The Ca2+ release-activated Ca2+ (CRAC) channel mediates store-operated calcium entry (SOCE), a ubiquitous pathway essential for many cell types, including immune cells. Three Orai (Orai1/2/3) proteins constitute the plasma membrane pore-forming units of CRAC channels that are activated by the endoplasmic reticulum (ER) Ca2+-sensing STIM1/2 proteins when ER Ca2+ stores are depleted. Orai1/2/3 are differentially expressed across primary cells with discernible differences in their structures and biophysical properties. Further, Orai1 has two alternatively translated isoforms: long mammalian-specific Orai1α and the 63-residue shorter Orai1β, which is evolutionarily older and conserved across vertebrates. Whether Orai1α/1β/2/3 produce unique cytosolic Ca2+ signatures that bias transcriptional responses through effectors like NFAT is unclear. Here, we used HEK293 cells engineered to express one native Orai isoform and show that all Orai isoforms couple to NFAT1/4 induction. The magnitude of NFAT1/4 induction for each Orai isoform matches that of SOCE, with the following profile: Orai1β>Orai1α>>Orai2>Orai3. Near-native re-expression of either Orai1α or Orai1β in primary murine Orai1 -/- CD4+ T cells restored SOCE, NFAT activation, cytokine production and promoted near identical transcriptional responses enriched for immune activation pathways. An analysis of genetic and clinical data of human individuals showed that homozygous null mutations selectively abolishing Orai1α are not associated with disease resembling CRAC channelopathy. Primary T cells from individuals homozygous or heterozygous for an Orai1α null mutation showed enhanced, rather than impaired, SOCE and NFAT induction. Our data indicate that NFAT activation and transcriptional outputs are primarily driven by the graded strength of SOCE mediated by each isoform of the Orai quartet.

The large amounts of calcium (Ca2+) stored in the endoplasmic reticulum (ER) and the controlled release of this Ca2+ store into the cytosol regulate many cellular functions, and altered ER Ca2+ homeostasis induces ER stress. Stromal-interacting molecules 1 and 2 (STIM1/2) are homologous ER-resident Ca2+ sensors that synergistically activate cytosolic Ca2+ influx through Orai channels to promote Ca2+-dependent changes in gene expression and ER Ca2+ refilling. Here, we demonstrated that reduced abundance of STIM2, but not that of STIM1, was associated with poor prognosis in colorectal cancer (CRC). STIM2-deficient CRC cells showed enhanced ER Ca2+ content in a manner dependent on the ER Ca2+ pump SERCA2, increased expression of genes associated with protein translation, and transcriptional and metabolic rewiring. STIM2 deficiency in CRC xenografts led to increased tumor size, invasion, and metastasis. STIM2 loss activated the expression of genes involved in ER stress responses in a manner dependent on the chaperone BiP and the transcription factor ATF4 and independent of Orai channels. These results suggest that loss of STIM2 may inform CRC prognosis.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes. Editor's evaluation This study shows that Orai1 and Orai3 are important elements of the calcium signaling machinery in mouse B cells and regulate downstream responses of proliferation, energetics, and metabolic reprogramming that occur after stimulation of the B cell antigen receptor. This work provides new information of interest to a broad range of readers in the fields of immunology, ion channels, and calcium signaling. https://doi.org/10.7554/eLife.84708.sa0 Decision letter eLife's review process Introduction Calcium (Ca2+) is an essential regulator of immune cell function (Prakriya and Lewis, 2015; Trebak and Kinet, 2019). Crosslinking of immunoreceptors like the T cell receptor (TCR) or B cell receptor (BCR) triggers a robust elevation in intracellular Ca2+ concentrations through the release of endoplasmic reticulum (ER) Ca2+ stores and a concomitant influx of Ca2+ from the extracellular space (Baba and Kurosaki, 2016; King and Freedman, 2009). In lymphocytes, Ca2+ entry across the plasma membrane (PM) is predominately achieved through CRAC channels, which constitute the ubiquitous SOCE (Prakriya and Lewis, 2015; Trebak and Kinet, 2019; Trebak and Putney, 2017; Vaeth et al., 2020). Stimulation of immunoreceptors coupled to phospholipase C isoforms results in the production of the secondary messenger inositol-1,4,5-trisphosphate (IP3), which triggers the release of ER Ca2+ through the activation of ER-resident IP3 receptors. A reduction in ER Ca2+ concentrations is sensed by stromal interaction molecule 1 (STIM1) and its homolog STIM2, resulting in a conformational change and clustering in ER-PM junctions where STIM molecules interact with and activate PM hexameric Orai channels (Orai1-3) to mediate SOCE (Lunz et al., 2019). Ca2+ entry through CRAC channels regulates immune cell function through a host of Ca2+-sensitive transcription factors including the NFAT, nuclear factor κB (NF-κB), c-Myc, and mTORC1 (Berry et al., 2018; Trebak and Kinet, 2019; Vaeth and Feske, 2018; Vaeth et al., 2020; Vaeth et al., 2017a). The coordination of these master transcriptional regulators is indispensable for innate and