The distinct role of STIM1 and STIM2 in the regulation of store-operated Ca2+ entry and cellular function

Stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum (ER) Ca²⁺ sensors that initiate store-operated Ca ²⁺ entry (SOCE). The roles of STIM1-mediated SOCE in cancer biology have been highlighted in different types of cancer, but that of STIM2 is unknown. By the model of cervical cancer, here we focus on the cooperative regulation of SOCE by STIM proteins and their distinct roles in cellular function. Immunofluorescent stainings of surgical specimens of cervical cancer show that STIM1 and STIM2 are abundant in tumor tissues, but STIM1 is the major ER Ca ²⁺ sensor identified in the invasive front of cancer tissues. STIM1 or STIM2 overexpression in cervical cancer SiHa cells induces an upregulated SOCE. Regarding cellular function, STIM1 and STIM2 are necessary for cell proliferation, whereas STIM1 is the dominant ER Ca ²⁺ sensor involved in cell migration. During SOCE, STIM1 is aggregated and translocated towards the Orai1-containing plasma membrane in association with the microtubule plus-end binding protein EB1. In contrast, STIM2 is constitutively aggregated without significant trafficking or association with microtubules. These results show the distinct role of STIM1 and STIM2 in SOCE and cellular function of cervical cancer cells.     

Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca(2+) entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+) homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.     

STIM and ORAI proteins in the nervous system

Stromal interaction molecules (STIM) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM proteins which, in turn, bind and open calcium channels in the plasma membrane formed by the proteins ORAI1, ORAI2, and ORAI3. The resulting store-operated calcium entry (SOCE), mostly controlled by the principal components STIM1 and ORAI1, has been particularly characterized in immune cells. In the nervous system, all STIM and ORAI homologs are expressed. This review summarizes current knowledge on distribution and function of STIM and ORAI proteins in central neurons and glial cells, i.e. astrocytes and microglia. STIM2 is required for SOCE in hippocampal synapses and cortical neurons, whereas STIM1 controls calcium store replenishment in cerebellar Purkinje neurons. In microglia, STIM1, STIM2, and ORAI1 regulate migration and phagocytosis. The isoforms ORAI2 and ORAI3 are candidates for SOCE channels in neurons and astrocytes, respectively. Due to the role of SOCE in neuronal and glial calcium homeostasis, dysfunction of STIM and ORAI proteins may have consequences for the development of neurodegenerative disorders, such as Alzheimer''s disease.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

STIM and ORAI proteins in the nervous system

Stromal interaction molecules (STIM) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM proteins which, in turn, bind and open calcium channels in the plasma membrane formed by the proteins ORAI1, ORAI2, and ORAI3. The resulting store-operated calcium entry (SOCE), mostly controlled by the principal components STIM1 and ORAI1, has been particularly characterized in immune cells. In the nervous system, all STIM and ORAI homologs are expressed. This review summarizes current knowledge on distribution and function of STIM and ORAI proteins in central neurons and glial cells, i.e. astrocytes and microglia. STIM2 is required for SOCE in hippocampal synapses and cortical neurons, whereas STIM1 controls calcium store replenishment in cerebellar Purkinje neurons. In microglia, STIM1, STIM2, and ORAI1 regulate migration and phagocytosis. The isoforms ORAI2 and ORAI3 are candidates for SOCE channels in neurons and astrocytes, respectively. Due to the role of SOCE in neuronal and glial calcium homeostasis, dysfunction of STIM and ORAI proteins may have consequences for the development of neurodegenerative disorders, such as Alzheimer's disease.     

Role of STIM1 in Regulation of Store-Operated Ca<Superscript>2+</Superscript> Influx in Pheochromocytoma Cells

Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate) can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive care. Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated Ca²⁺ entry (SOCE), an important component of [Ca²⁺]_i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C), or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate. Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca²⁺ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate: factors that can adversely affect neuronal function under pathological conditions.     

Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons

The interaction between Ca(2+) sensors STIM1 and STIM2 and Ca(2+) channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca(2+) levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca(2+) influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca(2+) levels in the ER and Ca(2+) leakage with the additional involvement of STIM1.     

Orai1 and STIM reconstitute store-operated calcium channel function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.     

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

The Ca²⁺ sensor STIM1 is crucial for activation of store-operated Ca²⁺ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca²⁺ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca²⁺ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.     

Novel Protein Kinase C-Mediated Control of Orai1 Function in Invasive Melanoma

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, a key driver of melanoma invasiveness, induces Ca²⁺ signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive cell lines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.     

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.     

