Table 1.
Effects of mutations on [3H]ryanodine binding to RyR2
| EC50 (μm) of Ca2+ activation | Adjusted p value | Basal activity (pmol/mg) | Adjusted p value | n number of separate experiments performed | |
|---|---|---|---|---|---|
| A. Mutations of Ca2+-coordinating residues | |||||
| RyR2 WT | 0.21 ± 0.02 | -- | 3.1 × 10−3 ± 1.7 × 10−3 | -- | 4 |
| E3848A | >1.0 × 104 | -- | 28.0 × 10−3 ± 2.8 × 10−3 | <0.0001 | 3 |
| E3848A/E3922A | -- | -- | 52.9 × 10−3 ± 4.1 × 10−3 | <0.0001 | 3 |
| H3850A | 0.78 ± 0.01 | <0.0001 | 0.4 × 10−3 ± 0.4 × 10−3 | 0.4700 | 3 |
| E3922A | 1.38 × 104 ± 0.25 × 104 | 0.0055 | 12.2 × 10−3 ± 3.1 × 10−3 | 0.0002 | 4 |
| Q3925A | 1.58 × 102 ± 0.24 × 102 | 0.0031 | 0.2 × 10−3 ± 0.2 × 10−3 | 0.3416 | 4 |
| T4931A | 0.28 ± 0.04 | 0.2359 | 1.5 × 10−3 ± 1.1 × 10−3 | 0.8839 | 3 |
| EC50 ANOVA summary: F = 426.3, p value < 0.0001; Basal activity ANOVA summary: F = 231.1, p value < 0.0001 | |||||
| B. Mutations of residues with negatively charged or oxygen-containing side chains | |||||
| RyR2 WT | 0.21 ± 0.02 | -- | 4.3 × 10−3 ± 2.1 × 10−3 | -- | 3 |
| T3929A | 0.29 ± 0.02 | 0.0071 | 3.5 × 10−3 ± 4.8 × 10−3 | 0.9998 | 3 |
| Q3932A | 0.28 ± 0.03 | 0.0231 | 7.2 × 10−3 ± 5.4 × 10−3 | 0.9964 | 3 |
| Q3933A | 0.18 ± 0.02 | 0.5725 | 17.3 × 10−3 ± 10.5 × 10−3 | 0.0869 | 3 |
| S3984A | 0.15 ± 0.03 | 0.0197 | 4.2 × 10−3 ± 1.7 × 10−3 | >0.9999 | 4 |
| N3989A | 0.24 ± 0.01 | 0.7655 | 4.4 × 10−3 ± 3.7 × 10−3 | >0.9999 | 3 |
| E4146A | 0.32 ± 0.03 | <0.0001 | 1.6 × 10−3 ± 2.2 × 10−3 | 0.9961 | 5 |
| Y4149S | 0.09 ± 0.02 | <0.0001 | 21.8 × 10−3 ± 5.7 × 10−3 | 0.0060 | 4 |
| T4934A | 0.29 ± 0.02 | 0.0035 | 9.7 × 10−3 ± 11.5 × 10−3 | 0.8642 | 3 |
| Q4936A | 0.12 ± 0.02 | 0.0004 | 70.1 × 10−3 ± 9.5 × 10−3 | <0.0001 | 3 |
| E4937A | 0.44 ± 0.01 | <0.0001 | 2.3 × 10−3 ± 2.5 × 10−3 | 0.9996 | 3 |
| EC50 ANOVA summary: F =59.8, p value < 0.0001; Basal activity ANOVA summary: F = 33.7, p value < 0.0001 | |||||
| C. Disease-associated mutations | |||||
| RyR2 WT | 0.21 ± 0.02 | -- | 5.8 × 10−3 ± 2.1 × 10−3 | -- | 3 |
| K3997E | 0.16 ± 0.01 | 0.0025 | 4.0 × 10−3 ± 0.3 × 10−3 | 0.9676 | 3 |
| M3999V | 0.06 ± 0.01 | <0.0001 | 27.0 × 10−3 ± 3.0 × 10−3 | <0.0001 | 3 |
| F4020L | 0.10 ± 0.01 | <0.0001 | 8.1 × 10−3 ± 1.1 × 10−3 | 0.8778 | 3 |
| N4097S | 0.15 ± 0.01 | 0.0002 | 4.6 × 10−3 ± 0.7 × 10−3 | 0.9971 | 3 |
| R4157Q | 0.14 ± 0.01 | <0.0001 | 8.4 × 10−3 ± 3.2 × 10−3 | 0.8026 | 3 |
| L4188P | 0.12 ± 0.02 | <0.0001 | 17.8 × 10−3 ± 6.4 × 10−3 | 0.0004 | 3 |
| T4196A | 0.11 ± 0.01 | <0.0001 | 20.3 × 10−3 ± 1.2 × 10−3 | <0.0001 | 3 |
| Q4201R | 0.11 ± 0.01 | <0.0001 | 22.4 × 10−3 ± 1.8 × 10−3 | <0.0001 | 3 |
| EC50 ANOVA summary: F =30.3, p value < 0.0001; Basal activity ANOVA summary: F = 28.4, p value < 0.0001 | |||||
Data are presented as mean ± S.D. The significance of differences in EC50 and basal activity between WT and mutants was evaluated by performing one-way ANOVA with Dunnett's multiple comparisons post hoc testing. A p value <0.05 was considered statistically significant.