Regulation Of Store-operated Ca2+ Entry Research Articles

Promoting effects of IL-13 on Ca 2+ release and store-operated Ca 2+ entry in airway smooth muscle cells

Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca 2+ signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca 2+ entry (SOCE) plays an important role in regulating Ca 2+ signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca 2+ fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca 2+ ([Ca 2+] i) level, Ca 2+ release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting β2 adrenergic agonist (β2 agonist) salbutamol suppressed IL-13-augumented basal [Ca 2+] i, Ca 2+ release and SOCE, whereas the long-acting β2 agonist salmeterol had no effect on altered Ca 2+ signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca 2+ release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca 2+ channel blockers SKF-96365 and NiCl 2, by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca 2+ release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and β2 agonists in inhibiting Ca 2+ signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.

Abstract 5661: Akt/mTOR is an Essential Downstream Pathway of Platelet-Derived Growth Factor Signaling in Pulmonary Artery Smooth Muscle Cells

Background: Platelet-derived growth factor (PDGF) is known to be a potent mitogen involved in the development and progression of pulmonary vascular remodeling. Recently, PDGF has attracted attention due to the inhibition of its receptor having a positive effect on hemodynamics, remodeling, and survival. Indeed, a PDGF receptor inhibitor is now undergoing clinical trials. It is, however, unclear which downstream pathway is important in pulmonary vascular remodeling. The Akt/mTOR pathway is central to many biological responses including cell growth, proliferation, survival, and metabolism. The involvement of the Akt/mTOR pathway in PDGF signaling was evaluated, in addition to a potential role in store-operated Ca 2+ entry (SOCE), which we previously reported to be important in cell proliferation and vascular remodeling in idiopathic and thromboembolic pulmonary hypertension. Methods and Results: In human pulmonary artery smooth muscle cells (PASMC) and fibroblasts, PDGF transiently phosphorylated Akt, mTOR and p70S6K (24.8-, 3.6-, 8.1-fold increase in 15 minutes, P<0.01, 0.001, 0.01, respectively). Interestingly, PDGF failed to induce Akt phosphorylation in pulmonary artery endothelial cells. Chronic treatment with PDGF (10 ng/ml) significantly enhanced the increase in cytosolic Ca 2+ due to cyclopiazonic acid (10 microM)-induced Ca 2+ mobilization (from 0.75±0.02 to 0.88±0.03 arbitrary unit (a.u.), P<0.001) and SOCE (from 1.03±0.02 to 1.51±0.05 a.u., P<0.001). When treated with rapamycin (10 nM), an inhibitor of mTOR, or Akt inhibitor (1 microM), SOCE was significantly reduced (to 1.28±0.05, 0.60±0.02 a.u., respectively, P <0.001). Conclusions: The data suggest that Akt/mTOR signaling is an important pathway in the regulation of SOCE in PASMC. A greater understanding of this pathway may enable the development of novel therapeutic approaches in patients with pulmonary vascular disease.

Essential role of STIM1 in the development of cardiomyocyte hypertrophy

Store-operated Ca2+ entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.

Store-operated Ca 2+ entry is sensitive to the extracellular Ca 2+ concentration through plasma membrane STIM1

Store-operated Ca 2+ entry (SOCE) is a major mechanism for Ca 2+ influx in platelets and other cells activated by a reduction in Ca 2+ concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca 2+, although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca 2+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn 2+ entry, which was inhibited by extracellular Ca 2+ in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25–139 of PM-STIM1 that contains the Ca 2+-binding domain, prevented the inactivation of Ca 2+ entry induced by extracellular Ca 2+. TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca 2+-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca 2+, an event that is likely to be mediated by interaction with Orai1.

STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.

Role of store-operated Ca2+ entry in adenosine-induced vasodilatation of rat small mesenteric artery

Store-operated Ca(2+) entry (SOCE) has recently been proposed to contribute to Ca(2+) influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular Ca(2+) concentration of fluo 3-loaded VSMCs. Adenosine (0.1-100 microM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca(2+) influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca(2+) levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A(2A) receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A(2A) receptors.

Structural And Mechanistic Insights Into Stim1-mediated Initiation Of Store Operated Calcium Entry.

Stromal interaction molecule-1 (STIM1) activates store operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. We have recently determined the solution structure of the Ca2+−sensing region of STIM1 consisting of the EF-hand and sterile α motif (SAM) domains (EF-SAM) (Stathopulos et al. Cell Oct 3rd issue, 2008). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, ‘hidden’ EF-hand or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+ sensing region of STIM is crucial to SOCE regulation. (Supported by CIHR and CFI).