adaptive immune cell effector function including entry into the cell cycle, clonal expansion, cytokine secretion, differentiation, and antibody production (Shaw and Feske, 2012; Trebak and Kinet, 2019; Vaeth et al., 2020). Orai1 has long been established as a central component of the native CRAC channel in all cell types studied. Although all three Orai isoforms are ubiquitously expressed across tissue types and can form functional CRAC channels when ectopically expressed, only recently have Orai2 and Orai3 emerged as regulators of native CRAC channel function (Emrich et al., 2022b; Tsvilovskyy et al., 2018; Vaeth et al., 2017b; Yoast et al., 2020a). Patients with inherited loss-of-function (LoF) mutations in Orai1 (e.g. R91W mutation) develop a CRAC channelopathy with symptoms including combined immunodeficiency, ectodermal dysplasia, muscular hypotonia, and autoimmunity (Feske et al., 2006; Lian et al., 2018; McCarl et al., 2009). While these patients display relatively normal frequencies of most immune cell populations, they are highly susceptible to reoccurring viral, bacterial, and fungal infections due to defects in T cell expansion, cytokine secretion, and metabolism (Vaeth et al., 2020). There are currently no reported patient mutations in Orai2 or Orai3 genes and the role these channels play within the immune system had largely been unclear. Interestingly, recent studies utilizing global Orai2 knockout mice have demonstrated that loss of Orai2 leads to enhanced SOCE and corresponding CRAC currents in bone marrow-derived macrophages, dendritic cells, T cells, enamel cells, and mast cells (Eckstein et al., 2019; Tsvilovskyy et al., 2018; Vaeth et al., 2017b), suggesting that Orai2 is a negative regulator of CRAC channel activity. Combined knockout in mice of both Orai1 and Orai2 in T cells led to a near ablation of SOCE and impaired humoral immunity, while ectopic expression of pore-dead mutants of Orai1 (E106Q) or Orai2 (E80Q) into individual Orai1-/- or Orai2-/- T cells blocked native SOCE (Vaeth et al., 2017b). Similarly, the generation of HEK293 cell lines lacking each Orai isoform individually and in combination resulted in altered Ca2+ oscillation profiles, CRAC currents, and NFAT isoform activation (Emrich et al., 2022b; Emrich et al., 2021; Yoast et al., 2020a; Yoast et al., 2020b). These data provide evidence that Orai2 and Orai3 exert negative regulatory effects on native CRAC channels possibly by forming heteromeric CRAC channels with Orai1. In contrast to T cells, much less is known regarding the role of SOCE in B lymphocytes. Early landmark work investigating B cell-specific STIM knockout mice established that STIM1 mediates the vast majority of SOCE in B cells, while only the combined deletion of both STIM1 and STIM2 resulted in substantial impairments in B cell survival, proliferation, and NFAT-dependent IL-10 secretion (Matsumoto et al., 2011). Unexpectedly, mice with STIM1/STIM2-deficient B cells show normal humoral immune responses to immunization with both T cell-dependent and independent antigens. These and other studies suggest that the severe immunodeficiency observed in CRAC channelopathy patients is due to impaired T cell responses (Matsumoto et al., 2011; Vaeth et al., 2016). Investigation of Orai1-/- mice on the mixed Institute for Cancer Research (ICR) background and Orai1R93W knock-in mice (the equivalent of human R91W mutation) demonstrated that SOCE is significantly attenuated in B cells (Gwack et al., 2008; McCarl et al., 2010). However, SOCE in B cells is only partially reduced with the loss of Orai1, suggesting that Orai2 and/or Orai3 mediate the remaining SOCE in B cell populations. A recent study showed that the deletion of Orai3 in mice does not affect B cell function and humoral immune responses (Wang et al., 2022), suggesting other Orai homologs compensate for the loss of Orai3. Thus, the contribution of each Orai isoform to native CRAC channel function and downstream signaling in B cells has remained obscure. In this study, we investigated the contributions of Orai isoforms to SOCE and its downstream signaling in B cells through multiple CRISPR/Cas9 knockout B cell lines and novel B cell-specific Orai knockout mice. Our findings demonstrate that the expression of each Orai isoform is dynamically regulated in response to B cell activation and that the magnitude of SOCE is unique among effector B cell populations. We show that the deletion of Orai1 alone does not alter BCR-evoked cytosolic Ca2+ oscillations, proliferation, and development. Unexpectedly, we show that Orai3 is involved in regulating B cell SOCE and downstream signaling functions. Deletion of both Orai1 and Orai3 strongly inhibits SOCE and hampers mitochondrial metabolism in B cells. Transcriptome and metabolomic analysis uncovered key signaling pathways that are regulated by SOCE and the phosphatase calcineurin for the efficient transition of B cells from a quiescent to a metabolically active state. Surprisingly, humoral immunity to influenza A virus infection of mice with B cell-specific deletion of both Orai1 and Orai3 was unaltered, suggesting that alternative Ca2+-independent signaling pathways activated in vivo through co-stimulatory receptors can overcome the loss of CRAC channel activity in B cells. Our data elucidate the role of SOCE in B cell functions and show that CRAC channel function in B lymphocytes is mediated by both Orai1 and Orai3. This knowledge is important for the potential targeting of CRAC channels in specific lymphocyte subsets in immune and inflammatory diseases. Results Orai channel isoforms are dynamically regulated in response to B cell activation Throughout their lifespan, B lymphocytes must undergo dramatic periods of metabolic adaptation whereby naïve, metabolically quiescent cells prepare to clonally expand and differentiate into effector populations (Akkaya and Pierce, 2019; Boothby and Rickert, 2017). Induction of these diverse metabolic programs is driven by downstream BCR-mediated Ca2+ signals in combination with Ca2+-independent costimulatory signals including CD40 activation and/or stimulation with various toll-like receptor (TLR) ligands (Akkaya et al., 2018; Baba and Kurosaki, 2016; Berry et al., 2020). Thus, using Seahorse assays we first evaluated how activation of wild-type mouse primary splenic B cells (isolated as described in methods) regulates oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as indicators of oxidative phosphorylation (OXPHOS) and glycolysis, respectively (Figure 1A and B). Naïve primary B cells from mouse spleens were isolated by negative selection and stimulated for 24 hr under five different conditions as follows: (1) control unstimulated cells (Unstim), (2) stimulation with anti-IgM antibodies (IgM) to activate the BCR, (3) stimulation with anti-CD40 (CD40), (4) stimulation with lipopolysaccharides (LPS), and (5) co-stimulation with anti-IgM and anti-CD40. B cell stimulation for 24 hr led to a robust increase in basal OCR, except for B cells stimulated with anti-CD40 alone where the increase in basal OCR was not statistically significant (Figure 1A and C). Figure 1B shows that B cells stimulated with either anti-IgM, anti-IgM +anti-CD40, or LPS become highly energetic and upregulate both OXPHOS and glycolytic pathways. However, B cells stimulated with anti-CD40 alone remain metabolically quiescent like control non-stimulated B cells (Figure 1B). We used the protonophore trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) to dissipate the mitochondrial membrane potential and calculate the maximal respiratory capacity of B cells. Consistent with results obtained for basal OCR, stimulated B cells showed a robust increase in maximal respiratory capacity except for cells stimulated with anti-CD40 alone (Figure 1D). Both total mitochondrial content and membrane potential measured with MitoTracker Green and TMRE staining, respectively, were substantially increased following anti-IgM, anti-IgM +anti-CD40, or LPS stimulation while both parameters were only slightly increased with stimulation with anti-CD40 alone (Figure 1E and F). While previous studies suggested that Orai1 regulates the majority of SOCE in primary B cells (Gwack et al., 2008; McCarl et al., 2010), the expression of different Orai isoforms both at rest and upon B cell activation is unknown. Utilizing the same five experimental conditions described above, we analyzed the mRNA expression of Orai1, Orai2, and Orai3 following 24 hr of stimulation. Stimulation with either anti-IgM or anti-IgM +anti-CD40 dramatically increased Orai1 expression, while the magnitude of this change following anti-CD40 or LPS stimulation was smaller and did not reach statistical significance (Figure 1G). Interestingly, stimulation conditions that strongly increased mitochondrial respiration (anti-IgM, anti-IgM +anti-CD40, LPS) all significantly reduced expression of Orai2 while stimulation with anti-CD40 alone had no significant effect on Orai2 expression (Figure 1H). The expression of Orai3 has significantly increased only with anti-IgM +anti-CD40 or LPS stimulation, but not with anti-IgM or anti-CD40 alone (Figure 1I). Figure 1 Download asset Open asset B cell activation dynamically regulates Orai channel expression. (A) Measurement of oxygen consumption rate (OCR) in primary B lymphocytes following 24 hr stimulation with anti-IgM (20 μg/mL), anti-CD40 (10 μg/mL), anti-IgM +anti-CD40, or LPS (10 μg/mL) using the Seahorse Mito Stress Test (n=3 biological replicates). (B) Energy map of maximal OCR and extracellular acidification rate (ECAR) following the addition of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (C, D) Quantification of basal (C) and maximal (D) respiration from Seahorse traces in (A) (One-way ANOVA with multiple comparisons to Unstimulated). (E, F) Measurement of (E) total mitochondrial content with the fluorescent dye MitoTracker Green and (F) mitochondrial membrane potential with TMRE following 24 hr stimulation (n=3 biological replicates; One-way ANOVA with multiple comparisons to Unstimulated). (G–I) Quantitative RT-PCR of (G) Orai1, (H) Orai2, and (I) Orai3 mRNA following 24 hr of stimulation with the stimuli indicated (n=3 biological replicates; one-way ANOVA with multiple comparisons to Unstimulated). All scatter plots and Seahorse traces are presented as mean ± SEM. For all figures, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. Figure 1—source data 1 Source data for Figure 1. https://cdn.elifesciences.org/articles/84708/elife-84708-fig1-data1-v2.xlsx Download elife-84708-fig1-data1-v2.xlsx Both Orai3 and Orai1 mediate SOCE in A20 B lymphoblasts Robust activation of primary B cells with anti-IgM/anti-CD40 co-stimulation resulted in upregulation of both Orai1 and Orai3 mRNA with downregulation of Orai2 (Figure 1G–I). We, therefore, reasoned that B cells may sustain long-term cytosolic Ca2+ signals through Orai1 and/or Orai3, while downregulation of Orai2 might relieve the inhibition of native CRAC channels, as recently demonstrated for activated T cells (Vaeth et al., 2017b). To gain insights into the molecular composition of CRAC channels in B cells, we first developed an in vitro system utilizing the mouse A20 B lymphoblast cell line to generate single and double Orai1 and Orai3 knockout cell lines with CRISPR/Cas9 technology. Two guide RNA (gRNA) sequences were utilized to cut at the beginning and end of the coding region of mouse Orai1 and Orai3 (mOrai1 and mOrai3), effectively excising the entirety of the genomic DNA for each Orai gene (Figure 2A). We generated multiple A20 clones that were lacking Orai1 and Orai3 individually and in combination and these knockout clones were validated through genomic DNA sequencing (Source data 1) and the absence of Orai1 or Orai3 transcripts by qPCR (Figure 2B–D). We measured SOCE in A20 cells after passive store depletion with thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum ATPase (SERCA). A20 cells lacking Orai1 demonstrated a large reduction in maximal SOCE by ~62%, while in cells lacking Orai3 SOCE was reduced by ~28% (Figure 2E and F). Importantly, the combined deletion of both Orai1 and Orai3 caused a near abrogation of SOCE by ~91% (Figure 2E and F). Furthermore, measurements of cytosolic Ca2+ oscillations induced by anti-IgG stimulation demonstrated that the oscillation frequency was substantially reduced with combined Orai1/Orai3 knockout, while only slightly reduced with the loss of either Orai1 or Orai3 individually (Figure 2G–K). These data suggest that both Orai3 and Orai1 were involved in optimal CRAC channel function in A20 B lymphoblasts. These data are consistent with the function of Orai channels in other cell types. Indeed, we recently utilized a series of single, double, and triple Orai CRISPR/Cas9 knockout HEK293 cell lines and showed that SOCE, but not Orai1, is required for agonist-induced Ca2+ oscillations (Yoast et al., 2020b). Under conditions of physiological agonist stimulation that causes modest ER store depletion while eliciting Ca2+ oscillations, Orai2 and Orai3 were sufficient to maintain cytosolic Ca2+ oscillations, while having relatively minor contributions to SOCE induced by maximal store depletion (Yoast et al., 2020b). Figure 2 Download asset Open asset Orai1 and Orai3 mediate the bulk of store-operated Ca2+ entry (SOCE) in A20 B lymphoblasts. (A) Cartoon schematic of the two gRNA CRISPR strategies we used to excise mouse Orai1 and Orai3 genes. (B–D) Quantitative RT-PCR of (B) Orai1, (C) Orai2, and (D) Orai3 mRNA in A20 Orai CRISPR clones (n=3 biological replicates). (E) Measurement of SOCE with Fura2 upon store depletion with 2 µM thapsigargin in 0 mM Ca2+ followed by re-addition of 2 mM Ca2+ to the external bath solution. (F) Quantification of peak SOCE in (E) (from left to right n=99, 100, 89, and 98 cells; Kruskal-Wallis test with multiple comparisons to WT A20). (G–J) Representative Ca2+ oscillation traces from 5 cells/condition measured with Fura2 upon stimulation with 10 μg/mL anti-IgG antibodies at 60 s (indicated by arrows) in the presence of 2 mM external Ca2+. (K) Quantification of total oscillations in 9 min from (G–J) (from left to right n=76, 79, 79, and 78 cells; Kruskal-Wallis test with multiple comparisons to WT A20). All scatter plots are presented as mean ± SEM. For all figures, *p<0.05; **p<0.01; ****p<0.0001; ns, not significant. Figure 2—source data 1 Source data for Figure 2. https://cdn.elifesciences.org/articles/84708/elife-84708-fig2-data1-v2.xlsx Download elife-84708-fig2-data1-v2.xlsx Orai1 is dispensable for cytosolic Ca2+ oscillations in primary B cells To determine the contribution of Orai1-mediated Ca2+ signals to primary B cell function, we generated B cell-specific Orai1 knockout (Orai1fl/fl Mb1-Cre/+) mice (Figure 3). Compared to Orai1fl/fl controls, the average surface expression of Orai1 was significantly reduced on B220+ B cells from Orai1fl/fl Mb1-Cre/+ mice by~70% (Figure 3A and B). The residual signal of ~30% above FMO (Figure 3A) is likely the result of the non-specific binding of the Orai1 antibody by its variable region to other Orai homologs (Orai2, Orai3), which partially share the peptide epitope recognized by the anti-Orai1 antibody. This residual Orai1 antibody signal is consistent with previous studies showing that Cre-mediated deletion of Orai1fl/fl alleles is very efficient in other cell types including T cells (Kaufmann et al., 2016), and neuronal progenitor cells (Somasundaram et al., 2014). Indeed, other lines of evidence indicate that Orai1 is deleted in B cells of Orai1fl/fl Mb1-Cre/+ mice: our qPCR data show that B cells from Orai1fl/fl Mb1-Cre/+ mice showed a near abrogation of Orai1 mRNA (to ~3% of control) compared to Mb1-Cre/+ control mice (Figure 3C) without any significant change in Orai2 (Figure 3D) or Orai3 (Figure 3E) mRNA expression. Further, Ca2+ measurements demonstrated that SOCE is reduced by ~69% in B cells isolated from Orai1fl/fl Mb1-Cre/+ mice (Figure 3F and G). We measured cytosolic Ca2+ oscillations in response to anti-IgM stimulation in the presence of 1 mM extracellular Ca2+. In agreement with previous reports and consistent with our data with A20 B lymphoblasts (Figure 2G and H), Orai1-deficient B cells display a comparable frequency of agonist-induced Ca2+ oscillations as control B cells (Figure 3H–J). Thus, while Orai1 knockout significantly decreased SOCE in primary B cells, it is dispensable for maintaining Ca2+ oscillations in response to physiological agonist stimulation. In control Mb1-Cre/+ mice, Orai1 expression was comparable between B220+ B cells and CD8+ T cells, with CD4+ T cells showing a slightly lower signal (Figure 3—figure supplement 1A, B ). After 24 hour stimulation of control B cells under the five conditions described above, Orai1-deficient B cells showed no apparent defects in activation as the expression of MHC-II was comparable to control B cells (Figure 3—figure supplement 1C–E), while expression of CD86 was slightly reduced with anti-IgM stimulation or anti-IgM +anti-CD40 co-stimulation (Figure 3—figure supplement 1F–H). Figure 3 with 1 supplement see all Download asset Open asset Orai1 is dispensable for BCR-induced Ca2+ oscillations in primary B cells. (A) Representative flow cytometry histogram of B cells isolated from Orai1fl/fl and Orai1fl/fl Mb1-Cre/+ mice. Splenocytes from naïve Orai1fl/fl and Orai1fl/fl Mb1-Cre/+ mice were fixed, permeabilized, and stained with rabbit anti-Orai1 polyclonal antibody (YZ6856, epitope: human ORAI1#275–291 intra-cellular C-terminal, cross-reacts with the mouse). The numbers inside the panel represent the mean fluorescence intensity (MFI) for the Orai1 antibody staining for each sample. (B) Quantification of Orai1 MFI minus fluorescence minus one (FMO) in B cells is shown. (n=4 biological replicates; unpaired T-test). (C–E) Quantitative RT-PCR of (C) Orai1, (D) Orai2, and (E) Orai3 mRNA in isolated B cells (n=6 biological replicates for each; Mann-Whitney test). (F) Measurement of SOCE with Fura2 upon store depletion with 2 µM thapsigargin in 0 mM Ca2+ followed by re-addition of 1 mM Ca2+ to the external bath solution. (G) Quantification of peak store-operated Ca2+ entry (SOCE) in (F) n=169 and 178 cells; Mann-Whitney test. (H–I) Representative Ca2+ oscillation traces from 5 cells/condition measured with Fura2 upon stimulation with 20 μg/mL anti-IgM antibodies at 1 min (indicated by arrows) in the presence of 1 mM external Ca2+. (J) Quantification of total oscillations in 9 min from (I, J) (n=97 and 111 cells; Mann-Whitney test). All scatter plots are presented as mean ± SEM. For all figures, **p<0.01; ****p<0.0001; ns, not significant. Figure 3—source data 1 Source Data for Figure 3. https://cdn.elifesciences.org/articles/84708/elife-84708-fig3-data1-v2.xlsx Download elife-84708-fig3-data1-v2.xlsx Loss of Orai1 does not alter B cell development While patients and mouse models with loss of function (LoF) mutations in Orai1 present with severe immunodeficiency due to impaired T cell function, the development of most immune cell populations is largely unaltered, suggesting SOCE is dispensable for initial immune cell selection and development (Gwack et al., 2008; Lacruz and Feske, 2015; McCarl et al., 2010). We evaluated B cell development within the bone marrow and spleen of Orai1fl/fl Mb1-Cre/+ mice (Figure 4). In agreement with previous reports that investigated B cell development in global Orai1 deficient mice, the development of early B cell progenitors in fractions A-F within the bone marrow were unaltered in Orai1fl/fl Mb1-Cre/+ mice (Figure 4A–C). Similarly, we observed no significant differences in peripheral transitional type 1 (T1), T2, and T3 immature B cells in the spleen (Figure 4D and F). Mature B cell populations in the spleen are predominately follicular B cells with a smaller fraction of marginal zone B cells, and these ratios were largely comparable between control and Orai1fl/fl Mb1-Cre/+ mice (Figure 4E and F). Figure 4 Download asset Open asset Orai1 is dispensable for B cell development. (A–B) Flow cytometric analysis of bone marrow populations for B cell fractions A (B220+CD43+HSA−BP-1−), B (B220+CD43+HSA+BP-1−), C (B220+CD43+HSA+BP-1+), D (B220+CD43−IgM−CD93+), E (B220+CD43−IgM+CD93+), and F (B220+CD43−IgM+CD93−). (C) Quantification of bone marrow populations in (A, B) (n=7 and six biological replicates; Mann-Whitney test). (D) Flow cytometric analysis of isolated populations in the spleen for B cell developmental stages T1 (B220+AA4.1+CD23−IgM+), T2 (B220+AA4.1+CD23+IgM+), and T3 (B220+AA4.1+CD23+IgM−). (E) Flow cytometric analysis of isolated populations in the spleen for marginal zone (MZ) B cells (B220+CD93−CD23−IgM+) and follicular (FO) B cells (B220+CD93−CD23+IgM+). (F) Quantification of splenic populations in (D, E) (n=7 and six biological replicates; Mann-Whitney test). All scatter plots are presented as mean ± SEM. Figure 4—source data 1 Source data for Figure 4. https://cdn.elifesciences.org/articles/84708/elife-84708-fig4-data1-v2.xlsx Download elife-84708-fig4-data1-v2.xlsx Both Orai3 and Orai1 synergistically contribute to SOCE in primary B cells To determine whether Orai3 regulates Ca2+ signals and function of primary B cells, we generated B cell-specific Orai3 knockout mice (Orai3fl/fl Mb1-Cre/+) and B cell-specific double Orai1/Orai3 knockout mice (Orai1/Orai3fl/fl Mb1-Cre/+). Primary B cells isolated from spleens of Orai1, Orai3, and Orai1/Orai3 knockout mice showed near complete ablation of their respective Orai isoform mRNA (to ~0.3–4% of control) with no compensatory changes in Orai2 mRNA expression (Figure 5A). As documented above (Figure 3F and G), depletion of ER Ca2+ stores with thapsigargin demonstrated that SOCE was significantly reduced in Orai1 knockout B cells (by ~69%), and this remaining Ca2+ entry was further reduced in Orai1/Orai3 double knockout B cells (by ~83%; Figure 5B and C). However, SOCE in single Orai3 knockout B cells was ~102% of control, which was not significantly different from control B cells. Figure 5 with 1 supplement see all Download asset Open asset Orai1 and Orai3 synergistically mediate store-operated Ca2+ entry (SOCE) in primary B cells. (A) Quantitative RT-PCR of Orai1, Orai2, and Orai3 mRNA in negatively isolated B cells from B cell-specific Orai knockout mice (n=3 biological replicates per genotype). (B) Measurement of SOCE in naïve B cells with Fura2 upon store depletion with 2 µM thapsigargin in 0 mM Ca2+ followed by re-addition of 1 mM Ca2+ to the external bath solution. Subsequently, SOCE was inhibited with the addition of 50 µM 2-APB at 13 min followed by 5 µM Gd3+ at 18 min. (C) Quantification of peak SOCE in (B) (from left to right n=200, 200, 199, and 149 cells; Kruskal-Wallis test with multiple comparisons to Mb1-Cre/+). (D) Quantification of the rate of 2-APB inhibition from 13 to 18 min. (E) Quantitative RT-PCR of Orai1, Orai2, and Orai3 mRNA in negatively isolated B cells from wild-type and Orai2-/- mice (n=3 and six biological replicates). (F) Measurement of SOCE in naïve B cells with Fura2 from wild-type and Orai2-/- mice. (G) Quantification of peak SOCE in (F) (n=147 and 240 cells; Mann-Whitney test). All scatter plots are presented as mean ± SEM. For all figures, ***p<0.001; ****p<0.0001; ns, not significant. Figure 5—source data 1 Source data for Figure 5. https://cdn.elifesciences.org/articles/84708/elife-84708-fig5-data1-v2.xlsx Download elife-84708-fig5-data1-v2.xlsx Orai channel isoforms demonstrate distinct pharmacological profiles and sensitivities to various CRAC channel modifiers (Bird and Putney, 2018; Zhang et al., 2020). One of the most extensively utilized CRAC channel modifiers is 2-aminoethyl diphenyl borate (2-APB) (Prakriya and Lewis, 2001), which at high (25–50 µM) concentrations strongly inhibits Orai1, partially inhibits Orai2, and potentiates Orai3 channel activity (DeHaven et al., 2008; Zhang et al., 2008; Zhang et al., 2020). After allowing Ca2+ entry to the plateau, 50 µM 2-APB was added, followed by the addition of 5 µM gadolinium (Gd3+) which potently blocks all Orai isoforms (Yoast et al., 2020b; Zhang et al., 2020). In wild-type B cells, 2-APB led to a gradual inhibition of SOCE over the course of 5 min, which completely returned to baseline following the addition of Gd3+. Interestingly, the remaining SOCE in Orai1 knockout B cells showed essentially no inhibition by 2-APB but was strongly inhibited by Gd3+. The lack of effect of 2-APB is likely due to the residual SOCE mediated by the remaining Orai3 isoform, which is resistant to inhibition by 2-APB. Furthermore, SOCE in Orai3 knockout B cells was inhibited at a significantly faster rate by 2-APB compared to wild-type B cells and was not further inhibited by Gd3+ (Figure 5B and D). The small amount of SOCE remaining in Orai1/Orai3 double knockout cells was reduced to baseline following the addition of 2-APB. This remaining SOCE in Orai1/Orai3 double knockout B cells prompted us to measure SOCE in B cells isolated from global Orai2 knockout mice (Orai2-/-). B cells from Orai2-/- mice showed complete loss of Orai2 mRNA with no apparent compensation in Orai1 or Orai3 mRNA (Figure 5E). SOCE stimulated by thapsigargin in B cells from Orai2-/- mice was enhanced by comparison to B cells from wild-type littermate controls (Figure 5F and G), suggesting that, as was shown in T cells (Vaeth et al., 2017b), Orai2 is also a negative regulator of SOCE in B cells. Thus, these experiments indicate that the remaining SOCE in B cells from Orai3/Orai1 double knockout mice is most likely mediated by Orai2. Given the modulation of Orai channel isoforms in response to B cell activation, we performed Ca2+ imaging recordings, similar to those in Figure 5, on primary B cells that were first activated for 48 hr with anti-IgM +anti-CD40 (Figure 5—figure supplement 1A–C). While similar trends were observed in experiments with naïve primary B cells, several exceptions were notable. SOCE in Orai3 knockout B cells was reduc

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis.

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in expression of Orai isoforms in response to B cell activation. We show that Orai3 and Orai1 are essential components of native CRAC channels in B cells and are critical for primary B cell proliferation and survival. The combined loss of Orai1 and Orai3 strongly impairs SOCE, nuclear factor for activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of B cells in response to antigenic stimulation. Our results clarify the molecular composition and cellular functions of SOCE in B lymphocytes. ### Competing Interest Statement The authors have declared no competing interest.

Antigen receptor stimulation triggers cytosolic Ca2+ signals, which activate transcriptional and metabolic programs critical for immune function. B-cell receptor (BCR) engagement causes rapid cytosolic Ca2+ rise through the ubiquitous store-operated calcium entry (SOCE) pathway. Slc8b1, which encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), extrudes Ca2+ out of the mitochondria and maintains optimal SOCE activity. Inhibition of NCLX in DT40 and A20 B lymphocyte lines was recently shown to impair cytosolic Ca2+ transients in response to antigen-receptor stimulation, however the downstream functional consequences of this impairment remain unclear. Here, we generated Slc8b1 knockout A20 B-cell lines using CRISPR/Cas9 technology and B-cell specific Slc8b1 knockout mice. Surprisingly, while loss of Slc8b1 in B lymphocytes led to reduction in SOCE, it had a marginal effect on mitochondrial Ca2+ extrusion, suggesting that NCLX is not the major mitochondrial Ca2+ extrusion mechanism in B cells. Furthermore, endoplasmic reticulum (ER) Ca2+ content and rates of ER depletion and refilling remained unaltered in Slc8b1 knockout B cells. Slc8b1 deficiency increased mitochondrial production of oxidants, reduced mitochondrial bioenergetics and altered mitochondrial ultrastructure. B-cell specific Slc8b1 knockout mice showed reduced germinal center B cell responses following foreign antigen and pathogen driven immune responses. Our studies provide novel insights into the function of Slc8b1 in germinal center B cells and its contribution to B-cell signaling and effector function.

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma.

Summary Invertebrates express one endoplasmic reticulum (ER)-resident Ca 2+ -sensing stromal-interaction molecule (Stim) and one Orai plasma membrane channel protein. Stim conveys store depletion to Orai, mediating the evolutionarily conserved Ca 2+ release-activated Ca 2+ (CRAC) current. The crucial role of their vertebrate homologues, STIM1 and Orai1 in mediating CRAC activity in mammals is well-established. However, mammals possess two STIM and three Orai isoforms and the choreography of their interactions under physiological receptor activation is unknown. We show that the five mammalian STIM1/2 and Orai1/2/3 isoforms have non-redundant functions. Yet, all five isoforms are always required together to ensure the graded diversity of mammalian Ca 2+ signaling events in response to the full spectrum of agonist strengths. Receptor-activated Ca 2+ signaling across the range of stimulus intensities requires functional interactions between not only STIM1/2 and Orai1/2/3, but also IP 3 R, ensuring that receptor-mediated Ca 2+ release is precisely tailored to Ca 2+ entry and activation of nuclear factor of activated T-cells (NFAT). This is orchestrated by two interdependent and counterbalancing paradigms: the N-termini Ca 2+ -binding ER-luminal domains of unactivated STIM1/2 inhibit IP 3 R-evoked Ca 2+ release. Gradual increase in agonist intensity leads to gradual STIM1/2 activation and relief of IP 3 R inhibition. Concomitantly, the cytosolic C-termini of activated STIM1/2 differentially interact with Orai1/2/3 proteins as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai proteins and IP 3 Rs at the ER-lumen and cytosol translate the strength of agonist stimulation to precise levels of Ca 2+ release, Ca 2+ entry and NFAT induction, ensuring the diversity and fidelity of complex mammalian Ca 2+ signaling. Highlights All five STIM/Orai and IP 3 R are always required together in mammalian Ca 2+ signalling Unactivated STIM1/2 inhibit IP 3 R and activated STIM1/2 cooperatively activate Orai1/2/3 STIM1 contribution increases and that of STIM2 decreases as agonist intensifies Graded IP 3 R disinhibition and Orai activation tailor receptor activity to NFAT induction

Mitochondrial Ca 2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca 2+ uptake by respiring mitochondria prevents Ca 2+ -dependent inactivation (CDI) of store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels and inhibits Ca 2+ extrusion to sustain cytosolic Ca 2+ signaling. However, how Ca 2+ uptake by the mitochondrial Ca 2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca 2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca 2+ entry (SOCE). Paradoxically, despite enhancing Ca 2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca 2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca 2+ oscillations, endoplasmic reticulum Ca 2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca 2+ , despite limiting CRAC channel activity.

