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. 2021 May 20;297(1):100808. doi: 10.1016/j.jbc.2021.100808

Figure 4.

Figure 4

Effect of disease-associated CTD mutations on SOICR activation and termination thresholds. Stable and inducible HEK293 cell lines expressing RyR2 WT and mutants 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 mutants 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 2 mM tetracaine to inhibit SOICR and then 20 mM caffeine to deplete the ER Ca2+ stores. Representative FRET recordings from RyR2 WT (A), P4920S (B), P4902L (C), E4950K (D), and G4955E (E) are shown. To minimize the influence of cyan fluorescent protein/yellow fluorescent protein cross talk, relative FRET measurements were used to calculate the activation threshold (F) and termination threshold (G) using the equations shown in A. FSOICR indicates the FRET level at which SOICR occurs, whereas Ftermi represents the FRET level at which SOICR terminates. The fractional Ca2+ release (H) was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal Fmax is defined as the FRET level after tetracaine treatment. The minimum FRET signal Fmin is defined as the FRET level after caffeine treatment. The store capacity (I) was calculated by subtracting Fmin from Fmax. Data shown are mean ± SD from six to seven separate experiments (WT, 89 cells; P4902S, 139 cells; P4902L, 128 cells; E4950K, 79 cells; and G4955E, 75 cells). ∗∗p < 0.01; ∗p < 0.05 versus WT. NS, not significant (one-way ANOVA with Dunnett's post hoc test, F = 38.3 and p < 0.0001 for the activation threshold (F); F = 17.9 and p < 0.0001 for the termination threshold (G); F = 29.4 and p < 0.0001 for the fractional Ca2+ release (H); F = 6.1 and p = 0.0011 for the store capacity (I)). CTD, C-terminal domain; ER, endoplasmic reticulum; HEK293, human embryonic kidney 293 cells; KRH, Krebs–Ringer–Hepes; RyR2, cardiac ryanodine receptor; SOICR, store overload–induced spontaneous Ca2+ release.