Explore the vast range of titles available.

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 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.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1–STIM1 interaction at ER–PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) channels are highly Ca.sup.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, Orai1[alpha], which comprises 301 amino acids, and a short variant, Orai1[beta], 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 Orai1[alpha] and Orai1[beta] exhibit different lipid raft distributions in resting cells when expressed individually, likely due to the presence of a caveolin-binding domain exclusively in Orai1[alpha]. 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. 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.sup.2+ mobilization was determined using fura-2 and G-GECO1.2 fused to Orai1[alpha] 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. Our results indicate that Orai1[alpha] and Orai1[beta] exhibit different lipid raft partitioning in resting cells when expressed individually, likely attributed to the presence of a caveolin-binding domain in Orai1[alpha]. 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 Orai1[beta] mutant has been found to interfere with Orai1[alpha]-mediated Ca.sup.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 Orai1[alpha] and Orai1[beta] although both variants form mostly independent channels. Our results indicate that while Orai1[alpha] and Orai1[beta] 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 Ca2+ entry (SOCE) is a mechanism controlled by the filling state of the intracellular Ca2+ stores, predominantly the endoplasmic reticulum (ER), where ER-resident proteins STIM1 and STIM2 orchestrate the activation of Orai channels in the plasma membrane, and Orai1 playing a predominant role. Two forms of Orai1, Orai1α and Orai1β, have been identified, which arises the question whether they are equally regulated by STIM proteins. We demonstrate that STIM1 preferentially activates Orai1α over STIM2, yet both STIM proteins similarly activate Orai1β. Under resting conditions, there is a pronounced association between STIM2 and Orai1α, implying their significance in maintaining basal Ca2+ levels within the cytosol and ER. STIM1 and STIM2 are also shown to influence the protein levels of the Orai1 variants, independent of Ca2+ influx, via lysosomal degradation. Interestingly, Orai1α and Orai1β appear to selectively regulate the protein level of STIM1, but not STIM2. These observations offer crucial insights into the regulatory dynamics between STIM proteins and Orai1 variants, enhancing our understanding of the intricate processes that fine-tune intracellular Ca2+ signaling.Competing Interest StatementThe authors have declared no competing interest.