Depletion Of ER Ca2 Research Articles

Capsaicin-insensitivity of TRPV1-R575D mutant located at the lipid-water-interface region can be rescued by either extracellular Ca2+-chelation or cholesterol reduction

TRPV1 acts as a unique polymodal ion channel having distinct structure and gating properties. In this context, TRPV1-R575D represents a special mutant located at the inner lipid-water-interface (LWI) region that has less possibility of interaction with membrane cholesterol. In control conditions, this lab-generated mutant of TRPV1 shows no “ligand-sensitivity”, reduced surface expression, reduced localization in the lipid rafts, yet induces high cellular lethality. Notably, the cellular lethality induced by TRPV1-R575D expression can be rescued by adding 5′I-RTX (a specific inhibitor of TRPV1) or by introducing another mutation in the next position, i.e. in TRPV1-R575D/D576R. In this work we characterized TRPV1-R575D and TRPV1-R575D/D576R mutants in different cellular conditions and compared with the TRPV1-WT. We report that the “ligand-insensitivity” of TRPV1-R575D can be rescued in certain conditions, such as by chelation of extracellular Ca2+, or by reduction of the membrane cholesterol. Here we show that Ca2+ plays an important role in the channel gating of TRPV1-WT as well as LWI mutants (TRPV1-R575D, TRPV1-R575D/D576R). However, chelation of intracellular Ca2+ or depletion of ER Ca2+ did not have a significant effect on the TRPV1-R575D. Certain properties related to channel gating of mutant TRPV1-R575D/D576R can be rescued partially or fully in a context -dependent manner. Cholesterol depletion also alters these properties. Our data suggests that lower intracellular basal Ca2+ acts as a pre-requisite for further opening of TRPV1-R575D. These findings enable better understanding of the structure-function relationship of TRPV1 and may be critical in comprehending the channelopathies induced by other homologous thermosensitive TRPVs.

Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes

In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Differential modulation of short-term plasticity at hippocampal mossy fiber and Schaffer collateral synapses by mitochondrial Ca2.

Presynaptic mitochondrial Ca2+ plays a critical role in the regulation of synaptic transmission and plasticity. The presynaptic bouton of the hippocampal mossy fiber (MF) is much larger in size than that of the Schaffer collateral (SC) synapse. Here we compare the structural and physiological characteristics of MF and SC presynaptic boutons to reveal functional and mechanistic differences between these two synapses. Our quantitative ultrastructural analysis using electron microscopy show many more mitochondria in MF presynaptic bouton cross-section profiles compared to SC boutons. Consistent with these results, post-tetanic potentiation (PTP), a form of presynaptic short-term plasticity dependent on mitochondrial Ca2+, is reduced by inhibition of mitochondrial Ca2+ release at MF synapses but not at SC synapses. However, blockade of mitochondrial Ca2+ release results in reduction of PTP at SC synapses by disynaptic MF stimulation. Furthermore, inhibition of mitochondrial Ca2+ release selectively decreases frequency facilitation evoked by short trains of presynaptic stimulation at MF synapses, while having no effect at SC synapses. Moreover, depletion of ER Ca2+ stores leads to reduction of PTP at MF synapses, but PTP is unaffected by ER Ca2+ depletion at SC synapses. These findings show that MF and SC synapses differ in presynaptic mitochondrial content as well as mitochondrial Ca2+ dependent synaptic plasticity, highlighting differential regulatory mechanisms of presynaptic plasticity at MF and SC synapses.

Blocking Store‐Operated Ca2+ Entry to Protect Cardiomyocytes from Epirubicin‐Induced Toxicity