Tightly Regulated Store-operated Ca2+ Entry In Healthy And Dystrophic Skeletal Muscle

Store-operated Ca2+ entry (SOCE) is a mechanism that allows the entry of extracellular Ca2+ upon depletion of the internal stores. This mechanism has been described in skeletal muscle and the two main molecular players, Stim1 and Orai1, have been identified. Previous work, mainly on myotubes, suggested that SOCE may become deregulated in dystrophic skeletal muscle and result in cellular Ca2+ overload. The final result of such a process would be activation of proteolytic enzymes, cell necrosis and/or apoptosis. To examine the regulation of SOCE in healthy and dystrophic muscle we examined the biochemistry and physiology of skeletal muscle from wild-type (wt) and mdx mice (8-20 weeks old). Western blotting of single fibres showed that Stim1 and Orai1 were expressed at 2-3 times higher levels in mdx compared with wt muscle (normalized to total myosin). Consistent with this, enzymatically isolated interossei fibres loaded with fluo-4AM and depleted of Ca2+ in a solution containing 0 Ca2+, 20 μM cyclopiazonic acid and 10 mM caffeine showed a 3-fold higher rate of Ca2+ entry into the depleted mdx fibres compared to wt upon re-addition of 2 mM Ca2+. However, this does not imply Ca2+ overload will occur via SOCE in dystrophic cells if deactivation is unaffected. To study SOCE kinetics, skinned fibres with t-system trapped fluo-5N were bathed in an internal solution with rhod-2 and continuously imaged in xyt mode on a confocal microscope. Intracellular Ca2+ release was induced by lowering cytoplasmic [Mg2+]. Transient t-system Ca2+-depletion and reuptake was preceded by transient SR Ca2+-release and reuptake in fibres from both mdx and wt mice. This indicates robust activation and deactivation mechanisms of SOCE in both wt and dystrophic muscle which prevent not only depletion but also overloading of the internal Ca2+-stores.

OAG induces an additional PKC-, PI3K-, and Rac2-mediated signaling pathway up-regulating NOX2 activity, independently of Ca2+ entry

The requirement of calcium ion (Ca(2)(+)) entry for neutrophil NADPH oxidase (NOX2) regulation is clearly established. However, its role in the signaling pathway leading to NOX2 activation is still elusive. 1-oleoyl-2-acetyl-sn-glycerol (OAG) causes an increase in NOX2 activity and has been shown to directly modulate Ca(2)(+) channels unrelated to the well-known store-operated Ca(2)(+) entry (SOCE) mechanism. In our study, we have investigated the potential role of OAG in Ca(2)(+) influx-mediated NOX2 activity in neutrophil-like-differentiated HL-60 cells to further characterize second signals involved in the regulation of NOX2. OAG inhibited fMLF- and thapsigargin-induced Ca(2)(+) entry, a phenomenon that was not restored by protein kinase C (PKC) or PI3K blockade. Addition of OAG resulted in a rapid decrease of maximal intracellular Ca(2)(+) concentration induced by thapsigargin. Both results suggest that OAG has an inhibitory effect, independent of PI3K and PKC, on the regulation of SOCE. In contrast to SOCE inhibition, OAG-induced NOX2 activation was mediated by PKC and PI3K. Our data establish that both kinases exert their effects through the regulation of Rac2 activity. In addition, OAG potentiated the effect of fMLF on the activation of NOX2 and led to a discernible activity of NOX2 upon thapsigargin stimulation. In conclusion, our results demonstrate that an additional PKC- and/or PI3K-dependent signal may act in synergy with Ca(2)(+) influx to trigger NOX2 activation.

Structural and Mechanistic Insights into STIM1-Mediated Initiation of Store-Operated Calcium Entry

Stromal interaction molecule-1 (STIM1) activates store-operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. Here, we present the atomic structure of the Ca2+-sensing region of STIM1 consisting of the EF-hand and sterile alpha motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+-sensing region of STIM is crucial to SOCE regulation.

Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.

Complex regulation of store-operated Ca2+ entry pathway by PKC-ε in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca 2+stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca 2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca 2+-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca 2+content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca 2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ, individually or simultaneously, accelerated Ca 2+ store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca 2+ influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca 2+ extrusion by PMCA or the Na +/Ca 2+ exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.

