Interplay between Mg2+and Ca2+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.