Figure 6. The drop in Orai1–Orai1 FRET is independent of expression levels of Orai1 but depends on the position of the fluorophores.

A, resting E_app values plotted against the intensity of Orai1–YFP in 83 cells. Cells were transfected with Orai1–YFP, Orai1–CFP, at 10, 25 or 100 ng per coverslip together with mCherry–STIM1 and E_app was measured prior to store depletion in 2 mm Ca²⁺ Ringer solution. B, the decline in E_app following store depletion is independent of the expression of Orai1–YFP. E_app was measured 300 s following the addition of 1 μm TG in Ca²⁺-free Ringer solution in cells transfected with Orai1–YFP/Orai1–CFP at 100 ng per coverslip (n = 69 cells). C, altering the amount of Orai1 DNA does not change the extent of decline in Orai1–Orai1 FRET following store depletion. HEK293 cells were transfected with Orai1–YFP/Orai1–CFP DNA at the indicated amounts per fluorophore. D, altering the position of CFP from C- to the N-terminus of Orai1 results in enhanced resting FRET (P < 0.001). Resting 3-cube E_app values in cells transfected with either Orai1–CFP and Orai1–YFP (both C-terminally tagged; n = 83 cells) or CFP–Orai1 (N-terminally tagged) and Orai1–YFP (C-terminally tagged; n = 34 cells) in 2 mm Ca²⁺. E, the decline in Orai1–Orai1 FRET following store depletion is reduced in cells expressing CFP–Orai1 + Orai1–YFP (P < 0.001).