The function of the calcium channel Orai1 in osteoclast development.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

Mechanisms and functions of calcium microdomains produced by ORAI channels, d-myo-inositol 1,4,5-trisphosphate receptors, or ryanodine receptors.

With the discovery of local Ca2+ signals in the 1990s the concept of 'elementary Ca2+ signals' and 'fundamental Ca2+ signals' was developed. While 'elementary Ca2+signals' relate to optical signals gained by activity of small clusters of Ca2+channels, 'fundamental signals' describe such optical signals that arise from opening of single Ca2+channels. In this review, we discuss (i) concepts of local Ca2+ signals and Ca2+ microdomains, (ii) molecular mechanisms underlying Ca2+ microdomains, (iii) functions of Ca2+ microdomains, and (iv) mathematical modelling of Ca2+ microdomains. We focus on Ca2+ microdomains produced by ORAI channels, D-myo-inositol 1,4,5-trisphosphate receptors, or ryanodine receptors. In summary, research on local Ca2+ signals in different cell models aims to better understand how cells use the Ca2+ toolkit to produce Ca2+ microdomains as relevant signals for specific cellular responses, but also how local Ca2+ signals as building blocks merge into global Ca2+ signaling.

Cardiomyocyte-Specific Deletion of Orai1 Reveals Its Protective Role in Angiotensin-II-Induced Pathological Cardiac Remodeling.

Pathological cardiac remodeling correlates with chronic neurohumoral stimulation and abnormal Ca2+ signaling in cardiomyocytes. Store-operated calcium entry (SOCE) has been described in adult and neonatal murine cardiomyocytes, and Orai1 proteins act as crucial ion-conducting constituents of this calcium entry pathway that can be engaged not only by passive Ca2+ store depletion but also by neurohumoral stimuli such as angiotensin-II. In this study, we, therefore, analyzed the consequences of Orai1 deletion for cardiomyocyte hypertrophy in neonatal and adult cardiomyocytes as well as for other features of pathological cardiac remodeling including cardiac contractile function in vivo. Cellular hypertrophy induced by angiotensin-II in embryonic cardiomyocytes from Orai1-deficient mice was blunted in comparison to cells from litter-matched control mice. Due to lethality of mice with ubiquitous Orai1 deficiency and to selectively analyze the role of Orai1 in adult cardiomyocytes, we generated a cardiomyocyte-specific and temporally inducible Orai1 knockout mouse line (Orai1CM-KO). Analysis of cardiac contractility by pressure-volume loops under basal conditions and of cardiac histology did not reveal differences between Orai1CM-KO mice and controls. Moreover, deletion of Orai1 in cardiomyocytes in adult mice did not protect them from angiotensin-II-induced cardiac remodeling, but cardiomyocyte cross-sectional area and cardiac fibrosis were enhanced. These alterations in the absence of Orai1 go along with blunted angiotensin-II-induced upregulation of the expression of Myoz2 and a lack of rise in angiotensin-II-induced STIM1 and Orai3 expression. In contrast to embryonic cardiomyocytes, where Orai1 contributes to the development of cellular hypertrophy, the results obtained from deletion of Orai1 in the adult myocardium reveal a protective function of Orai1 against the development of angiotensin-II-induced cardiac remodeling, possibly involving signaling via Orai3/STIM1-calcineurin-NFAT related pathways.

Modulation of Orai1 by cationic peptides triggers their direct cytosolic uptake.

At concentrations exceeding 10 µM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake.

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.

Induction of store-operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ.

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.

ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain.

KEY POINTS: Gain-of-function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM. ABSTRACT: Skeletal muscle differentiation relies on store-operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1-/- /Stim2-/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1.

Stromal-Interacting Molecule 1 (Stim1)/Orai1 Modulates Endothelial Permeability in Ventilator-Induced Lung Injury.

