Figure 4
Effect of tunicamycin (TM) and dantrolene (DAN) on Ca2+ release characteristics and CaM-RyR2 interaction in WT and RYR2V3599K neuronal cells. (A) Representative images of intracellular Ca2+ before and after addition of ionomycin (10 μM). (B) Summarized data of baseline Ca2+, increased cytosolic [Ca2+] after addition of ionomycin (Δ[Ca2+]), rate of release (slope). Values for individual mice are plotted together with mean ± SEM. N = 25–34 cells from 3 mice. Parentheses indicate the number of mice. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with post-hoc Tukey’s multiple comparison test). (C) Representative images of endogenous CaM and RyR2 in neuronal cells. Multiple experiments were performed simultaneously with a single control experiment; (Left) immuno-staining of RyR2 (red), immuno-staining of CaM (green) and merged image. (Right) the summarized data of the Pearson’s and Manders’ coefficients. Scale bars: 10 μm. Values for individual mice are plotted together with mean ± SEM. N = 21–27 cells from 4–6 mice. Parentheses indicate the number of mice. *p < 0.05, ***p < 0.001 (one-way ANOVA with post-hoc Tukey’s multiple comparison test in WT, unpaired t test in RYR2V3599K). (D) Representative immunoblots of the RyR2-bound CaM–SANPAH (a photoreactive crosslinker; N-succinimidyl-6-[4′-azido-2′-nitrophenylamino]). (Bottom) Summarized data of CaM binding to RyR2 as a function of the concentration of CaM–SANPAH. The CaM binding was expressed as the ratio to the maximum binding at 1,024 nM CaM. Dotted line means CaM binding curve in WT. Data represent means ± SEM of 3 mice. Parentheses indicate the number of mice. Full-length blots are presented in Supplementary Fig. 12 **p < 0.01 (one-way ANOVA with post-hoc Tukey’s multiple comparison test in WT, unpaired t test in RYR2V3599K).
