Fig. 2: Substitution of Orai1-A254 by charged residues triggers constitutive activity.

A Schematics showing side and top views of the TM3/TM4-interface along with TM2′ of the next subunit highlighting the tested residue A254 (white) opposed to V181 (light gray). B Time courses of Ca²⁺ current densities after whole-cell break-in of Orai1-A254X (X = K/E/Q) in the absence of STIM1. C Block diagram of maximal whole-cell current densities of mutants recorded in (B) compared to Orai1 (wt) in the absence (−) and presence (+) of STIM1. D Normalized current/voltage (I/V) relationships of Orai1-A254K in the absence (−) and presence (+) of STIM1 were taken at maximum current levels. Inlet represents reversal potential (Vrev) of wt STIM1 + Orai1 currents versus respective constitutive mutant currents. Single values are indicated as gray circles. E The average number of HEK293 cells that exhibit nuclear NFAT localization determined upon co-expression (CFP-NFAT) with YFP-Orai1 (wt) or YFP-Orai1-A254X (X = K/E/Q) mutants in the absence of STIM1 after 24 h in 2 mM Ca²⁺ containing media. For the analysis, 47–85 images of cells containing in total 72–245 cells were used. F Representative images of HEK293 cells co-expressing corresponding mutants with CFP-NFAT in the presence of 2 mM Ca²⁺ (Scale bar, 10 μm). For the presented bar diagrams, Welch-ANOVA and Kruskal–Wallis ANOVA were employed for statistical analyses with differences considered statistically significant at p < 0.05 (see Supplementary Data 1). Statistical differences of wt Orai1 compared to all Orai1-A254X mutants in the absence of STIM1 is indicated with asterisks.