Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca²⁺ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca²⁺ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca²⁺ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H₂O₂ induces an initial Ca²⁺ increase, which in prostate cancer cells is blocked at high concentrations of H₂O₂. Upon depletion of intracellular Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H₂O₂ compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector T_H cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca²⁺ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca²⁺ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.     

A Novel Native Store-operated Calcium Channel Encoded by Orai3: SELECTIVE REQUIREMENT OF Orai3 VERSUS Orai1 IN ESTROGEN RECEPTOR-POSITIVE VERSUS ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS*

Store-operated calcium (Ca²⁺) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca²⁺ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca²⁺ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ current (I_CRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca²⁺ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I_CRAC in estrogen receptor-positive (ER⁺) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER⁻) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER⁺ breast cancer cells represent the first example where the native SOCE pathway and I_CRAC are mediated by Orai3. Future studies implicating Orai3 in ER⁺ breast cancer progression might establish Orai3 as a selective target in therapy of ER⁺ breast tumors.     

TRPC1 and ORAI1 channels in colon cancer

Colon cancer cells, like other types of cancer cells, undergo the remodeling of the intracellular Ca²⁺ homeostasis that contributes to cancer cell hallmarks including enhanced cell proliferation, migration, and survival. Colon cancer cells display enhanced store-operated Ca²⁺ entry (SOCE) compared with their non-cancer counterparts. Colon cancer cells display an abnormal expression of SOCE molecular players including Orai1 and TRPC1 channels, and the stromal interacting molecule (STIM) 1 and 2. Interestingly, upregulation of Orai1 and TRPC1 channels and their contribution to SOCE are associated with cancer malignancy in colon cancer cells. In a specific cellular model of colon cancer, whereas in non-cancer colon cells SOCE is composed of the Ca²⁺ release activated (CRAC) currents, in colon cancer cells SOCE is composed of CRAC- and cationic, non-selective store operated (SOC) currents. Former SOCs are mediated by TRPC1 channels. Moreover, colon cancer cells also display dysregulation of the expression of 1,4,5-triphosphate receptors (IP₃R) that could contribute to the enhanced SOCE. Another important factor underlying the enhanced SOCE is the differential mitochondrial modulation of the CRAC and SOC currents in non-cancer and colon cancer cells. In colon cancer cells, mitochondria take up more Ca²⁺ that prevent the Ca²⁺-dependent inactivation of the SOCs, leading to sustained Ca²⁺ entry. Notably, the inhibition of SOCE in cancer colon cells abolishes their cancer hallmarks. Robust evidence has shown the efficiency of non-steroidal anti-inflammatory drugs (NSAIDs) and difluoromethylornithine (DFMO) to reverse the enhanced cell proliferation, migration, and apoptosis resistance of cancer cells. In colon cancer cells, both NSAIDs and DFMO decrease SOCE, but they target different molecular components of SOCE. NSAIDs decrease the Ca²⁺ uptake by mitochondria, limiting their ability to prevent the Ca²⁺-dependent inactivation of the SOCs that underlie SOCE. On the other hand, DFMO inhibits the expression of TRPC1 channels in colon cancer cells, eliminating their contribution to SOCE. The identification of players of SOCE in colon cancer cells may help to better understand the remodeling of the Ca²⁺ homeostasis in cancer. Importantly, the use of different pharmacological tools that target different SOCE molecular players in colon cancer cells may play a pivotal role in designing better chemoprevention strategies.     

Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca²⁺ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.     

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca²⁺ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca²⁺ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na⁺ that is critical in activating the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) causing enhanced mitochondrial Na⁺ uptake and Ca²⁺ efflux. Omission of extracellular Na⁺ prevents the cytosolic Na⁺ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na⁺ and Ca²⁺ signals to effect mitochondrial redox control over SOCE.     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.     

Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells

The molecular nature of calcium (Ca²⁺)-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca²⁺ entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca²⁺ influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca²⁺-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.     

