Fig. 2. CRAC current in HEK 293 cells expressing Orai1 and Stim1.

(A) Currents elicited in HEK 293 cells expressing Orai1 and Stim1 in whole-cell voltage-clamp at a holding potential of −50 mV. Cells were treated with Ca²⁺-free external solution (solution II) containing NPPB (30 μM) and thapsigargin (1 μM) for 10 min to deplete ER Ca²⁺ and block CaCC. HEK 293 cells were dialyzed with Cs⁺-rich solution containing BAPTA (solution VI). When cells were dialyzed with scrambled CC2 peptide (10 μM), reintroduction of 2 mM [Ca²⁺]_o caused development of inward current. Ramp potentials from −80 to +60 mV were run before and after addition of 2 mM [Ca²⁺]_o [examples noted by • and • in (A)]. (B) Currents elicited by ramp potentials [• (black trace) is control, and • (blue trace) is after addition of 2 mM [Ca²⁺]_i] in (A). (C) Currents elicited in HEK 293 cells expressing Orai1 and Stim1 in whole-cell voltage-clamp at a holding potential of −50 mV. Cells were treated with Ca²⁺-free external solution (solution II) containing NPPB (30 μM) and thapsigargin (1 μM) for 10 min to deplete ER Ca²⁺. HEK 293 cells were dialyzed with Cs⁺-rich solution containing BAPTA (solution VI). (D) Current responses to ramp potentials [• (black trace) is control, and • (red trace) is after addition of 2 mM Ca²⁺] in (C). (E) Summary data showing the effects of scrambled CC2 (scCC2) peptide and CC2 peptide on current elicited by reintroducing 2 mM [Ca²⁺]_o at −80 mV. Data are means ± SEM (n = 5 cells for each group; ***P < 0.001, Student’s two-tailed t test). (F) STIM1 sequence in several species and the sequences of the CC2 and scrambled CC2 peptides.