Figure 7. The Orai-ANSGA mutation restores ion selectivity to the Orai1-V102C mutant and exhibits no CDI.

(a) Current–voltage relationship for Orai1-V102C expressed in HEK cells (without STIM1 expression) with cytosolic Ca²⁺ buffered to 90 nM with EGTA (stores replete), revealing a reversal potential of ∼25 mV reflecting the decreased Ca²⁺ selectivity of the pore mutant. (b) I–V for Orai1-V102C expressed in HEK-STIM1 cells with 10 mM BAPTA in the pipette solution to deplete Ca²⁺ stores, revealing right-shifted reversal potential similar to WT Orai1. (c) I–V for the Orai1-V102C-ANSGA construct expressed in HEK cells without expressed STIM1 with Ca²⁺ buffered to 90 nM (stores replete) as in a, revealing that the reversal potential is shifted right-ward similar to the shift induced by STIM1 and store emptying. (d) I–V for the Orai1-V102C-ANSGA expressed in HEK-STIM1 cells with 10 mM BAPTA-induced store emptying, revealing that the right shift by ANSGA is not further altered by STIM1. (e) Summary of the reverse potential data shown in a–d. Values are means±s.e.m. of number of individual cells shown. (f,g) Representative CRAC currents elicited by 200 ms hyperpolarization in HEK-STIM1-YFP cells expressing CFP-Orai1 with 10 mM EGTA (f) or 10 mM BAPTA (g) in the pipette solution. (h,i) The same CRAC current measurements undertaken with HEK cells expressing CFP-Orai1-ANSGA with 10 mM EGTA (h) or 10 mM BAPTA (i) in the pipette solution. (j) CRAC current measurements undertaken with HEK-STIM1 cells co-expressing CFP-Orai1-ANSGA with 10 mM EGTA in the pipette solution. (k) Summary of quantified CDI defined as the current recorded at 185 ms divided by the current recorded at 2 ms after each pulse. Orai1-WT (EGTA), n=6; Orai1-WT (BAPTA), n=5; Orai1-ANSGA (EGTA), n=8; Orai1-ANSGA (BAPTA), n=7. All values are means±s.e.m. (l) Protocol for CDI measurement: after maximum current was reached, holding potential steps from +30 to −60, −80, −100 and −120 mV were applied with 5 s intervals between steps. The currents were recorded in 20 mM Ca²⁺ Ringer's solution.