Biophysical properties of Orai1-GCaMP6f and GFP-Orai1 STIM1-operated currents. Representative whole-cell recordings from HEK 293A cells transiently cotransfected at a ratio of 10:1 with mCherry-STIM1 plus either Orai1-GCaMP6f (A–D, top; n = 6 cells) or GFP-Orai1 (E–H, bottom; n = 6 cells). (A and E) Leak-subtracted time course, following break-in to achieve whole-cell recording at time = 0, of Orai1-GCaMP6f (A) and GFP-Orai1 (E) STIM1-operated current recorded in response to ramp stimuli from −120 mV (black points) to +100 mV (gray points). (B, C, F, and G) Current-voltage relationship of Orai1-GCaMP6f (B and C) and GFP-Orai1 (F and G) current recorded in 20 mM Ca²⁺ (B and F) and Na⁺ DVF (C and G) solutions, respectively. (D and H) Ca²⁺-dependent inactivation of Orai1-GCaMP6f (D) and GFP-Orai1 (H) current during −120 mV hyperpolarizing pulse from a holding potential of 0 mV, recorded in 20 mM Ca²⁺ external solution. Bars indicate exchange of external solutions (see Table 1 for solution recipes). Arrows in A and D indicate the time points for I-V curves in B, C, F, and G. Lines with arrowheads indicate biophysical parameters presented in Table 2.