Figure 1. Constitutively-active Orai1 Ca²⁺ signals and NFAT activation induced by mutations detected through cancer database screening.

(A) Model of the human Orai1 protein structure, with Orai1 single-point mutants found in human tumors in conserved positions highlighted, as determined from large-scale cancer genomics data sets. (B) Percentage of RBL cells that express NFAT-driven RFP (average + SEM), measured 24h after co-transfection of the NFAT reporter and cancer-associated Orai1 mutants from (A), wild-type Orai1, or constitutively-active Orai1-V102A and Orai1-L138F mutants. Significantly increased number of RBL cells expressing NFAT-driven RFP (p < 0.05 by t-test) is indicated by the red bars and a star. Analogous experiments were performed in a nominally Ca²⁺ free bath solution for co-expression of NFAT reporter and wild-type Orai1 or Orai1-L138F, indicated by blue bars (n = 3 – 8 cell images, from at least 3 separate transfections). (C) Representative images of the co-expression of two cancer-associated Orai1 mutants (Orai1-A137V, Orai1-G247S) and wild-type Orai1 with the NFAT reporter in RBL mast cells. Scale bar, 10 µm. (D) Resting cytosolic Ca²⁺ concentrations in Fura-2-loaded HEK cells overexpressing wild-type Orai1 or cancer-associated Orai1 mutants (n = 31 – 70 cells, from 3 – 4 individual transfections). Significantly increased Ca²⁺ concentrations (p < 0.05 by t-test) are indicated by red bars and a star. (E) Time course of whole cell patch-clamp experiment in HEK cells co-expressing STIM1 and Orai1 (black), Orai1-A137V (blue), or Orai1-A137V alone (red) in a 10 mM extracellular Ca²⁺ (n = 7 – 10 cells, from at least 2 individual transfections). Store-operated currents were activated by 20 mM EGTA in patch pipette. (F-H) Representative current-voltage relationships for STIM1 and Orai1 expressing cells (F), Orai1-A137V (G), or STIM1 and Orai1-A137V (H) in a 10 mM Ca²⁺ (black) or Na⁺-based, divalent-free (Na-DVF, red) solution.