Fig 2. The G357S mutation decreases both activation and termination thresholds for SOICR.

Stable, inducible HEK293 cell lines expressing RyR2 WT (A) or the G357S mutant (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER and induced using tetracycline before the experiment. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mM) to induce SOICR. FRET recordings from representative cells (total 72 for WT and 77 for G357S) are shown. To minimize the influence by YFP/CFP cross-talk, we used relative FRET measurements for calculating 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. The fractional Ca²⁺ release (E) 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. The store capacity (F) was calculated by subtracting F_min from F_max. Data shown are mean ± SEM (n = 5–6) (*, p<0.01 vs WT; NS, not significant).