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

Previous cryo-electron micrographs suggested that the skeletal muscle Ca²⁺ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca²⁺ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [³H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca²⁺/Mg²⁺-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca²⁺-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca²⁺/Mg²⁺-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca²⁺-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca²⁺/Mg²⁺-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.