Epirubicin (EPI) is one of widely used anthracycline chemotherapy drugs, yet its cardiotoxicity severely limits its clinical application. Altered intracellular Ca2+ homeostasis has been linked to epirubicin‐induced cell death and hypertrophy. While store‐operated Ca2+ entry (SOCE) recently has been linked with cardiac hypertrophy and heart failure, its role in epirubicin‐induced cardiotoxicity remains unknown.METHODHL‐1, a cardiomyocyte cell line derived from adult mouse atria, was used in this study. Intracellular ROS was measured by dihydroethidium(DHE) fluorescence and intracellular Ca2+ was monitored by ratiometric dye Fura‐2 on a spectrofluorometer (excitation λ = 350/385nm and emission λ = 510nm; Photon Technology International, NJ). Upon depletion of ER Ca2+ stores by 10μM thapsigargin (TG) in 0mM Ca2+ extracellular solution, SOCE was activated when extracellular solution was rapidly exchanged to 2mM Ca2+. Apoptosis assay was conducted by nuclei staining, F‐actin phalloidin staining and Western Blotting of caspase‐3. While cell size was compared in HL‐1 cells with different treatments using Image J, the mRNA of Orai1, Orai3, STIM1 and B‐type natriuretic peptide (BNP) were also compared using real time qPCR.RESULTSIncubation of 1uM EPI for 30min induced significant ROS production in cultured HL‐1 cells compared with vehicle control (10410±1492 vs. 2762±939, p<0.0001). EPI induced an enhanced SOCE (0.1214±0.0104 vs. 0.0682±0.0062, n=32, p=0.0008), likely through increased expression of SOCE components Orai1, Orai3, and STIM1 compared with control cells. Apoptosis was observed upon EPI treatment, indicated by the condensed and fragmented nuclei, degradation of F‐actin and increased cleavage of caspase‐3 protein. EPI treatment also resulted in larger cell size (3894±120 μm2) compared with non‐treated control cells (2452±57 μm2, n=581, p<0.0001). The hypertrophy marker BNP was up‐regulated upon EPI treatment as well. BTP‐2, a known SOCE blocker, could block this EPI‐induced SOCE and rescue HL‐1 cells from EPI‐induced apoptosis. BTP‐2 also prevented HL‐1 cells from EPI‐induced hypertrophy in terms of cell size (see Figure , 2548±69 μm2) and also reduction of BNP expression.CONCLUSIONEpirubicin upregulated SOCE, induced apoptosis and hypertrophy in HL‐1 cardiomyocytes. Blocking SOCE by BTP‐2 could at least partially rescue HL‐1 cells from epirubicin‐induced apoptosis and hypertrophy.Support or Funding Information(1) NIH S10 OD025230 to Dr. Zui Pan(2) University of Texas at Arlington Center for Research and Scholarship Pilot Grant for Trainee to Xian LiuFigure 1

SEPT7-mediated regulation of Ca2+ entry through Orai channels requires other septin subunits.

Orai channels are plasma membrane resident Ca2+ channels that allow extracellular Ca2+ uptake after depletion of ER-Ca2+ stores by a process called store-operated Ca2+ entry (SOCE). Septins of the SEPT2 subgroup act as positive regulators of SOCE in human nonexcitable cells. SEPT2 subgroup septins form the central core of hetero-hexameric or hetero-octameric complexes with SEPT6, SEPT7 and SEPT9 subgroup septins. The presence of fewer septin encoding genes coupled with ease of genetic manipulation allows for better understanding of septin subgroup function in Drosophila. Our earlier findings show that although dSEPT7 reduction does not alter Orai-mediated Ca2+ entry during SOCE, it results in constitutive activation of Orai channels in resting neurons. Here, we have investigated the role of other septin subgroup members in regulating Orai channel activation in Drosophila neurons by both cellular and functional assays. We show that dSEPT1, a SEPT2 subgroup septin can exist in a complex with dSEPT2 and dSEPT7 in the central nervous system (CNS) of Drosophila. Our findings suggest that the nature of septin filaments and heteromers obtained after reducing septins of different subgroups alters their ability to regulate Orai channel opening. The molecular mechanisms underlying this complex regulation of Orai function by septins require further cellular investigations.

STIM1 Knockout Enhances PDGF-Mediated Ca2+ Signaling through Upregulation of the PDGFR⁻PLCγ⁻STIM2 Cascade.

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR–PLCγ–STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ–IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.

Cell surface GRP78 activation by anti-GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.

31 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increases PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titers were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Abstract 4594: Involvement of receptors tyrosine kinase in the modulation of store-operated calcium entry

Abstract A multitude of studies show that alterations in Ca2+ signalling initiate or support the development of hallmarks of cancer. In particular, the role of store-operated Ca2+ entry (SOCE) in tumorigenesis and tumour progression has been subject to intense investigation. SOCE is the main mechanism by which external Ca2+ enters into the cell. It is initiated by depletion of ER Ca2+ stores and mediated by several proteins such as STIM1, an ER transmembrane protein sensing Ca2+ within the ER and ORAI1, a plasma membrane Ca2+ channel. We recently showed that SOC inhibition interfered with EGFR-dependent signalling in non-small cell lung carcinoma cells. In this study, we observed that STIM1 depletion reduced neuregulin-dependent proliferation of breast cancer cells. Since neuregulin binds to ErbB3 and/or ErbB4 and therefore activates ErbB2, this result prompted us to investigate whether ErbB proteins might modulate SOCE. We observed that lapatinib, a dual inhibitor of EGFR and ErbB2, dramatically inhibited SOCE. As expected, lapatinib also inhibited phosphorylation of EGFR, ErbB2 and downstream pathways of both receptors. Specific inhibition of EGFR by erlotinib or gefitinib had no effect on SOCE. In contrast, specific inhibition of ErbB2 by CP724714 mimicked the effects of lapatinib. We also investigated the role of downstream pathways of ErbB2 in the modulation of SOCE. Inhibition of the MAPK and the JAK-STAT pathways does not modify the amplitude of SOCE. Contrariwise, LY294002 and MK2206, two inhibitors of the PI3K pathway, dramatically reduced SOCE. Interestingly, in silico analysis showed that STIM1 might be phosphorylated by Akt. All these results suggest that SOCE is important in the proper function of ErbB2-dependent signalling. This raises the possibility to target molecular mediators of SOCE in order to interfere with the proliferation of cancer cells. Citation Format: Noémie Emeriau. Involvement of receptors tyrosine kinase in the modulation of store-operated calcium entry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4594.

