Figure 4. The Orai3 M4x Leu profile reveals the peptide mediates STIM1-induced Orai3 channel activation.

(A) Time course of E-FRET interactions between STIM1-YFP co-expressed with either full-length WT CFP-Orai3, CFP-Orai3 L282D or L285D single mutants, CFP-Orai3 L282D/L285D double mutant, or CFP control, before and after store depletion with 2.5 μM ionomycin.

(B) Summary data for E-FRET analyses in (A), showing the change in E-FRET from baseline (0 s) to the peak after ionomycin addition (400 s).

(C) Cytosolic Ca²⁺ signals measured by fura-2 ratiometric Ca²⁺ imaging in HEK-Orai1/2/3^TKO transiently expressing STIM1-mCherry together with either WT CFP-Orai3 (green) or L282D (red), L285D (blue), or L282D/L285D (purple) mutants of CFP-Orai3 (purple). Cells in Ca²⁺-free medium were treated with 2.5 μM ionomycin followed by 1 mM Ca²⁺ add back.

(D) Summary statistics for the average peak of store-operated Ca²⁺ entry in (C).

(E) I/V relationship of whole-cell I_CRAC measurements in HEK-Orai1/2/3^TKO cells transfected with the same CFP-Orai3 constructs shown in (C) together with STIM1-YFP.

(F) Ca²⁺ levels in fura2-loaded HEK-Orai1/2/3^TKO cells transiently expressing either WT CFP-Orai3 (green); L282D (red), L285D (blue), or L282D/L285D (purple) mutants; or negative control LK-CFP (black). Constitutive Ca²⁺ entry and 2-APB responses were in Ca²⁺-free solution, replaced with 1 mM Ca²⁺ followed by 50 μM 2-APB, as shown.

(G) Summary statistics for the 2-APB-induced Ca²⁺ entry peak in the experiments in (F).

(H) I/V relationship of 2-APB-induced whole-cell current measurements for HEK-Orai1/2/3^TKO cells transfected with the same CFP-Orai3 constructs shown in (F). One-way ANOVA analyses were under taken on results in (B), (D), and (G). Ca²⁺ entry shown are means ± SEM (****p < 0.0001) and representative results from at least three independent experiments.