Figure 4. Activation of the Orai1 CAR mutant results in decreased SOCE-induced cytosolic Ca²⁺ concentrations and NFAT signaling.

(A) Time course of cytosolic Ca²⁺ concentration measured by Fura-2 microscopy for HEK 293 cells coexpressing mCherry-tagged STIM1 and eYFP-tagged wild-type or mutant Orai1, or mock-transfected cells, or and unstimulated cells that were not exposed to thapsigargin (TG). In Ca²⁺-free extracellular solution, ER stores were depleted with thapsigargin (TG) at 3 min and 0.3 mM Ca²⁺ was added at 12 min (n = 12 - 25). STIM + Orai1 data were compared to STI + Orai-D110A at XX by t-test and determined significantly different (p <0.05) upon addition of extracellular Ca²⁺. (B) Representative images of GFP-NFAT localization in mock-transfected HEK cells or HEK cells coexpressing Cherry-tagged STIM1 and the indicated eYFP-tagged Orai1 before (upper panel) or 30 minutes after exposure to 1 μM TG (lower panel) in a 0.3 mM Ca²⁺-containing bath solution. (C) Time course showing the ratio of nucleus to cytosolic GFP-NFAT fluorescence intensity for cells coexpressing GFP-NFAT with the indicated transfected proteins. ER store depletion was induced by 1 μM TG at 5 minutes in a Ca²⁺-free solution and 0.3 mM Ca²⁺ was added at 10 minutes (n = 5 – 12). The ratios for NFAT + STIM1 + Orai1 were compared to those for NFAT + STIM1 + Orai1-D110A using a t-test at XX and determined significantly different (p < 0.05) in a 0.3 mM Ca²⁺ solution (D) Shown are quantitative analysis and representative images of activation of an NFAT-controlled reporter gene (NFAT reporter) in RBL mast cells expressing RFP under the control of an NFAT-regulated promoter, CFP-tagged STIM1, and eYFP-tagged wild-type or mutant Orai1 that were exposed to 100 nM TG for 3.5 hours in 0.3 mM or 1.8 mM Ca²⁺-containing medium. Representative images are shown on the left, the percent of cells positive for RFP fluorescence are shown in the middle (n = 34 – 86 cells), and the intensity of RFP fluorescence is shown on the right (n = 34 – 86 cells). Data were analyzed by t-test for statistical significance (p < 0.05) as indicated by the stars. (E) Representative HEK 293 cells show CFP-tagged STIM1 cluster formation with eYFP-tagged Orai1 or eYFP-tagged Orai1-D110A upon store depletion with 1 μM TG and presence of 2mM Ca²⁺ solution. R-factor as a measure of the linear correlation between STIM1 and Orai1, as well as STIM1 and Orai1-D110A before and after store depletion by TG (n = 28 – 39). (F) The NFAT reporter gene, CFP-tagged STIM1 and eYFP-tagged Orai1 or eYFP-tagged Orai1-D110A coexpressed in WM3734 melanoma cells were treated with 100 nM thapsigargin for one hour in a 0.4 mM Ca²⁺-containing media. Intensity of NFAT driven RFP expression was determined in those cells that exhibited STIM1 and Orai1 or Orai1-D110A expression 24h hours after thapsigargin treatment. Data were analyzed by t-test for statistical significance (p < 0.05) and determined that Orai1-D110A intensity is significantly decreased compared to wild-type Orai1.