Interplay between Mg ²⁺ and Ca ²⁺ at multiple sites of the ryanodine receptor

RyR1 is an intracellular Ca ²⁺ channel important in excitable cells. Ca ²⁺ activates it at low concentrations and inhibits it at high concentrations. Mg ²⁺ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg ²⁺ -mediated inhibition, the molecular-level mechanisms remain unclear. We determined two cryo-EM structures of RyR1 with Mg ²⁺ up to 2.8 Å resolution, identifying multiple Mg ²⁺ binding sites. Mg ²⁺ inhibits at the known Ca ²⁺ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg ²⁺ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg ²⁺ inhibition is overcome during excitation.