BACKGROUND Increased endothelial permeability is involved in ventilator-induced lung injury (VILI). Stim1/Orai1 mediates store-operated Ca2+ activation, which modulates endothelial permeability. However, the underlying mechanisms of the Stim1/Orai1 pathway in VILI are poorly understood. MATERIAL AND METHODS Wistar rats were exposed to low tidal volume (7 mL/kg) or high tidal volume (40mL/kg) ventilation. Human Lung Microvascular Endothelial Cells (HULEC) were subjected to 8% or 18% cyclic stretching (CS). BTP2 pretreatment was performed. Lung wet/dry weight ratio, histological changes of lung injury, and bronchoalveolar lavage fluid (BALF) protein were measured. Endothelial permeability and intracellular calcium concentration were evaluated in HULECs. Protein expression was determined by Western blotting. RESULTS High tidal volume mechanical ventilation-induced lung injury (such as severe congestion and hemorrhage) and BTP2 pretreatment protected lungs from injury. The expression of Stim1, Orai1, and PKCα, lung wet/dry weight ratio, and BALF protein level significantly increased in the high tidal volume group compared to the control group and low tidal volume group. Importantly, BTP2 pretreatment alleviated the above-mentioned effects. Compared with exposure to 8% CS, the protein levels of Stim1, Orai1, and PKCα in HULECs significantly increased after exposure to 18% CS for 4 h, whereas BTP2 pretreatment significantly inhibited the increase (P<0.05). BTP2 pretreatment also suppressed increase of endothelial permeability and the intracellular calcium induced by 18% CS (P<0.05). CONCLUSIONS When exposed to high tidal volume or large-magnitude CS, Stim1 and Orai1 expression are upregulated, which further activates calcium-sensitive PKCα and results in calcium overload, endothelial hyperpermeability, and, finally, lung injury.

LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca2+ entry.

Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-ß family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes. KEY MESSAGES: •Endometrial receptivity is negatively regulated by LEFTY2. •LEFTY2 inhibits the expression of key murine receptivity genes, including Cox2, Bmp2 and Wnt4, and blocks embryo implantation. •LEFTY2 downregulates the expression of Orai1 and inhibits SOCE. •LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit COX2, BMP2, and WNT4 expression in endometrial cells. •Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.

The role of the store-operated calcium entry channel Orai1 in cultured rat hippocampal synapse formation and plasticity.

KEY POINTS: The role of non-synaptic calcium entry in the formation and functions of dendritic spines was studied in dissociated cultured rat hippocampal neurons. Orai1, a store-operated calcium channel, is found in dendritic spines. Orai1 co-localizes in dendritic spines with STIM2 under conditions of lower [Ca2+ ]o. Orai1 channels are associated with the formation of new dendritic spines in response to elevated [Ca2+ ]o. Lack of Orai1, either by transfection with a dominant negative construct or with small interfering RNA to Orai1, results in retarded dendritic spines, an increase in density of filopodia, lower synaptic connectivity and the ability to undergo plastic changes. These results highlight a novel role for Orai1 in synapse formation, maturation and plasticity. ABSTRACT: The possible role of store operated calcium entry (SOCE) through the Orai1 channel in the formation and functions of dendritic spines was studied in cultured hippocampal neurons. In calcium store-depleted neurons, a transient elevation of extracellular calcium concentration ([Ca2+ ]o ) caused a rise in [Ca2+ ]i that was mediated by activation of the SOCE. The store depletion resulted in an increase in stromal interacting molecule 2 (an endoplasmic calcium sensor) association with Orai1 in dendritic spines. The response to the rise in [Ca2+ ]o was larger in spines endowed with a cluster of Orai1 molecules than in spines devoid of Orai1. Transfection of neurons with a dominant negative Orai1 resulted in retarded maturation of dendritic spines, a reduction in synaptic connectivity with afferent neurons and a reduction in the ability to undergo morphological changes following induction of chemical long-term potentiation. Similarly, small interfering RNA (siRNA)-treated neurons had fewer mature dendritic spines, and lower rates of mEPSCs compared to scrambled control siRNA-treated neurons. Thus, influx of calcium through Orai1 channels facilitates the maturation of dendritic spines and the formation of functional synapses in central neurons.

