Fig. 1: Exchanging Orai1-V181 by residues with charged side chains triggers robust constitutive activity.

A Schematics showing a side view of the entire Orai1-channel complex as well as side and top views of the TM3/TM4-interface along with TM2′ of the next subunit highlighting the tested residue V181. Time courses of Ca²⁺ current densities after whole-cell break-in of Orai1-V181X (X = K/E/R/Q/D/H) (B), (X = G/A/C/S/M) (C), (X = F/W/Y/I/L) (D) in the absence of STIM1. E–G Block diagrams of maximal whole-cell current densities of mutants recorded in (B–D) compared to Orai1 (wt) in the absence (−) and presence (+) of STIM1. Normalized current/voltage (I/V) relationships of Orai1-V181K (H), Orai1-V181A (I), and Orai1-V181W (J) in the absence (−) and presence (+) of STIM1 were taken at maximum current levels. Insets represent the Vrev of wt STIM1 + Orai1 currents versus the respective constitutive mutant currents. Single values are indicated as gray circles. K The average number of HEK293 cells that exhibit NFAT localization to the nucleus determined upon co-expression (CFP-NFAT) with YFP-Orai1 (wt), or corresponding mutants shown in (H–J) after 24 h in 2 mM Ca²⁺-containing media. For the analysis, 72–97 images of cells containing in total 72–328 cells were used. L Representative images of HEK293 cells co-expressing YFP-Orai1 (wt) or corresponding mutants shown in (K), respectively, with CFP-NFAT in the presence of 2 mM Ca²⁺ (Scale bar, 10 μm). For the presented bar diagrams, ANOVA (one-way/Kruskal–Wallis ANOVA) or Welch-ANOVA was 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-V181X mutants in the absence of STIM1 is indicated with asterisks.