Effect of anti-GRP78 autoantibodies on prostate cancer progression through tissue factor procoagulant activity.

258 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increase PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumour growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titres were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumour growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Activation of calcium-sensing receptor increases TRPC3/6 expression in T lymphocyte in sepsis

Sepsis is a systemic inflammatory response syndrome induced by infection. T Lymphocytes play an important role in this disease. Transient receptor potential (TRP) channels and calcium-sensing receptors (CaSR) are expressed in lymphocytes to promote intracellular Ca2+ release. However, data about the link between CaSR and TRP channels in septic T lymphocytes are few. In this study, by Ca2+ imaging and Western blotting, we found that in septic rat peripheral blood T lymphocytes expressions of TRPC3 and TRPC6 proteins are higher. The SR/ER Ca2+ ATPase inhibitor thapsigargin (TG) and CaSR agonist NPS R-568 also increased expressions of TRPC3 and TRPC6 proteins, which were reversed by PLC-IP3 channel blocker U73122 and TRPC channels inhibitor SKF96365. By Ca2+ imaging, we found that the depletion of ER Ca2+ stores by TG elicited a transient rise in cytoplasmic Ca2+, followed by sustained increase depending on extracellular Ca2+. But, SKF96365, not Verapamil (L-type channels inhibitor) and NiCl2 (Na+/Ca2+ exchanger inhibitor), inhibited the relatively high [Ca2+]i. NPS R-568 also resulted in the same effect, and the duration of [Ca2+]i increase was eliminated completely by U73122 and was reduced in the absence of [Ca2+]o. NPS R-568 and TG increased the apoptotic ratio of septic T lymphocytes, which can be suppressed by SKF96365 and U73122. These results suggested that CaSR activation promoted the expression of TRPC3 and TRPC6 and enhanced T lymphocytes apoptosis through PLC-IP3 signaling pathway in sepsis.

Phosphorylation of TRPC4 as a regulatory mechanism of Isoc

Calcium signaling mediates a number of cellular events, and in many non‐excitable cells, store‐operated calcium entry (SOCE) represents the major Ca2+ event for signaling. In SOCE, depletion of ER Ca2+ stores leads to extracellular Ca2+ entry through SOC channels. Pulmonary artery endothelial cells (PAECs) exhibit a Ca2+‐selective SOCE current termed Isoc. Activation of Isoc in PAECs leads to formation of inter‐endothelial cell gaps. TRPC proteins 1 and 4 contribute to Isoc channel architecture. The region encompassing the protein 4.1 binding site and adjacent proline‐rich region on the TRPC4 subunit are essential for Isoc channel activation. Isoc inactivation is phosphorylation‐ and Ca2+‐dependent but the identity of kinases/phosphatases and their targets are still being explored. TRPC4 is a promising target for phosphoregulation of Isoc. Here, we examine the phosphorylation status of TRPC4 following activation of thapsigargin‐induced SOCE in PAECs. Following store depletion, TRPC4 phosphorylation increases as evidenced by western blot. In the presence of a calcineurin phosphatase inhibitor, baseline and thapsigargin‐induced TRPC4 phosphorylation is increased, and thapsigargin‐induced inter‐endothelial gap formation is reduced. These data suggest TRPC4 phosphorylation plays a role in Isoc regulation. Supported by 1F32HL112565–01 and 5R00HL089361

Silencing of the Charcot–Marie–Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2 + homeostasis by reducing store-operated Ca2 + entry

GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca2+ stores. GDAP1 silencing does not affect mitochondrial Ca2+ uptake, ER-Ca2+, or Ca2+ flow from ER to mitochondria, but reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) following mobilization of ER-Ca2+ and SOCE-driven Ca2+ entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca2+ uptake in mitochondria.