CRAC channel gating and its modulation by STIM1 and 2-aminoethoxydiphenyl borate.

Ca2+ release-activated Ca2+ (CRAC) channels play an essential role in the immune system. The pore-forming subunit, Orai1, is an important pharmacological target. Here, we summarize the recent discoveries on the structure-function relationship of Orai1, as well as its interaction with the native channel opener STIM1 and chemical modulator 2-aminoethoxydiphenyl borate (2-APB). We first introduce the critical structural elements of Orai1, which include a Ca2+ accumulating region, ion selectivity filter, hydrophobic centre, basic region, extended transmembrane Orai1 N-terminal (ETON) region, transmembrane (TM) regions 2 and 3, P245 bend, 263 SHK265 hinge linker and L273-L276 hydrophobic patch. We then hypothesize the possible mechanisms by which STIM1 triggers the conformational transitions of TM regions and exquisitely shapes the ion conduction pathway during generation of the CRAC current (Icrac ) with high Ca2+ selectivity. Finally, we propose mechanisms by which 2-APB modulates Icrac . On the STIM1-activated Orai1 channel, a low dose of 2-APB acts directly, dilating its extremely narrow pore diameter from 3.8 to 4.6 Å, increasing its unitary channel conductance, and potentiating the Icrac . Further elucidation of the structure of the opened CRAC channel and a better understanding of structure-function relationship will benefit the future development of novel immune modulators.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BACKGROUND: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. METHODS: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. RESULTS: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. CONCLUSIONS: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Molecular modulators of store-operated calcium entry.

Three decades ago, store-operated Ca(2+) entry (SOCE) was identified as a unique mechanism for Ca(2+) entry through plasma membrane (PM) Ca(2+)-permeable channels modulated by the intracellular Ca(2+) stores, mainly the endoplasmic reticulum (ER). Extensive analysis of the communication between the ER and the PM leads to the identification of the protein STIM1 as the ER-Ca(2+) sensor that gates the Ca(2+) channels in the PM. Further analysis on the biophysical, electrophysiological and biochemical properties of STIM1-dependent Ca(2+) channels has revealed the presence of a highly Ca(2+)-selective channel termed Ca(2+) release-activated Ca(2+) channel (CRAC), consisting of Orai1 subunits, and non-selective cation channels named store-operated channels (SOC), including both Orai1 and TRPC channel subunits. Since the identification of the key elements of CRAC and SOC channels a number of intracellular modulators have been reported to play essential roles in the stabilization of STIM-Orai interactions, collaboration with STIM1 conformational changes or mediating slow Ca(2+)-dependent inactivation. Here, we review our current understanding of some of the key modulators of STIM1-Orai1 interaction, including the proteins CRACR2A, STIMATE, SARAF, septins, golli and ORMDL3.

The role of Orai-STIM calcium channels in melanocytes and melanoma.

Calcium signalling within normal and cancer cells regulates many important cellular functions such as migration, proliferation, differentiation and cytokine secretion. Store operated Ca(2+) entry (SOCE) via the Ca(2+) release activated Ca(2+) (CRAC) channels, which are composed of the plasma membrane based Orai channels and the endoplasmic reticulum stromal interaction molecules (STIMs), is a major Ca(2+) entry route in many cell types. Orai and STIM have been implicated in the growth and metastasis of multiple cancers; however, while their involvement in cancer is presently indisputable, how Orai-STIM-controlled Ca(2+) signals affect malignant transformation, tumour growth and invasion is not fully understood. Here, we review recent studies linking Orai-STIM Ca(2+) channels with cancer, with a particular focus on melanoma. We highlight and examine key molecular players and the signalling pathways regulated by Orai and STIM in normal and malignant cells, we expose discrepancies, and we reflect on the potential of Orai-STIMs as anticancer drug targets. Finally, we discuss the functional implications of future discoveries in the field of Ca(2+) signalling.