Fig. 4.

Mutations at negatively charged and polar residues affect Ca²⁺ coordination in the putative Ca²⁺-binding site. a–c Ca²⁺-dependent [³H]ryanodine binding of RyR2 carrying mutations in the putative Ca²⁺-binding site; E3847 (a), E3921 (b) and Q3924 (c). Assays were carried out in high salt medium without Mg²⁺ to remove inactivation via the low-affinity Ca²⁺/Mg²⁺ site. Data are given as mean ± SEM (n = 4). d Summary of pCa₅₀ values of WT and the mutants. Data are given as mean (horizontal bar) and individual values (circles). &, not determined. ***p < 0.0001 vs WT using one-way ANOVA followed by Dunnett’s post hoc test. Mutations differentially reduced or lost Ca²⁺ sensitivity for activation. e The architecture of putative Ca²⁺-binding site (5T15). The possible coordination of oxygen atoms to Ca²⁺ are indicated by orange dashed lines, and possible hydrogen bond stabilizing the coordination geometry by green dashed lines