CPVT-associated cardiac ryanodine receptor mutation G357S with reduced penetrance impairs Ca2+ release termination and diminishes protein expression

Figures

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  Fig 1. Effect of the CPVT-associated RyR2 G357S mutation on the propensity for store overload-induced Ca2+ release (SOICR). Stable, inducible HEK-293 cells expressing RyR2 WT (A) or RyR2 G357S (B) were loaded with 5μM Fura-2 AM in KRH buffer. The cells were then perfused continuously with KRH buffer containing increasing levels of extracellular Ca2+ (0–2 mM) to induce SOICR. Fura-2 ratios were recorded using epifluorescence single cell Ca2+ imaging. C, the percentage of RyR2 WT (898 cells) and the G357S mutant (846 cells) cells that displayed Ca2+ oscillations at various extracellular Ca2+ concentrations (n = 7). Data shown are mean ± SEM (*, p<0.01 vs WT; NS, not significant).

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  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. FSOICR indicates the FRET level at which SOICR occurs, whereas Ftermi 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 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 (F) was calculated by subtracting Fmin from Fmax. Data shown are mean ± SEM…

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  Fig 3. The G357S mutation markedly reduces the level of RyR2 expression in HEK293 cells. Stable, inducible HEK293 cells expressing RyR2 WT or the G357S mutant were collected and lysed after induction for 24 h. The same amount of HEK293 cell lysate protein was used for Western blotting (WB) (A, B) using the anti-RyR antibody (34c) and anti-β-actin antibody or for [3H]ryanodine binding in the presence of 500 mM KCl, 5 nM [3H]ryanodine, 100 μM Ca2+, 2.5 mM ATP, and 2.5 mM caffeine (C). The expression level and the maximum [3H]ryanodine binding of the G357S mutant was normalized to that of RyR2 WT. Data shown are mean ± SEM (n = 3) (*, p<0.05 vs WT).

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  Fig 4. The G357S mutation does not affect cytosolic Ca2+ dependent activation of [3H]ryanodine binding. [3H]ryanodine binding to cell lysates prepared from HEK293 cells expressing RyR2 WT or the G357S mutant was carried out at various Ca2+ concentrations (~0.2 nM-0.1 mM), 500 mM KCl and 5 nM [3H]ryanodine. (A) [3H]ryanodine bound to RyR2 WT or G357S at various Ca2+ concentrations was normalized to its own maximal binding (100%). (B) [3H]ryanodine bound at various Ca2+ concentrations was normalized to the maximal binding to RyR2 WT (100%). The data points shown are mean ± SEM from three separate experiments.

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  Fig 5. The G357S mutation has little effect on cytosolic Ca2+-regulated Ca2+ release. Stable, inducible HEK293 cell lines expressing RyR2 WT (A) or G357S (B) were transfected with the FRET-based ER luminal Ca2+-sensing protein D1ER and induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed, and perfused with intracellular-like medium plus increasing levels of free Ca2+ (0.1, 0.2, 0.4, 1 and 10 μM). FRET recordings from representative cells (total 89 for WT and 87 for G357S) are shown. To minimize the influence by YFP/CFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca2+ level (defined in A) (C).The dashed lines (F0.1-F10) indicate the steady state FRET levels after perfusing with each Ca2+ concentration (0.1, 0.2, 0.4, 1, or 10 μM). 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 (D) was calculated by subtracting Fmin from Fmax. Data shown are mean ± SEM (n = 4–6).

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  Fig 6. The G357S mutation reduces the stability of the ABC domains of RyR2. A. Crystal structure of the mouse RyR2ABC (residues 1–547), showing three domains and a central chloride anion that links the three domains together. B. Details showing G357S. The backbone conformation is allowed for glycine, but not serine. C. Thermal melting curves indicating thermal stability of WT RyR2ABC vs. G357S RyR2ABC. The melting temperatures, obtained by taking the maxima of the first derivatives, are 42.8°C ± 0.17°C for RyR2ABC WT and 39.8°C ± 0.17°C for RyR2ABC G357S (errors indicating SEM). Statistical significance was determined using an unpaired two-tailed t-test with a 95% confidence level, with P = 0.0002. D. Ramachandran plot for the RyR2ABC crystal structure, with backbone phi angles on the X-axis, and psi angles on the Y-axis. The shaded areas indicate preferred regions for non-glycine residues. Glycine residues are indicated with squares, with G357 indicated by an arrow. E. Cryo-EM structure of RyR [42, 43], showing the location of the corresponding glycine. One subunit, at the ‘front’ of the viewer, has been left out for clarity. The N-terminal region, the Central domains, and the S6 inner…