Novel role for STIM1 as a trigger for calcium influx factor production

STIM1 has been recently identified as a Ca(2+) sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ER-delimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile alpha-motif (SAM) domain. We demonstrate that 1) molecular knockdown or overexpression of STIM1 results in corresponding impairment or amplification of CIF production and 2) inherent deficiency in the ER-delimited CIF production and SOCE activation in some cell types can be a result of their deficiency in STIM1 protein; expression of a wild-type STIM1 in such cells was sufficient to fully rescue their ability to produce CIF and SOCE. We found that glycosylation sites in the ER-resident SAM domain of STIM1 are essential for initiation of CIF production. We propose that after STIM1 loses Ca(2+) from EF hand, its intraluminal SAM domain may change conformation, and via glycosylation sites it can interact with and activate CIF-producing machinery. Thus, CIF production appears to be one of the earliest STIM1-dependent events in the ER lumen, and impairment of this process results in impaired SOCE response.     

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca²⁺ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser⁶²¹ and Thr⁶²⁶ were O-GlcNAcylated and that Thr⁶²⁶ was O-GlcNAcylated in the steady state but Ser⁶²¹ was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser⁶²¹. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser⁶²¹. These data suggest that both decrease in O-GlcNAcylation at Thr⁶²⁶ and increase in O-GlcNAcylation at Ser⁶²¹ in STIM1 lead to impairment of SOCE activity through decrease in Ser⁶²¹ phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.     

Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.     

17β-Estradiol Inhibits Phosphorylation of Stromal Interaction Molecule 1 (STIM1) Protein: IMPLICATION FOR STORE-OPERATED CALCIUM ENTRY AND CHRONIC LUNG DISEASES*

Sex plays a significant role in the development of lung diseases including asthma, cancer, chronic bronchitis, and cystic fibrosis. In cystic fibrosis, 17β-estradiol (E2) may inhibit store-operated Ca²⁺ entry (SOCE) to impinge upon airway secretions, leaving females at greater risk of contracting lung infections. Stromal interaction molecule 1 (STIM1)-mediated SOCE is essential for cell homeostasis and regulates numerous processes including cell proliferation, smooth muscle contraction, and secretion. E2 can signal nongenomically to modulate Ca²⁺ signaling, but little is known of the underlying mechanisms. We found that E2 exposure inhibited STIM1 translocation in airway epithelia, preventing SOCE. This correlated with a decrease in STIM1-STIM1 FRET and STIM1 mobility in E2-exposed HEK293T cells co-expressing estrogen receptor α. We also examined the role of STIM1 phosphorylation in E2-mediated inhibition of STIM1 mobility. STIM1 is basally phosphorylated at serine 575, which is required for SOCE. Exposure to E2 significantly decreased STIM1 serine phosphorylation. Mutating serine 575 to an alanine blocked STIM1 phosphorylation, reduced basal STIM1 mobility, and rendered STIM1 insensitive to E2. These data indicate that E2 can signal nongenomically by inhibiting basal phosphorylation of STIM1, leading to a reduction in SOCE.     

Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry

Store-operated Ca2+ entry (SOCE) is likely the most common mode of regulated influx of Ca2+ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca2+ current (I CRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca2+ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I(CRAC)-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca2+ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.     

Role of STIM1/Orai1-mediated store-operated Ca²⁺ entry in airway smooth muscle cell proliferation

Hyperplasia of airway smooth muscle cells (ASMCs) is a characteristic change of chronic asthma patients. However, the underlying mechanisms that trigger this process are not yet completely understood. Store-operated Ca(2+) (SOC) entry (SOCE) occurs in response to the intracellular sarcoplasma reticulum (SR)/endoplasmic reticulum (ER) Ca(2+) store depletion. SOCE plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs. Stromal interaction molecule (STIM)1 has been proposed as an ER/SR Ca(2+) sensor and translocates to the ER underneath the plasma membrane upon depletion of the ER Ca(2+) store, where it interacts with Orai1, the molecular component of SOC channels, and brings about SOCE. STIM1 and Orai1 have been proved to mediate SOCE of ASMCs. In this study, we investigated whether STIM1/Orai1-mediated SOCE is involved in rat ASMC proliferation. We found that SOCE was upregulated during ASMC proliferation accompanied by a mild increase of STIM1 and a significant increase of Orai1 mRNA expression, whereas the proliferation of ASMCs was partially inhibited by the SOC channel blockers SKF-96365, NiCl(2), and BTP-2. Suppressing the mRNA expression of STIM1 or Orai1 with specific short hairpin RNA resulted in the attenuation of SOCE and ASMC proliferation. Moreover, after knockdown of STIM1 or Orai1, the SOC channel blocker SKF-96365 had no inhibitory effect on the proliferation of ASMCs anymore. These results suggested that STIM1/Orai1-mediated SOCE is involved in ASMC proliferation.     

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.     

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.