FIGURE 9.

Effect of V4880A on SOICR activation and termination thresholds. A and B, stable, inducible HEK293 cell lines expressing RyR2 WT (A) and V4880A (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER 48 h before single-cell FRET imaging. Expression of the RyR2 WT and mutant was induced 24 h before imaging. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mm) to induce SOICR. This was followed by the addition of 1.0 mm tetracaine to inhibit SOICR and then 20 mm caffeine to deplete the ER Ca²⁺ stores. FRET recordings from representative RyR2 WT (total 57 cells, A) and mutant V4880A cells (total 53 cells, B) are shown. C and D, to minimize the influence of CFP/YFP cross-talk, we used relative FRET measurements to calculate the activation threshold (C) and termination threshold (D) using the equations shown in A. F_SOICR indicates the FRET level at which SOICR occurs, whereas F_termi represents the FRET level at which SOICR terminates. E, the fractional Ca²⁺ release was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. F, the store capacity was calculated by subtracting F_min from F_max. Data shown are mean ± S.E. (n = 4; *, p < 0.05 versus WT; NS, not significant).