Reciprocal Regulation of Store-Dependent and Independent CRAC Channel Gating by STIM1 and 2-Apb

Ca2+ release-activated Ca2+ (CRAC) channels generate Ca2+ signals important for gene expression, the release of inflammatory mediators, and cell motility. Under physiological conditions, CRAC channels are activated by depletion of ER Ca2+ stores via physical interactions with the ER Ca2+ sensor, STIM1. However, recent studies indicate that exogenously over-expressed Orai3 CRAC channels can also be directly activated in a store- and STIM1-independent fashion by the compound, 2-aminoethyldiphenyl borate (2-APB). Whether 2-APB also regulates store-operated gating of Orai3 channels and if STIM1 can modulate direct activation by 2-APB is unknown. Here, we report that 2-APB regulates STIM1 gating of Orai3 channels in several respects. Low concentrations potentiate Orai3 currents in a store- and STIM1-dependent manner, whereas high doses strongly inhibit store-operated Orai3 currents. Inhibition by 2-APB occurs concurrently with changes in several STIM1-dependent Orai3 channel properties, including fast inactivation and potentiation, suggesting that high doses of 2-APB affect STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resist direct activation by 2-APB. The resistance to 2-APB activation varies in direct proportion to 2-APB-induced inhibition of the store-operated Orai3 conductance, suggesting that 2-APB has to first uncouple STIM1 from Orai3 channels before causing direct activation. Collectively, our results indicate that the store-dependent and independent modes of CRAC channel activation are reciprocally regulated, such that channels bound to STIM1 resist 2-APB gating, while 2-APB suppresses STIM1-gating.

CRAC Channel Function Through ORAI1, STIM1 and STIM2 is Required for Proinflammatory Effector T Cell Function in Autoimmunity

ORAI1 constitutes the pore forming subunit of the Ca2+ release activated Ca2+ (CRAC) channel, which is responsible for store-operated Ca2+ entry (SOCE) in many cell types. ORAI1 is activated by stromal interaction molecules (STIM) 1 and 2 following immunoreceptor triggering and depletion of ER Ca2+ stores. ORAI1, STIM1 and also STIM2 are essential mediators of SOCE and CRAC channel function in human and murine T lymphocytes. A role for ORAI1 and STIM1 in T cells in vivo has been inferred from in vitro studies of cells from human immunodeficient patients with mutations in ORAI1 and STIM1 and mice with targeted deletion of Orai1, Stim1 and Stim2 genes. Investigating the role of SOCE in T cells in the context of autoimmunity and inflammation, we find that mice with T-cell specific deletion of Stim1 or Stim2 are protected from experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. T cells lacking STIM1 or STIM2 failed to produce the proinflammatory cytokines IL-17 and IFNγ, which are crucial for the induction of EAE. SOCE is also required for the proliferation and homeostasis of IL-17 producing Th17 cells. ORAI1 is similarly critical for several aspects of T cell mediated immunity in vivo. Immune responses that depend on Th1, Th2 and Th17 cells were severely attenuated in Orai1-deficient mice expressing a non-functional form of the CRAC channel. Orai1-deficient mice tolerated skin allotransplants significantly longer than wildtype littermates. Furthermore, T cells from Orai1-deficient mice failed to induce colitis in a T-cell dependent adoptive transfer model of inflammatory bowel disease as they lacked production of IL-17, IFNγ and TNFα. Taken together, our findings demonstrate that ORAI1, STIM1 and STIM2 are critical mediators of proinflammatory T cell function and autoimmunity.

Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.

An Orai1 Activating Minimal Fragment Of Stim1

In immune cells generation of sustained Ca2+ levels is mediated by the Ca2+ release activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of ER Ca2+ store leads to STIM1 multimerization into discrete punctae that co-cluster with ORAI1 thereby triggering coupling to and activation of ORAI1 channels. The C-terminus of STIM1 is sufficient for the activation of ORAI1 currents independent of store depletion. Here we unmasked an ORAI activating minimal fragment (OAMF) within STIM1 C-terminus that exhibited enhanced interaction with ORAI1 and resulted in three-fold increased Ca2+ currents. STIM1-OAMF still showed the ability of a homomeric interaction similar to longer fragments as well as the full-length form of STIM1 C-terminus. In contrast, further deletion of a thirty amino acid region resulted in a substantial reduction of homomeric interaction concomitant to a loss of coupling to as well as activation of ORAI1. In aggregate, we have identified two key regions within STIM C-terminus that govern ORAI1 activation. (Supported by PhD-Program W1201 from the FWF)

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.

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca2+ stores, following the release of Ca2+ during intracellular signalling, triggers the Ca2+ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca2+]i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca2+ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca2+ entry in the form of motes.

Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (ΔΨm) began to decrease in SL-1 cells at 4 h post infection and Δψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was downregulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrionmediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.