Multiple Closed States of the Ryanodine Receptor Determined by CRYOEM

Abstract

Ryanodine receptors (RyRs) are intracellular ion channels involved in Ca2+ release from internal stores in excitable cells. These channels are the largest channels known and are homotetramers, sizing ∼2,26 MDa. The 3D structure of RyR1 in it open and close states was determined previously, revealing that the ion gate opening mechanism rely on long-range conformational changes over 100 Å. The RyR gating properties are highly regulated by Ca2+, Mg2+, ATP, and FKBP12. The native conformation of RyR1 in presence of physiological concentrations of Mg2+ and ATP is unknown. Here we determine the 3D structure of RyR1 in non-activating conditions (submicromolar Ca2+) in the presence of Mg2+ and an ATP analog, but in a flexible conformation by absence of FKPBP12. This new structure was determined using cryoEM and image processing. The resulting 3D structure is in the closed conformation when compared to 3D reconstructions of RyR1 in open and closed conditions in presence of FKBP12 determined previously. In addition, from the comparison among several 3D reconstructions, we establish new conformation-function correlations. We find that the rhomboid structures formed by domains 2-4-5-6 situated far away from the ion gate move as a whole during gating, and define a “flexion angle” that appears to be correlated with the degree of opening of the channel, whereby the flexion angle after adding Mg2+ and ATP shifts by 3 degrees towards the closed state. In conclusion this research suggests that physiological concentrations of Mg2+ and ATP shift the RyR1 conformation toward the closed conformation and also suggests that the closed conformation encompasses sub-states.