycin A-induced cell death. These data demonstrate that extensive ATP depletion signals oncosis through ER Ca(2+) release, a sustained increase in Ca(2+)(f) and calpain activation. Depletion of ER Ca(2+) stores prior to toxicant exposure prevents increases in Ca(2+)(f) and oncosis.     

Mitochondria adjust Ca(2+) signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca(2+) signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca(2+) signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca(2+) entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca(2+) machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca(2+) imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca(2+) signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca(2+) handling system directing more Ca(2+) into mitochondria via microdomains of high [Ca(2+)] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca(2+)-ATPase-mediated Ca(2+) uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca(2+) fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

The novel endoplasmic reticulum (ER)-targeted protein HAP induces cell apoptosis by the depletion of the ER Ca(2+) stores

HAP, a novel human apoptosis-inducing protein, was identified to localize exclusively to the endoplasmic reticulum (ER) in our previous work. In the present work, we reported that ectopic overexpression of HAP proteins caused the rapid and sustained elevation of the intracellular cytosolic Ca(2+), which originated from the reversible ER Ca(2+) stores release and the extracellular Ca(2+) influx. The HeLa cells apoptosis induced by HAP proteins was not prevented by establishing the clamped cytosolic Ca(2+) condition, or by buffering of the extracellular Ca(2+) with EGTA, suggesting that the depletion of ER Ca(2+) stores rather than the elevation of cytosolic Ca(2+) or the extracellular Ca(2+) entry contributed to HAP-induced HeLa cells apoptosis. Caspase-3 was also activated in the process of HAP-triggered apoptotic cell death.     

A quantitative kinetic model for ATP-induced intracellular Ca2+ oscillations

A quantitative kinetic model is proposed to simulate the ATP-induced intracellular Ca(2+) oscillations. The quantitative effect of ATP concentration upon the oscillations was successfully simulated. Our simulation results support previous experimental explanations that the Ca(2+) oscillations are mainly due to interaction of Ca(2+) release from the endoplasmic reticulum (ER) and the ATP-dependent Ca(2+) pump back into the ER, and the oscillations are prolonged by extracellular Ca(2+) entry that maintains the constant Ca(2+) supplies to its intracellular stores. The model is also able to simulate the sudden disappearance phenomenon of the Ca(2+) oscillations observed in some cell types by taking into account of the biphasic characteristic of the Ca(2+) release from the endoplasmic reticulum (ER). Moreover, the model simulation results for the Ca(2+) oscillations characteristics such as duration, peak [Ca(2+)](cyt), and average interval, etc., lead to prediction of some possible factors responsible for the variations of Ca(2+) oscillations in different types of cells.     

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

Dysregulation of Ca2+ signaling in astrocytes from mice lacking amyloid precursor protein

The relationship between altered metabolism of the amyloid-β precursor protein (APP) and Alzheimer's disease is well established but the physiological roles of APP still remain unclear. Here, we studied Ca(2+) signaling in primary cultured and freshly dissociated cortical astrocytes from APP knockout (KO) mice and from Tg5469 mice overproducing by five- to sixfold wild-type APP. Resting cytosolic Ca(2+) (measured with fura-2) was not altered in cultured astrocytes from APP KO mice. The stored Ca(2+) evaluated by measuring peak amplitude of cyclopiazonic acid [CPA, endoplasmic reticulum (ER) Ca(2+) ATPase inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium was significantly smaller in APP KO astrocytes than in wild-type cells. Store-operated Ca(2+) entry (SOCE) activated by ER Ca(2+) store depletion with CPA was also greatly reduced in APP KO astrocytes. This reflected a downregulated expression in APP KO astrocytes of TRPC1 (C-type transient receptor potential) and Orai1 proteins, essential components of store-operated channels (SOCs). Indeed, silencer RNA (siRNA) knockdown of Orai1 protein expression in wild-type astrocytes significantly attenuated SOCE. SOCE was also essentially reduced in freshly dissociated APP KO astrocytes. Importantly, knockdown of APP with siRNA in cultured wild-type astrocytes markedly attenuated ATP- and CPA-induced ER Ca(2+) release and extracellular Ca(2+) influx. The latter correlated with downregulation of TRPC1. Overproduction of APP in Tg5469 mice did not alter, however, the stored Ca(2+) level, SOCE, and expression of TRPC1/4/5 in cultured astrocytes from these mice. The data demonstrate that the functional role of APP in astrocytes involves the regulation of TRPC1/Orai1-encoded SOCs critical for Ca(2+) signaling.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Stanniocalcin 2 is a negative modulator of store-operated calcium entry

