Figure 6. The Orai3 M4x peptide overcomes a powerful STIM1 coupling mutant and mediates an important action of 2-APB.

(A) Constitutive Ca²⁺ entry in fura-2-loaded HEK-STIM1/2^DKO cells transiently expressing CFP-Orai3 with either WT YFP-SOAR dimer (red), the YFP-SOAR-F394H homodimer mutant (blue), or YFP vector alone (black). A total of 1 mM Ca²⁺ was added to cells in Ca²⁺-free solution, as shown.

(B) Summary statistics for the Ca²⁺ entry shown in (A).

(C) E-FRET analysis of HEK293 cells transiently expressing either WT YFP-SOAR dimer (red) or the YFP-SOAR-F394H homodimer mutant (blue), together with either WT LK-CFP-O3M4x or its L285D mutant.

(D) SOCE in fura-2-loaded HEK cells transiently expressing CFP-Orai3 with either WT STIMI-YFP (red), the STIM1-YFP-F394H homodimer mutant (blue), or YFP control (black). Store depletion was with 2.5 μM ionomycin, followed by 1 mM Ca²⁺ addback, and 50 μM 2-APB, as shown.

(E) Enlarged detail for the 2-APB-mediated Ca²⁺ responses shown in (D) in cells expressing either STIM1-YFP-F394H or YFP control.

(F) Summary statistics for Ca²⁺ entry experiments represented in (D).

(G) Time course E-FRET measurements for HEK-Orai1/2/3^TKO cells transiently co-expressing CFP-Orai3 with either WT STIM1-YFP (red) or STIM1-YFP F394H mutant (blue). Additions of 2.5 μM ionomycin and 50 μM 2-APB were as shown.

(H) Average change in E-FRET from baseline (before Ca²⁺) to store-depleted peak (iono), and additional E-FRET peak change after the 2-APB addition, from experiments in (G).

(I) E-FRET time course measured in HEK-Orai1/2/3^TKO cells transiently co-expressing the Orai3-M4x peptide with either STIM1-YFP (red) or STIM1-YFP-F394H mutant (blue). Additions of 2.5 μM ionomycin and 50 μM 2-APB were as shown.

(J) Summary statistics for E-FRET results shown in (I). Changes in FRET are shown from baseline (Ca²⁺-free) to store-depleted peak (iono) and additional FRET peak after 2-APB.

One-way ANOVA analysis was performed in (B) and (F). Ca²⁺ entry results are means ± SEM (****p < 0.0001). Results are representative of at least three independent experiments