The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCß2.

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1ß, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1ß lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1ß is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCß activation. Our results indicate that Orai1α interacts with PKCß2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1ß.

Orai1 is the pore-forming subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1ß, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca2+ concentration, as demonstrated by cell loading with the fast intracellular Ca2+ chelator dimethyl BAPTA in the absence of extracellular Ca2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1ß to the plasma membrane when expressed individually; by contrast, when Orai1ß is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1ß in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca2+ store depletion.

Role of Orai-family channels in the activation and regulation of transcriptional activity.

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5-trisphosphate receptors. Then, a reduction of the endoplasmic reticulum intraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membrane Ca2+ channels comprised by Orai proteins. STIM1/Orai-mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T-cells, the cAMP-response element binding protein, the nuclear factor κ-light chain-enhancer of activated B cells, c-fos, and c-myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+ -dependent signaling pathways.

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1ß.

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP3 generation, which results in intracellular Ca2+ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1ß, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1ß, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ 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 Orai1ß, 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 Ca2+ 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 Orai1ß, 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 Orai1ß 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 Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

The Ca2+ Sensor STIM in Human Diseases.

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.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.