Decreased SOCE in patient T cells and loss of STIM1 gating in cells expressing ORAI1-H134P mutation. (A and B) Calcium measurements in in vitro–expanded T cells from the patient (Pat), his father (Fa), and mother (Mo) using Fura-2 AM. Representative time course of Fura-2 fluorescence ratios (340/380 nm) over time (left). Quantified area under the curve (AUC) and slope of Ca²⁺ influx at times indicated by circles (right). Data are from at least four independent experiments. (A) Cells were placed in 0 mM Ca²⁺ Ringer’s solution, and SOCE was induced by anti-CD3 cross-linking, followed by the addition of 1 mM Ca²⁺ Ringer’s solution, and stimulation with 1 µM ionomycin (iono). For anti-CD3 AUC: P = 0.6584; 0.0012; 0.0223. For anti-CD3 slope: P = 0.7256; 0.0002; <0.0001. For iono AUC: P = 0.9146; <0.0001; 0.0001. For iono slope: P = 0.5677; <0.0001; <0.0001. (B) Cells were placed in 0 mM Ca²⁺ Ringer’s solution, and SOCE was induced by stimulation with 1 µM TG, followed by the addition of 1 mM Ca²⁺. AUC: P = 0.3418; <0.0001; <0.0001. Slope: P = 0.9719; <0.0001; <0.0001. (C–E) Whole-cell patch-clamp recordings of HEK293 cells. CRAC currents were activated by passive depletion of ER Ca²⁺ stores with 8 mM BAPTA in the patch pipette and recorded in the whole-cell recording configuration. The plots show the peak whole-cell current at −100 mV (in pA) during steps to −100 mV applied every second following whole-cell break-in. For each condition, the I-V curves are shown on the right. The I-Vs are averages of 6-10 ramps collected between 50 and 60 s (C), 5 and 15 s (H134P ORAI1+STIM1), and 110 and 115 s (WT ORAI1+STIM1) (D). (C) Cells were transfected with ORAI1-H134P alone. Time course of current (left) and I-V relationship (right). (D) Cells were transfected with STIM1 together with either ORAI1-WT or ORAI1-H134P. (E) Current densities normalized to the cell capacitance of ORAI1-WT and ORAI1-H134P with or without STIM1 measured at whole-cell break-in (t = 0 in C) or after store depletion (t = 100 s in D). P < 0.001. (F and G) FRET measurements in HEK293 cells transfected with CFP-CAD together with either ORAI-WT-YFP, ORAI1-H134P-YFP, or ORAI1-L273D-YFP. (F) Representative confocal images from a total of 52 cells (WT), 22 cells (L273D), and 43 cells (H134P). Scale bars are 5 µm. (G) Quantification of FRET efficiencies. P ≤ 0.0001; 0.519. Statistical significance was calculated using ordinary one-way ANOVA (A–D) and one-way ANOVA followed by unpaired Student’s t test (E and G). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; I-V, current–voltage.