The regulation of cellular Ca(2+) homeostasis is essential for innumerable physiological and pathological processes. Stanniocalcin 1, a secreted glycoprotein hormone originally described in fish, is a well-established endocrine regulator of gill Ca(2+) uptake during hypercalcemia. While there are two mammalian Stanniocalcin homologs (STC1 and STC2), their precise molecular functions remain unknown. Notably, STC2 is a prosurvival component of the unfolded protein response. Here, we demonstrate a cell-intrinsic role for STC2 in the regulation of store-operated Ca(2+) entry (SOCE). Fibroblasts cultured from Stc2 knockout mice accumulate higher levels of cytosolic Ca(2+) following endoplasmic reticulum (ER) Ca(2+) store depletion, specifically due to an increase in extracellular Ca(2+) influx through store-operated Ca(2+) channels (SOC). The knockdown of STC2 expression in a hippocampal cell line also potentiates SOCE, and the overexpression of STC2 attenuates SOCE. Moreover, STC2 interacts with the ER Ca(2+) sensor STIM1, which activates SOCs following ER store depletion. These results define a novel molecular function for STC2 as a negative modulator of SOCE and provide the first direct evidence for the regulation of Ca(2+) homeostasis by mammalian STC2. Furthermore, our findings implicate the modulation of SOCE through STC2 expression as one of the prosurvival measures of the unfolded protein response.     

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.     

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.     

On the role of TRPC1 in control of Ca2+ influx, cell volume, and cell cycle

Ca(+) signaling plays a crucial role in control of cell cycle progression, but the understanding of the dynamics of Ca(2+) influx and release of Ca(2+) from intracellular stores during the cell cycle is far from complete. The aim of the present study was to investigate the role of the free extracellular Ca(2+) concentration ([Ca(2+)](o)) in cell proliferation, the pattern of changes in the free intracellular Ca(2+) concentration ([Ca(2+)](i)) during cell cycle progression, and the role of the transient receptor potential (TRP)C1 in these changes as well as in cell cycle progression and cell volume regulation. In Ehrlich Lettré Ascites (ELA) cells, [Ca(2+)](i) decreased significantly, and the thapsigargin-releasable Ca(2+) pool in the intracellular stores increased in G(1) as compared with G(0). Store-depletion-operated Ca(2+) entry (SOCE) and TRPC1 protein expression level were both higher in G(1) than in G(0) and S phase, in parallel with a more effective volume regulation after swelling [regulatory volume decrease (RVD)] in G(1) as compared with S phase. Furthermore, reduction of [Ca(2+)](o), as well as two unspecific SOCE inhibitors, 2-APB (2-aminoethyldiphenyl borinate) and SKF96365 (1-(β-[3-(4-methoxy-phenyl)propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride), inhibited ELA cell proliferation. Finally, Madin-Darby canine kidney cells in which TRPC1 was stably silenced [TRPC1 knockdown (TRPC1-KD) MDCK] exhibited reduced SOCE, slower RVD, and reduced cell proliferation compared with mock controls. In conclusion, in ELA cells, SOCE and TRPC1 both seem to be upregulated in G(1) as compared with S phase, concomitant with an increased rate of RVD. Furthermore, TRPC1-KD MDCK cells exhibit decreased SOCE, decreased RVD, and decreased proliferation, suggesting that, at least in certain cell types, TRPC1 is regulated during cell cycle progression and is involved in SOCE, RVD, and cell proliferation.     

Effects of aging on inositol 1,4,5-triphosphate-induced Ca(2+) release in unfertilized mouse oocytes

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca(2+)-ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383-390). We undertook the present study to further examine the effects of oocyte aging on Ca(2+) release from the inositol 1,4,5-triphosphate (InsP(3))-sensitive Ca(2+) channels of the ER membrane, because not only Ca(2+) reuptake, but also Ca(2+) release from the ER, substantially affect Ca(2+) oscillations in fertilized oocytes. A transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was induced by photolysis of caged InsP(3) microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca(2+)](i) significantly decreased in the aged oocytes. Reduced ER Ca(2+) release in the aged oocyte may not be attributable to aging-related desensitization of the InsP(3)-sensitive Ca(2+) channels in the ER because concentrations of caged InsP(3) for half maximal [Ca(2+)](i) increase were identical for fresh and aged oocytes. The peak [Ca(2+)](i) response following administration of 5 microM thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca(2+) stores. We conclude from these results that reduction of Ca(2+) release from the InsP(3)-sensitive Ca(2+) stores in the aged oocyte arises from depletion of the ER Ca(2+) stores with aging. These aging-related changes in Ca(2+) release and reuptake may account for alterations in Ca(2+) oscillations in aged fertilized oocytes.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells.

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.     

Regulation of inositol 1,4,5-trisphosphate receptor-mediated calcium release by the Na/K-ATPase in cultured renal epithelial cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.     

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.