Role of Ca 2+ -Independent Phospholipase A 2 and Store-Operated Pathway in Urocortin-Induced Vasodilatation of Rat Coronary Artery

Urocortin has been shown to produce vasodilatation in several arteries, but the precise mechanism of its action is still poorly understood. Here we demonstrate the role of store operated Ca2+ entry (SOCE) regulated by Ca2+-independent phospholipase A2 (iPLA2) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentration-dependent (IC50 approximately = 4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA2 activation, a crucial step for SOC channel activation, and prevents Ca2+ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA2, exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA2 mRNA and proteins expression in rat coronary smooth muscle cells. In summary, we propose a new mechanism of vasodilatation by urocortin which involves the regulation of iPLA2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery.

Relocalization of STIM1 for Activation of Store-operated Ca2+ Entry Is Determined by the Depletion of Subplasma Membrane Endoplasmic Reticulum Ca2+ 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.

Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

Gene expression profiles were generated using cDNA microarray technology for clones of human embryonic kidney (HEK)-293 cells selected to have either high or low levels of store-operated Ca2+ entry (SOCE). For five high clones, three low clones, and control HEK-293 cells, duplicate Affymetrix U133A human gene arrays were run after extraction of total RNA from cells growing in the presence of serum. Of the approximately 22,000 genes represented on the microarray, 58 genes had readings at least twofold higher, while 32 genes had readings at least twofold lower, in all five high SOCE clones compared with control HEK-293 cells. In the low SOCE clones, 92 genes had readings at least twofold higher, while 58 genes had readings at least twofold lower, than in HEK-293 cells. Microarray results were confirmed for 18 selected genes by real-time RT-PCR analysis; for six of those genes, predicted changes in the low SOCE clone were confirmed by an alternative method, monitoring mRNA levels in HEK-293 with SOCE decreased by expression of small interfering (si)RNA to canonical transient receptor potential protein-1. Genes regulated by SOCE are involved in signal transduction, transcription, apoptosis, metabolism, and membrane transport. These data provide insight into the physiological role of SOCE. In addition, a potential regulator of SOCE, insulin receptor substrate (IRS)-2, has been identified. A reduction of IRS-2 levels by siRNA methods in two high clones dramatically reduced SOCE, whereas overexpression of IRS-2 in a low SOCE clone elevated SOCE.

Calcium-Dependent Facilitation and Graded Deactivation of Store-Operated Calcium Entry in Fetal Skeletal Muscle

Activation of store-operated Ca 2+ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca 2+ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca 2+ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca 2+ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca 2+ into the sarcoplasmic reticulum (SR). A novel Ca 2+-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca 2+. Our data suggest that cytosolic Ca 2+ can facilitate SOCE whereas SR luminal Ca 2+ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca 2+ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca 2+ entry in response to physiological demand. Such Ca 2+ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.

Ca2+-Calmodulin-dependent Protein Kinase II Potentiates Store-operated Ca2+ Current

A rise in intracellular Ca2+ (Ca2+i) mediates various cellular functions ranging from fertilization to gene expression. A ubiquitous Ca2+ influx pathway that contributes significantly to the generation of Ca2+i signals, especially in non-excitable cells, is store-operated Ca2+ entry (SOCE). Consequently, the modulation of SOCE current affects Ca2+i dynamics and thus the ensuing cellular response. Therefore, it is important to define the mechanisms that regulate SOCE. Here we show that a rise in Ca2+i potentiates SOCE. This potentiation is mediated by Ca2+-calmodulin-dependent protein kinase II (CaMKII), because inhibition of endogenous CaMKII activity abrogates Ca2+i-mediated SOCE potentiation and expression of constitutively active CaMKII potentiates SOCE current independently of Ca2+i. Moreover, we present evidence that CaMKII potentiates SOCE by altering SOCE channel gating. The regulation of SOCE by CaMKII defines a novel modulatory mechanism of SOCE with important physiological consequences.

Calmodulin Regulates Ca 2+-Dependent Feedback Inhibition of Store-Operated Ca 2+ Influx by Interaction with a Site in the C Terminus of TrpC1

The mechanism involved in [Ca 2+] i-dependent feedback inhibition of store-operated Ca 2+ entry (SOCE) is not yet known. Expression of Ca 2+-insensitive calmodulin (Mut-CaM) but not wild-type CaM increased SOCE and decreased its Ca 2+-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664–793 (TrpC1ΔC) also attenuated Ca 2+-dependent inactivation of SOCE. CaM interacted with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1ΔC. Two CaM binding domains, aa 715–749 and aa 758–793, were identified. Expression of TrpC1Δ758–793 but not TrpC1Δ715–749 mimicked the effects of TrpC1ΔC and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca 2+-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE.