Native Orai1 is the primary driver of NFAT1/4 nuclear localization.

NFAT1-GFP (A–D) and NFAT4-GFP (E-L) nuclear translocation in HEK293 Orai double knockout (DKO) cells stimulated with either 2 μM thapsigargin (Tg; A-H) or 10 μM CCh (I-L) in the presence of 2 mM extracellular Ca²⁺. Panels represent time-lapse traces of NFAT nuclear/cytoplasmic fluorescence ratios (A, E, I), quantification of maximal NFAT nuclear/cytoplasmic fluorescence ratios (B, F, J), quantification of NFAT translocation rates (C, G, K) and representative images of NFAT-GFP nuclear localization at the indicated times post stimulation (D, H, L). Data are expressed as mean ± SEM of the nuclear/cytoplasmic GFP fluorescence ratio. Parametric data were analyzed using one-way ANOVA with Dunnett’s post hoc test, and nonparametric data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Scale bar, 10 μm.

Both Orai1α and Orai1β drive NFAT1/4 nuclear localization.

(A) Schematic illustrating alternative translation initiation of Orai1 mRNA, producing Orai1α (301 aa) and Orai1β (238 aa). NFAT1-GFP (B-E) and NFAT4-GFP (F-M) nuclear translocation in HEK293 Orai triple knockout (TKO) cells reconstituted with native levels of Orai1α and Orai1β isoforms expressed under the control of the chicken thymidine kinase (TK) promoter. Cells were stimulated with either 2 μM thapsigargin (Tg, B-I) or 10 μM CCh (J-M) in the presence of 2 mM extracellular Ca²⁺. Panels represent time-lapse traces of NFAT nuclear/cytoplasmic fluorescence ratios (B, F, J), quantification of maximal NFAT nuclear/cytoplasmic fluorescence ratios (C, G, K), quantification of NFAT translocation rates (D, H, L) and representative images of NFAT-GFP nuclear localization at the indicated times post stimulation (E, I, M). Data are expressed as mean ± SEM of the nuclear/cytoplasmic GFP fluorescence ratio. Parametric data were analyzed using one-way ANOVA with Dunnett’s post hoc test, and nonparametric data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Direct Orai2 versus Orai3 comparisons were analyzed using the Mann-Whitney test and are reported in the legend to avoid overcrowding the panels: C, P < 0.0001; D, P < 0.0001; G, P < 0.0001; H, P < 0.0001; K, P = 0.3567; L, P = 0.0683. Scale bar, 10 μm.

The magnitude of NFAT activation mirrors that of Ca²⁺ influx driven by each Orai isoform.

(A) SOCE traces in HEK293 Orai TKO cells reconstituted with each of the four Orai isoforms C-terminally tagged with CFP and expressed from TK-driven constructs. Recordings were performed on cells with equivalent levels of CFP fluorescence. Cells were stimulated with 2 μM thapsigargin (Tg) in Ca²⁺-free medium, followed by re-addition of 2 mM extracellular Ca²⁺; 5 μM Gd³⁺ was applied to block CRAC currents and 10 μM Ionomycin was added at the end of the recordings. (B) Quantification of maximal SOCE amplitude mediated by each Orai isoform by comparison to mock-transfected HEK293 TKO cells. (C) Immunoblot confirming Orai1 knockout in MCF7 cells (MCF7-O1KO) compared with wildtype (WT) controls; GAPDH served as a loading control. (D) SOCE recordings in WT MCF7 cells and MCF7-O1KO cells. (E) Quantification of SOCE peak in WT MCF7 cells and MCF7-O1KO cells from D. (F) Time lapse of NFAT1 nuclear translocation following thapsigargin (Tg) stimulation in MCF7 WT, MCF7-O1KO, and MCF7 WT cells treated with 20 μM BAPTA-AM. (G) Quantification of maximal NFAT1 nuclear/cytoplasmic ratios in each condition from F. Data are expressed as mean ± SEM of the nuclear/cytoplasmic GFP fluorescence ratio. Parametric data were analyzed using one-way ANOVA with Dunnett’s post hoc test, and nonparametric data were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. Scale bar, 10 μm.

NFAT activation by Orai1α and Orai1β depends on localized Ca²⁺ signals near the channel pore.

(A) Schematic illustrating the fast Ca²⁺ chelator BAPTA and the slower chelator EGTA differentially buffer local versus global Ca²⁺ signals. (B–C) SOCE recordings in HEK293 Orai TKO cells expressing native levels of Orai1β (B) or Orai1α (C) driven by the TK promoter, showing that both chelators reduced global Ca²⁺ influx. (D–I) NFAT1 nuclear translocation in cells reconstituted with either Orai1β (D-F) or Orai1α (G-I), which was suppressed by BAPTA-AM but not by EGTA-AM loading. (D, G) Time course of NFAT1 nuclear/cytoplasmic fluorescence ratio following thapsigargin (Tg) stimulation in both conditions. (E, H) Quantification of maximal NFAT1 nuclear translocation. (F, I) Representative images of NFAT1-GFP nuclear localization at indicated times after Tg stimulation in control, EGTA-AM–treated, and BAPTA-AM–treated cells. Data are expressed as mean ± SEM of the nuclear/cytoplasmic GFP fluorescence ratio. Parametric data were analyzed using one-way ANOVA with Dunnett’s post hoc test, and nonparametric data were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. Scale bar, 10 μm.

Both Orai1α and Orai1β support NFAT signaling and cytokine production in primary CD4⁺ T cells.

CD4⁺ T cells from Orai1fl/fl Cd4Cre mice were transduced with retroviral constructs encoding Orai1α-IRES-Ametrine, Orai1β-IRES-Ametrine, or empty vector (MIA). Cells were expanded under Th0 differentiation conditions (anti-IL-4/IFN-γ) and retrovirally transduced for 2 days, then polarized under Th1 condition (IL-12 plus anti-IL-4) for an additional 2 days. (A) Analysis of SOCE in CD4+ T cells from Orai1fl/flCd4Cre mice transduced with Orai1α, Orai1β or empty vector. Cells were loaded with Indo-1 and stimulated with TG in Ca2+-free ringer solution followed by addition of 1 mM Ca2+. SOCE was measured by flow cytometry. Shown are the slope of SOCE, area under the curve (AUC) and peak. Data are from 11 mice and four repeat experiments with one technical replicate per condition. (B) ImageStream analysis of endogenous NFAT1 localization using an anti-NFAT1 antibody. Representative images show brightfield (BF), CD4, Ametrine, NFAT1, and DAPI channels in Orai1-deficient and Orai1α or Orai1β-rescued T cells, either unstimulated or stimulated with 1 μM ionomycin to deplete ER stores. (C) Quantification of NFAT1 nuclear translocation, expressed as similarity scores between NFAT1 and DAPI signals. (D) Cytokine production following PMA/ionomycin stimulation. Representative flow plots of TNF, IFN-γ, and IL-2 in Orai1-deficient T cells transduced with either Orai1α, Orai1β, or empty vector gated on Ametrinedim cells. (E) Quantification of mean fluorescence intensities (MFI) of TNF, IFN-γ, and IL-2 in Orai1α- and Orai1β-rescued T cells. Data are from 3 mice and two repeat experiments with one technical replicate per condition. Statistical analysis in panels (A, E) were analyzed by ordinary one-way ANOVA multiple comparison. All results are expressed as means ± SEM. *P < 0.05 was considered as significant.

Orai1α and Orai1β orchestrate near-identical transcriptional programs in CD4⁺ T cells.

Bulk RNA sequencing of CD4⁺ T cells from Orai1fl/fl Cd4Cremice were transduced with Orai1α, Orai1β, or empty vector (EV) and either left unstimulated (0h) or stimulated with anti-CD3/anti-CD28 for 6h. (A) BigWig tracks of ectopically expressed Orai1α and Orai1β mRNA mapped to the murine Orai1 RefSeq (NM_175423). (B) Principal component analysis (PCA) of all RNA-seq samples (left) and of stimulated samples only (right). (C–E) Analysis of differential expressed genes (DEG). (C) Volcano plots comparing Orai1α vs EV controls, Orai1β vs EV controls, and Orai1α vs Orai1β. (D) Venn diagram showing overlap of DEGs in Orai1α and Orai1β transduced T cells. (E) Dot plot of the log₂ fold-changes in gene expression in Orai1α vs Orai1β-expressing CD4⁺ T cells. (F) Bubble plot of dysregulated pathways in Orai1α and Orai1β-expressing CD4⁺ T cells compared to EV controls using the Pathway Interaction Database (PID). (G) Heatmap of the top 50 upregulated genes in stimulated Orai1α transduced CD4⁺ T cells. Data in A-G are from three samples per condition. Genes in (C-E) were considered significant if the adjusted P value (Padj) was <0.01. The significance of pathways in (F) was calculated using a right-tailed Fisher’s exact test. Z scores in (G) were calculated using the average expression across all samples. Significance was adjusted using the Benjamini-Hochberg method. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001.

Human ORAI1 variants predicted to delete Orai1α protein expression lack clinical features associated with CRAC channelopathy syndrome.

(A) Schematic representation of Orai1 channel topology and loss-of-function (LoF, red) and gain-of-function (GoF, green) mutations reported in patients. Mutations are localized in regions shared by Orai1α and Orai1β. Also indicated are the locations of two frameshift mutations (blue) in the Orai1α-specific N-terminal region. (B) Immunoblot analysis of WT Orai1 and the p.P43Tfs*45 variant ectopically expressed in HEK293 cells and detected with anti-Orai1 polyclonal antibody (Cat # is O8264, Sigma-Aldrich). Indicated are the molecular weights of Orai1α and Orai1β isoforms. (C) Population genetic analysis of Orai1 variants in UK Biobank (UKBB), gnomAD, Qatar Biobank (QBB), and All of Us (AoU) datasets. Allele frequencies (UKBB, gnomAD, QBB,AoU) and number of homozygous carriers (gnomAD, QBB, AoU) of each variant. (D) Prevalence of diagnostic codes in UKBB associated with symptoms of CRAC channelopathy syndrome (infection, ectodermal disease, myopathy) in 24 individuals homozygous for Orai1 p.P43TfsX45 and p.P46SfsX42 variants compared to 984 controls. (E) Distribution of disease burden (total and for specific diagnostic categories) in individuals homozygous for Orai1 p.P43Tfs*45 and p.P46Sfs*42 variants compared to controls.

Functional studies of Orai1α deficient human T cells.

(A) Confocal images illustrating the nuclear translocation of endogenous NFAT1 (green) induced by 1 μM Thapsigargin (Tg) in cells derived from wild-type, heterozygous and homozygous individuals for the Orai1α null variant (p.P43Tfs45). (B) Quantification of endogenous NFAT1 translocation expressed as nuclear to cytoplasmic ratio before and after store depletion with (Tg). Each point is an individual cell. (C) SOCE recordings in T cells loaded with the Ca2+ dye Fura2. Store depletion is induced in the absence of extracellular Ca2+ using Tg (1 μM), extracellular Ca2+ (2 mM) is added after 20 min to trigger Ca2+ influx through SOCE. (D) Quantification of peak SOCE amplitude after Ca2+ addition in T cells from the three genotypes. Data are expressed as mean ± SEM, statistical significance was assessed using one-way ANOVA with Dunnett’s post hoc test.

Near-native expression of Orai proteins in Orai-TKO HEK293 cells.

(A) Representative fluorescence images of HEK293 Orai-TKO cells expressing either Orai1α, Orai1β, Orai2 or Orai3 using TK promoter-driven expression plasmids. Also shown are images of HEK293 Orai-TKO cells expressing Orai1α and Orai1β when expression is driven by the stronger cytomegalovirus (CMV) promoter. (B-C) Comparison of CFP fluorescence intensity in cells transfected with Orai1α-CFP or Orai1β-CFP driven by the TK promotor, showing comparable expression between isoforms. (D) Confocal fluorescence images confirming low expression of the four Orai isoforms using TK promoter-driven expression plasmids. Data are expressed as mean ± SEM. Parametric data were analyzed using one-way ANOVA with Dunnett’s post hoc test, and nonparametric data were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. (E) Western blot showing endogenous Orai1 in HEK293 cells, lack of Orai1 expression in HEK293 Orai TKO cells, and expression of Orai1α-CFP or Orai1β-CFP following their reconstitution in the HEK293 TKO background. Orai1α-CFP and Orai1β-CFP migrate at higher molecular weights than endogenous Orai1 due to CFP tagging, with Orai1α-CFP appearing above Orai1β-CFP, consistent with its longer N-terminus. HSP70 was used as a loading control.

Endogenous expression levels of Orai1α and Orai1β across polarized CD4⁺ T-cell subsets.

(A) Naïve CD4⁺ T cells were isolated from mouse spleen and lymph nodes by negative selection and activated with anti-CD3/CD28 under lineage-skewing conditions to generate Th1, Th2, iTreg, cTh17, and pTh17 cells. (B) Orai1α and Orai1β protein expression was assessed by Western blotting in naïve and polarized CD4⁺ T-cell subsets. (C) Quantification shows the relative Orai1β/Orai1α expression ratios across conditions.

High level of expression of Orai1α or Orai1β suppresses SOCE.

(A) Retroviral transduction of CD4⁺ T cells from Orai1fl/flCd4Cremice with Orai1α-IRES-Ametrine, Orai1β-IRES-Ametrine or empty vector (EV) containing the Ametrine reporter. (B) Representative flow cytometry plots showing ectopic Orai1 expression (APC) in transduced (Ametrine⁺) CD4⁺ T cells. Fixed and permeabilized cells were stained with a C-terminal anti-Orai1 antibody. (C) Gating strategy used for SOCE measurements in CD4⁺ T cells in E and Figure 5A. Live, CD4⁺ T cells were gated on Ametrinehigh (top 25th percentile) or Ametrinelow (bottom 25th percentile) populations. (D) Mean fluorescence intensity (MFI) of Orai1 expression in Ametrinelow T cells (as in panel C) transduced with Orai1α, Orai1β, or EV. Fixed and permeabilized cells were stained with a C-terminal anti-Orai1 antibody. (E) SOCE measured in Ametrinehigh CD4+ T cells transduced with Orai1α, Orai1β, or EV and loaded with Indo-1. Cells were stimulated with thapsigargin (TG) in Ca2+ free buffer followed by readdition of 1 mM Ca2+ and analyzed by flow cytometry. Data are from 11 mice and four repeat experiments with one technical replicate per condition. Statistical analysis in panels (D, E) by ordinary one-way ANOVA multiple comparison. All results are expressed as means ± SEM. ns, not significant (P > 0.05).

STIM1 clusters in T cells from wildtype and Orai1α null heterozygote and homozygote individuals.

(A) STIM1 clusters in T cells derived from WT, heterozygous and homozygous individuals for the Orai1α null variant (P43T). Top row: Airy Scan images of the clusters induced by thapsigargin were analyzed using 3D Spot Segmentation and the volume of each cluster measured. Bottom row: typical examples of the cluster pattern at the bottom of the cells recorded in the three genotypes. (B) Summary of the average cluster volume plotted on a per cell basis in the three genotypes. Data are shown as Mean ± SEM with statistical significance assessed using one-way ANOVA followed by Dunnett post-hoc test.

The Orai1α -specific N-terminus has low evolutionary conservation.

(A, B) Phylogenetic tree (A) and multiple sequence alignment (B) showing the sequence similarity of the Orai1α-specific N-terminus across different species. Sequences were aligned using UniProt alignment tools. Conserved residues are highlighted. (C) Percent identity matrix showing pairwise sequence identities for the Orai1α-specific N-terminus across different species.

Antibodies used for flow cytometric analysis of murine T cells.