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Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca 2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca 2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca 2+ -dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca 2+ store depletion and their subsequent modulation via slow Ca 2+ -dependent inactivation. Conclusion: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.

Myocardial infarction (MI) causes massive loss of cardiac myocytes and injury to the coronary microcirculation, overwhelming the limited capacity of cardiac regeneration. Cardiac repair after MI is finely organized by complex series of procedures involving a robust angiogenic response that begins in the peri-infarcted border area of the infarcted heart, concluding with fibroblast proliferation and scar formation. Efficient neovascularization after MI limits hypertrophied myocytes and scar extent by the reduction in collagen deposition and sustains the improvement in cardiac function. Compelling evidence from animal models and classical in vitro angiogenic approaches demonstrate that a plethora of well-orchestrated signaling pathways involving Notch, Wnt, PI3K, and the modulation of intracellular Ca2+ concentration through ion channels, regulate angiogenesis from existing endothelial cells (ECs) and endothelial progenitor cells (EPCs) in the infarcted heart. Moreover, cardiac repair after MI involves cell-to-cell communication by paracrine/autocrine signals, mainly through the delivery of extracellular vesicles hosting pro-angiogenic proteins and non-coding RNAs, as microRNAs (miRNAs). This review highlights some general insights into signaling pathways activated under MI, focusing on the role of Ca2+ influx, Notch activated pathway, and miRNAs in EC activation and angiogenesis after MI.

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ entry that regulates several cellular functions. SOCE remodeling mediated by changes in the expression and/or function of the Orai channels results in the reorganization of intracellular Ca 2+ homeostasis leading to a variety of pathologies, including cancer. Notably, a significant alteration of Orai function has been reported in breast cancer cells, where the dysregulation of the Notch1 signaling pathway plays a role in the development and progression of cancer hallmarks. Here, we have investigated the possible role of Notch1 in the regulation of the expression of Orai1 and Orai3 in different breast cancer cell lines. Expression of the active form of Notch1, as well as cell stimulation with the Notch1 agonist Jagged-1 (Jag-1), demonstrates a differential role of Notch1 in the regulation of Orai expression in non-tumoral breast epithelial cells and triple negative or luminal breast cancer cells. The role of Notch1 was confirmed using DAPT, a γ-secretase inhibitor that prevents activation of the Notch pathway. Modulation of Orai1 and Orai3 expression by Notch1 was paralleled by changes in SOCE. The effect in Orai expression mediated by activation of Notch1 signaling pathway was mimicked by the expression of HEY1 or the non-phosphorylatable HEY1-S68A mutant; by contrast, expression of the phosphomimetic HEY1-S68D mutant was without effect on Orai expression. Understanding the Notch1-HEY1-Orai axis might provide insights into the development of subtype-specific therapeutic strategies targeting breast cancer.

Orai1 is the pore-forming subunit of the Ca 2+ -release activated Ca 2+ channels that mediate store-operated Ca 2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca 2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells.

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario.

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.

Store‐operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+‐release‐activated Ca2+ channels constituted by Orai family members, with predominance of calcium release‐activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT‐29 and Caco‐2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT‐29 and Caco‐2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta‐66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC. Colorectal cancer (CRC) cells exhibit altered store‐operated Ca2+ entry (SOCE), showing enhanced Ca2+ entry and expression of Orai1 and STIM1 proteins, which also increases cell migration. Exposure of CRC cells to Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics reduces the expression of these proteins, resulting in a decrease of SOCE and, consequently, cell migration.

Background Ca 2+ release-activated Ca 2+ (CRAC) channels are highly Ca 2+ selective plasma membrane channels formed by the hexameric assembly of Orai subunits, with a predominant role for Orai1. Two Orai1 variants have been identified, Orai1α, which comprises 301 amino acids, and a short variant, Orai1β, lacking the first N-terminal 63 or 71 amino acids; however, little is known about their possible heteromerization to form CRAC channels. Here we show that Orai1α and Orai1β exhibit different lipid raft distributions in resting cells when expressed individually, likely due to the presence of a caveolin-binding domain exclusively in Orai1α. However, when both variants are co-expressed, they show a similar distribution predominantly in the lipid raft domains, indicating potential interaction between the two Orai1 forms. Methods A lipid raft isolation protocol in combination with Western blotting assay was conducted to detect the expression of each Orai1 variants in the isolated membrane fractions. Ca 2+ mobilization was determined using fura-2 and G-GECO1.2 fused to Orai1α fluorescence. Evidence of physical interaction between both Orai1 variants was provided using co-immunoprecipitation, APEX2 peroxidase-catalyzed proximity labeling, Förster resonance energy transfer (FRET) and super-resolution microscopy. Results Our results indicate that Orai1α and Orai1β exhibit different lipid raft partitioning in resting cells when expressed individually, likely attributed to the presence of a caveolin-binding domain in Orai1α. However, when both variants are co-expressed, they show a similar distribution predominantly in the lipid raft domains, indicating potential interaction between the two Orai1 forms. Expression of a dominant-negative Orai1β mutant has been found to interfere with Orai1α-mediated Ca 2+ entry. Using co-immunoprecipitation, APEX2 peroxidase-catalyzed proximity labeling, Förster resonance energy transfer (FRET) and super-resolution microscopy our results indicate that there is certain interaction between Orai1α and Orai1β although both variants form mostly independent channels. Conclusions Our results indicate that while Orai1α and Orai1β mostly form separate CRAC channels, a small subset of both Orai1 variants combine to form heteromeric channels. These findings provide new insights on the nature of CRAC channels.

Store-operated Ca(2+) entry (SOCE) is an ubiquitous mechanism for Ca(2+) entry in eukaryotic cells. This route for Ca(2+) influx is regulated by the filling state of the intracellular Ca(2+) stores communicated to the plasma membrane channels by the proteins of the Stromal Interaction Molecule (STIM) family, STIM1, and STIM2. Store-dependent, STIM1-modulated, channels include the Ca(2+) release-activated Ca(2+) channels, comprised of subunits of Orai proteins, as well as the store-operated Ca(2+) (SOC) channels, involving Orai1, and members of the canonical transient receptor potential family of proteins. Recent studies have revealed the expression of splice variants of STIM1, STIM2, and Orai1 in different cell types. While certain variants are ubiquitously expressed, others, such as STIM1L, show a more restricted expression. The splice variants for STIM and Orai1 proteins exhibit significant functional differences and reveal that alternative splicing enhance the functional diversity of STIM1, STIM2, and Orai1 genes to modulate the dynamics of Ca(2+) signals.

The STIM family of proteins plays a crucial role in a plethora of cellular functions through the regulation of store-operated Ca2+ entry (SOCE) and, thus, intracellular calcium homeostasis. The two members of the mammalian STIM family, STIM1 and STIM2, are transmembrane proteins that act as Ca2+ sensors in the endoplasmic reticulum (ER) and, upon Ca2+ store discharge, interact with and activate the Orai/CRACs in the plasma membrane. Dysregulation of Ca2+ signaling leads to the pathogenesis of a variety of human diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and immune disorders. Therefore, understanding the mechanisms underlying Ca2+ signaling pathways is crucial for developing therapeutic strategies targeting these diseases. This review focuses on several rare conditions associated with STIM1 mutations that lead to either gain- or loss-of-function, characterized by myopathy, hematological and immunological disorders, among others, and due to abnormal activation of CRACs. In addition, we summarize the current evidence concerning STIM2 allele duplication and deletion associated with language, intellectual, and developmental delay, recurrent pulmonary infections, microcephaly, facial dimorphism, limb anomalies, hypogonadism, and congenital heart defects.