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1−/− and heptaTRPC knockout mice

Store operated Ca²⁺ entry (SOCE) is the most important Ca²⁺ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca²⁺ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca²⁺ release and Ca²⁺ entry in responsive cells while GHRH and CRH only induce Ca²⁺ entry. SOCE has been shown to contribute to Ca²⁺ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1−/− or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca²⁺ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1−/− mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca²⁺-oscillations related to electrical activity were not affected in the Orai1−/− mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca²⁺ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1−/−. In addition, Ca²⁺ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca²⁺ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca²⁺-oscillations and Ca²⁺ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.     

Emerging roles of Orai3 in pathophysiology

Calcium (Ca²⁺) is a ubiquitous second messenger that regulates a plethora of physiological functions. Deregulation of calcium homeostasis has been reported in a wide variety of pathological conditions including cardiovascular disorders, cancer and neurodegenerative diseases. One of the most ubiquitous pathways involved in regulated Ca²⁺ influx into cells is the store-operated Ca²⁺ entry (SOCE) pathway. In 2006, Orai1 was identified as the channel protein that mediates SOCE in immune cells. Orai1 has two mammalian homologs, Orai2 and Orai3. Although Orai1 has been the most widely studied Orai isoform, Orai3 has recently received significant attention. Under native conditions, Orai3 was demonstrated to be an important component of store-independent arachidonate-regulated Ca²⁺ (ARC) entry in HEK293 cells, and more recently of a store-independent leukotrieneC₄-regulated Ca²⁺ (LRC) entry pathway in vascular smooth muscle cells. Recent studies have shown upregulation of Orai3 in estrogen receptor-expressing breast cancers and a critical role for Orai3 in breast cancer development in immune-compromised mice. Orai3 upregulation was also shown to contribute to vascular smooth muscle remodeling and neointimal hyperplasia caused by vascular injury. Furthermore, Orai3 has been shown to contribute to proliferation of effector T-lymphocytes under oxidative stress. In this review, we will discuss the role of Orai3 in reported pathophysiological conditions and will contribute ideas on the potential role of Orai3 in native Ca²⁺ signaling pathways and human disease.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

Pore opening mechanism of CRAC channels

Three decades ago, James W. Putney Jr. conceptualized the idea of store-operated calcium entry (SOCE) to explain how depletion of endoplasmic reticulum (ER) Ca²⁺ stores evokes Ca²⁺ influx across the plasma membrane. Since the publication of this highly influential idea, it is now established that SOCE is universal among non-excitable and probably even many types of excitable cells, and contributes to numerous effector functions impacting immunity, muscle contraction, and brain function. The molecules encoding SOCE, the STIM and Orai proteins, are now known and our understanding of how this pathway is activated in response to ER Ca²⁺ store depletion has advanced significantly. In this review, we summarize the current knowledge of how Orai1 channels are activated by STIM1, focusing on recent work supporting a hydrophobic gating mechanism for the opening of the Orai1 channel pore.     

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca²⁺ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca²⁺ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca²⁺-dependent exocytosis of inflammatory mediators. We show that the Ca²⁺ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca²⁺ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2^−/− PMCs at high (2 mM) extracellular Ca²⁺ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1 mM) Ca²⁺ concentration. Likewise, the density of CRAC currents, Ca²⁺-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca²⁺ transients, store-operated Ca²⁺ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca²⁺ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

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.     

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF

Store-operated Ca²⁺ entry (SOCE) is a functionally relevant mechanism for Ca²⁺ influx present in electrically excitable and non-excitable cells. Regulation of Ca²⁺ entry through store-operated channels is essential to maintain an appropriate intracellular Ca²⁺ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca²⁺-dependent inactivation mediated by two temporally separated mechanisms: fast Ca²⁺-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca²⁺ with residues in the channel pore while slow Ca²⁺-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca²⁺]_cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P₂-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca²⁺ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca²⁺ signals.     

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca²⁺ entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca²⁺-independent phospholipase A₂ β (iPLA₂β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA₂β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA₂β but not STIM1. These data confirm the role of iPLA₂β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.     

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca²⁺ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca²⁺ increase generated by an interplay between ER-Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca²⁺ depletion and binds Orai1 to activate Ca²⁺ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca²⁺ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca²⁺ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca²⁺ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca²⁺ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca²⁺ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca²⁺ influx.