Interplay between Mg²⁺ and Ca²⁺ at multiple sites of the ryanodine receptor

RyR1 is an intracellular Ca²⁺ channel important in excitable cells such as neurons and muscle fibers. Ca²⁺ activates it at low concentrations and inhibits it at high concentrations. Mg²⁺ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg²⁺-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg²⁺ up to 2.8 Å resolution, identifying multiple Mg²⁺ binding sites. Mg²⁺ inhibits at the known Ca²⁺ 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, Mg²⁺ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg²⁺ inhibition is overcome during excitation.