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

Abstract

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.

Structure-based mechanism of RyR channel operation by calcium and magnesium ions

“…The median of D1 values measured in the RyR1-C structures is too large for the residues to interact, while those of the open, primed, or inactivated RyR1 structures are in the range allowing for proper interaction between residues in a suitable rotamer configuration. Of interest is the RyR1-Mg structure 7umz, defined as "ACP/10mM free Mg 2+ -closed state" (Nayak et al, 2024), obtained at a very high concentration of Mg 2+ ions that are supposed to stabilize the inactivated state (Laver et al, 1997). This structure provided D1 similar to those found in primed, open, and inactivated states.…”

“…The recent RyR1 structure 7umz, obtained in the presence of 10 mM Mg 2+ , was defined as the closed state since, since the activation site resembled that in the absence of bound Ca 2+ despite the presence of a bound Mg 2+ ion (Nayak et al, 2024). However, the low flexion angle (-2.8°), and the configuration of the EF-hand region relative to the S23 loop reported by (Nayak et al, 2024), as well as in Figure 2 and Figure 3 of this work, resemble the primed and the inactivated state at the same time. The position of the EF-hand region relative to the central domain differs from RyR1 in both the closed and the Ca 2+inactivated states, similar as RyR2 structures (Figure 4).…”

“…This could explain in part why the RyR2 structure 6jiy obtained at 5 mM Ca 2+ had the pore open (diameter of 17 Å) (Gong et al, 2019), at odds with the expectation to find the channel in the inactivated state with a closed pore at so high Ca 2+ concentration. Confoundingly, even at 3.7 mM Ca 2+ or 5-10 mM Mg 2+ concentrations in the reconstitution media the Ca or Mg atoms were observed only at the Ca-activation site, near the bound ATP molecule, or in the ion flux pathway of RyR1 but not elsewhere (Nayak and Samso, 2022;Nayak et al, 2024). This indicates that the potential inhibition site has an affinity for divalent ions smaller than ATP (Kd ≈200 µM, (Bers et al, 1994)).…”

“…With increasing concentrations of the activators (Ca 2+ , ATP derivatives, or xanthine derivatives), the configuration of the tetramers shifted from the closed state to the primed and open state (des Georges et al, 2016;Dashti et al, 2020). At a very high Ca 2+ or Mg 2+ concentration, the inactivated state was observed (Nayak and Samso, 2022;Nayak et al, 2024). Under similar conditions, analogous C, P, and O states were observed also in RyR2 (Chi et al, 2019); unfortunately, the structure of RyR2 in the inactivated state has not been published yet.…”

“…The complex structure of RyR tetramer may adopt a large number of configurations (des Georges et al, 2016;Dashti et al, 2020). In RyR1, four discrete structural states were discerned under different cytosolic conditions -closed (C), primed (P), open (O), and inactivated (I) (des Georges et al, 2016;Nayak and Samso, 2022;Nayak et al, 2024). With increasing concentrations of the activators (Ca 2+ , ATP derivatives, or xanthine derivatives), the configuration of the tetramers shifted from the closed state to the primed and open state (des Georges et al, 2016;Dashti et al, 2020).…”