Effect of the H29D mutation on the threshold for SOICR in HEK293 cells.

<p>Stable, inducible HEK293 cell lines expressing RyR2 WT or H29D were transfected with the FRET-based ER luminal Ca²⁺ sensing protein D1ER 48 h before single cell FRET imaging. The expression of RyR2 WT and H29D 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 (A) and H29D (B) cells (total 68 cells each) are shown. The activation threshold (C) and termination threshold (D) were determined using the equations shown in panel A. F_SOICR indicates the FRET level at which SOICR occurs, while 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 = 6).