Fig. 3: ORAI3 and ORAI2 interact with STIM1 under basal conditions.

a Measurements of ER Ca²⁺ release (in 0 mM Ca²⁺) and SOCE (in 2 mM Ca²⁺) upon store depletion with 2 µM thapsigargin (Tg) in ORAI-TKO cells (untransfected controls) and in ORAI-TKO cells individually rescued with either ORAI1, ORAI2, or ORAI3 driven by the “weak” thymidine kinase (tk) promoter; all traces are plotted as mean ± SEM. b–d Scatter blot representing quantification of the magnitude (b) and slope (c) of SOCE and fluorescence intensity (d) of transfected tk-driven YFP-ORAI isoforms in a (for b–d from left to right n = 112, 116, 67, and 120; n-values correspond to individual cells). e–g Representative Ca²⁺ oscillations in response to 10 µM carbachol (Cch; added where indicated by arrow) measured using Fura2 over the course of 15 min in ORAI-TKO cells transfected with individual tk-driven ORAI isoforms. Cells were maintained in HBSS containing 2 mM Ca²⁺ for the duration of the experiments and stimulated with 10 µM Cch at 1 min (as indicated by arrow in “e”). Representative traces from five cells/condition were chosen to represent the datasets as a whole. h–j Quantification of total oscillations/14 min (h), % of oscillating cells (n = 51(ORAI1), 94(ORAI2), and 100(ORAI3); data represent individual cells) (i), and % of plateau cells (j) for data in e–g (for i, j, n = 4 for all conditions; data represent independent experiments). k Fluorescence intensity of transfected tk-driven YFP-ORAI isoforms for data in e–j) (n = 120 (ORAI1), 119 (ORAI2), and 118 (ORAI3); data represent individual cells). l E-FRET represented as mean ± SEM between STIM1-YFP and either CFP-ORAI isoform at rest and after addition of 2 µM thapsigargin (Tg) in HBSS containing 2 mM Ca²⁺. m–p Scatter blots representing quantification of basal E-FRET (m), the magnitude (n), and slope (o) of E-FRET change after addition of thapsigargin and YFP/CFP fluorescence ratios (p) from data in l (for m–p, n = 39(ORAI1), 46 (ORAI2), and 60 (ORAI3); data represent individual cells). All data are represented as mean ± SEM. Panels b–d, h, k, m–p were statistically analyzed using the Kruskal–Wallis one-way ANOVA with multiple comparisons. Panels i, j were analyzed using one-way ANOVA with multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant). All comparisons were made to tk-ORAI1.