Structural and functional interactions between the EF hand domain and S2–S3 loop in the type-1 ryanodine receptor ion channel

Figures

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  Figure 1. Molecular dynamics simulations of RyR1 structure to identify hydrogen bond interactions between EF-hand and S2–S3 domains. The membrane-embedded open state of RyR1 (PDB: 5TAL) was simulated using GROMACS to investigate potential hydrogen bond interactions between the EF hand and S2–S3 loop domains. A, our molecular dynamics simulation revealed up to six hydrogen bond interactions, with five hydrogen bonds being the maximum observed in a single trajectory frame. B, the EF hand residue K4101 exhibited proximity to D4730 in the S2–S3 loop. C–E, the S2–S3 loop residue R4736 interacted with (C) E4075, (D) Q4078, and (E) D4079 in the EF hand domain. F, a snapshot of the domain interface between the EF hand and S2–S3 loop is shown, highlighting the presence of five hydrogen bonds except for the R4736 (NH1)-D4079 (OD1) interaction. RyR, ryanodine receptor; PDB, Protein Data Bank.

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  Figure 2. Ca2+dependent activity changes of WT and mutant RyR1s. Activities of WT and mutant RyR1s were determined in the presence of various concentration of Ca2+ by [3H]ryanodine binding methods. A and B, Ca2+ dependent of bound [3H]ryanodine of K4101- and D4730-RyR1 mutants are shown together with WT-RyR1 in Panel A and B, respectively. C and D, half maximal Ca2+ concentrations for Ca2+ activation (EC50) and inhibition (IC50) are shown in Panel C and D, respectively. Data are mean ± SD (n=5–9). ∗p < 0.05 and ∗∗p < 0.01 compared with WT-RyR1 by one-way ANOVA followed by Tukey’s test of six different genotypes of RyR1. RyR, ryanodine receptor.

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  Figure 3. Mg2+-dependent inhibitions of WT and mutant RyR1s. Activities of WT and mutant RyR1s were determined in the presence of 165 μM Ca2+ and various concentrations of Mg2+ by [3H]ryanodine binding methods. A and B, Mg2+-dependent of bound [3H]ryanodine of K4101- and D4730-RyR1 mutants are shown together with WT-RyR1 in Panel A and B, respectively. C, half maximal Ca2+ concentrations for Mg2+ inhibition (IC50) are shown. Data are mean ± SD (n=4–9). ∗p < 0.05 and ∗∗p < 0.01 compared with WT-RyR1 by one-way ANOVA followed by Tukey’s test of six different genotypes of RyR1. RyR, ryanodine receptor.

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  Figure 4. Caffeine-dependent activations of WT and mutant RyR1s. Activities of WT and mutant RyR1s were determined in the presence of 0.26 μM Ca2+ and various concentrations of caffeine by [3H]ryanodine binding methods. A and B, caffeine-dependent of bound [3H]ryanodine of K4101- and D4730-RyR1 mutants are shown together with WT-RyR1 in Panel A and B, respectively. Caffeine effect on K4101E mutant at 0.06 μM Ca2+ is shown as a dotted blue line in Panel A. Significant activations by caffeine in each genotype were determined by Kruskal-Wallis one-way ANOVA on Ranks followed by Dunn’s test for WT and K4101E or by one-way ANOVA followed by Tukey’s test for the other mutant RyR1s. All the mutant except K4101E at 0.26 μM Ca2+ (solid blue line) were significantly activated by 10 mM and 20 mM caffeine (p < 0.05). Significant activations of K4101E at 0.26 μM Ca2+ were only observed with 3 mM caffeine. C, half maximal Ca2+ concentrations for caffeine activation (EC50) are shown. Data are mean ± SD (n=4–7). EC50 values of the mutants (not including K4101E) were not significantly different from WT-RyR1 by either one-way ANOVA or Kruskal-Wallis one-way ANOVA on Ranks. RyR, ryanodine receptor.

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  Figure 5. In silico mutagenesis of the interactions between EF and S2–S3 domains in RyR1. PyMol was used to perform in silico mutagenesis based on the structure shown in Figure 1B. A–F, six different mutations on amino acids involved in hydrogen bond interactions (K4101, D4730, and R4736) were tested to observe changes in hydrogen bond interactions. The methods used for in silico mutagenesis are described in Experimental procedures. RyR, ryanodine receptor.

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  Figure 6. Ca2+-dependent activity changes of WT and mutant RyR2s.A, schematic of WT and mutant RyR2s. B and C, activities of WT and mutant RyR2s were determined in the presence of various concentrations of Ca2+ by [3H]ryanodine binding methods. Ca2+ dependent of bound [3H]ryanodine of WT-, G4663S-, and double SD-RyR2 are compared in Panel B. G4663S-RyR2 and coexpression of G4663S- and H4763P-RyR2 at the phosphor-mimicking conformation are compared to double SD mutant in Panel C. D and E, half maximal Ca2+ concentrations for Ca2+ activation (EC50) and inhibition (IC50) are shown in Panel D and E, respectively. Data are mean ± SD (n=4–6). EC50 values of the mutants were not significantly different among the five groups by either one-way ANOVA or Kruskal-Wallis one-way ANOVA on Ranks. Significant differences of IC50 values among five groups were tested by one-way ANOVA followed by Tukey’s test. ∗p < 0.05 and ∗∗p < 0.01 compared with double SD-RyR2. RyR, ryanodine receptor.

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  Figure 7. Proposed model of Ca2+-dependent inhibition of RyR1. Structural analysis revealed that a high concentration of Ca2+ leads to a reduction in the distance between EF hand and S2–S3 loop domains, resulting in the inhibited state of RyR1. Our present study proposes that this mechanism includes two pairs of hydrogen bond interactions between the amino acids highlighted as purple dots. Skeletal disease-associated mutation sites on the domain interface are shown as gray dots. RyR, ryanodine receptor.