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release-activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5 mu M), 2-APB (5 mu M and 50 mu M), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.

The essential role of ORAI1 channels in receptor-evoked Ca 2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca 2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca 2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca 2+ -dependent inactivation. This broadened bandwidth of Ca 2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca 2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.

Summary Despite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca 2+ (mtCa 2+ ) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa 2+ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa 2+ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na + /Ca 2+ exchanger NCLX ( SLC8B1 ) is decreased in colorectal tumors and is associated with advanced-stage disease in patients. We reveal that downregulation of NCLX leads to mtCa 2+ overload, mitochondrial depolarization, mitophagy, and reduced tumor size. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, the expression of epithelial-to-mesenchymal transition (EMT), hypoxia, and stem cell pathways. Mechanistically, mtCa 2+ overload leads to an increase in mitochondrial reactive oxygen species (mtROS) which activates HIF1α signaling supporting the metastatic behavior of tumor cells lacking NCLX. Our results reveal that loss of NCLX expression is a novel driver of metastatic progression, indicating that control of mtCa 2+ levels is a novel therapeutic approach in metastatic colorectal cancer. Highlights The expression of NCLX is decreased in colorectal tumors and is associated with advanced-stage disease in patients. NCLX plays a dichotomous role in colorectal tumor growth and metastasis. NCLX downregulation causes mitophagy and reduced colorectal cancer tumor growth. NCLX downregulation induces stemness, chemoresistance and metastasis through mtCa 2+ /ROS/HIF1α signaling axis. Graphical Abstract Significance Mitochondrial Ca 2+ (mtCa 2+ ) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa 2+ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca 2+ uniporter (MCU). mtCa 2+ uptake by the MCU is followed by a ∼100-fold slower mtCa 2+ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na + /Ca 2+ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa 2+ homeostasis that cannot easily be compensated by another Ca 2+ transport mechanism. This represents the first study investigating the role of NCLX in tumorigenesis and metastasis. We demonstrate for the first time that colorectal cancers exhibit loss of NCLX expression and that this is associated with advanced-stage disease. Intriguingly, decreased NCLX function has a dichotomous role in colorectal cancer. Thus, we reveal that NCLX loss leads to reduced primary tumor growth and overall tumor burden in vivo . Yet, the consequential increases in mtCa 2+ elicit pro-survival, hypoxic and gene transcription pathways that enhance metastatic progression. This dichotomy is a well-established feature of chemoresistant and recurrent tumor cells including cancer stem cells. Moreover, the downstream changes elicited by NCLX loss are reminiscent of mesenchymal colorectal cancer subtypes that display poor patient survival. Our data indicate that the demonstrated changes to the mtCa 2+ /mtROS/HIF1α signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.

Store operated calcium (Ca2+) entry is an important homeostatic mechanism in cells, whereby the release of Ca2+ from intracellular endoplasmic reticulum stores triggers the activation of a Ca2+ influx pathway. Mediated by Orai1, this Ca2+ influx has specific and essential roles in biological processes as diverse as lactation to immunity. Although pharmacological inhibitors of this Ca2+ influx mechanism have helped to define the role of store operated Ca2+ entry in many cellular events, the lack of isoform specific modulators and activators of Orai1 has limited our full understanding of these processes. Here we report the identification and synthesis of an Orai1 activity enhancer that concurrently potentiated Orai1 Ca2+ -dependent inactivation (CDI). This unique enhancer of Orai1 had only a modest effect on Orai3 with weak inhibitory effects at high concentrations in intact MCF-7 breast cancer cells. The Orai1 enhancer heightened vascular smooth muscle cell migration induced by platelet-derived growth factor and the unique store operated Ca2+ entry pathway present in skeletal muscle cells. These studies show that IA65 is an exemplar for the translation and development of Orai isoform selective agents. The ability of IA65 to activate CDI demonstrates that agents can be developed that can enhance Orai1-mediated Ca2+ influx but avoid the cytotoxicity associated with sustained Orai1 activation. IA65 and/or future analogues with similar Orai1 and CDI activating properties could be fine tuners of physiological processes important in specific disease states, such as cellular migration and immune cell function.

ORAI1 constitutes the store-operated Ca 2+ release-activated Ca 2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca 2+ -sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca 2+ -dependent inactivation (CDI), protecting against Ca 2+ toxicity. Here we identify a spatially-restricted Ca 2+ /cAMP signaling crosstalk critical for mediating CDI. Binding of Ca 2+ -activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca 2+ . Ca 2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca 2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca 2+ signals and NFAT activation.