Figure 6. CRAC channels activate NFAT-dependent gene expression in NPCs. A, NPCs were nucleofected with GFP-NFAT1 and imaged 24–48 h later. Images were acquired in 2 mm extracellular Ca2+, 15 min after application of 1 μm TG, in a Ca2+-free solution, and 15 min after re-addition of 2 mm Ca2+. Where indicated, BTP-2 (1 μm) was applied for 3–5 h before the experiment. B, Summary of the percentage of cells showing nuclear translocation of GFP-NFAT1. (E13 and P2; one-way ANOVA, p = 0.0003, followed by unpaired t test between indicated test conditions, *p < 0.05, **p < 0.01, ***p < 0.001, n = 93–133 cells from 3–4 experiments). C, NFAT luciferase activity stimulated by depletion of ER Ca2+ stores is significantly suppressed by pretreatment with the CRAC channel inhibitors 2-APB (50 μm), SKF96365 (20 μm), and BTP-2 (0.5 μm) in the presence of low external Ca2+ and by cyclosporine A (1 μm; E13; one-way ANOVA, p < 0.0001; unpaired t test between indicated test conditions, **p < 0.01, ***p < 0.001, representative of 3 cultures). D, E, NFAT luciferase activity is diminished by siRNA treatment against STIM1 and Orai1 (P1; one-way ANOVA, p < 0.0001; unpaired t test, **p < 0.01, representative of 3 cultures; D) and in NPCs derived from Orai1R93W/R93W mice (E18; one-way ANOVA, p < 0.0001; unpaired t test, **p < 0.01; E). F, Muscarine stimulates nuclear translocation of GFP-NFAT1, which is abolished by BTP-2 (1 μm; E13, p < 0.01, unpaired t test, n = 219–461 cells from 4 experiments). G, Theoretical [Ca2+]i profiles as a function of distance from an open CRAC channel. The [Ca2+] profiles were calculated as described previously (Neher, 1986; Stern, 1992). The single-channel CRAC current was assumed to be 5 fA. H, [Ca2+]i elevations produced by SOCE in EGTA- or BAPTA-treated cells. NPCs were loaded with 20 μm EGTA-AM or 20 μm BAPTA-AM for 25–35 min at 37°C. (E13, ***p < 0.001, unpaired t test, n = 58–73 cells; from 2 experiments). I, Nuclear translocation is not significantly affected by intracellular EGTA, but is suppressed by BAPTA (E13 and E14; one-way ANOVA, p = 0.0002; unpaired t test, p = 0.73 for EGTA, and ***p < 0.001 for BAPTA-treated cells, n = 200–233 cells from 3 experiments).