Ca²⁺-inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

Activation of the intracellular Ca²⁺channel ryanodine receptor (RyR) triggers a cytosolic Ca²⁺surge, while elevated cytosolic Ca²⁺inhibits the channel in a negative feedback mechanism. Cryo-EM carried out under partially inactivating Ca²⁺conditions revealed two conformations of RyR1, an open state and an inactivated state, resolved at 4.0 and 3.3 Å resolution, respectively. RyR1s were embedded in nanodiscs with two lipids resolved at each inter-subunit crevice. Ca²⁺binding to the high affinity site engages the central (CD) and C-terminal domains (CTD) into a quasi-rigid unit, which separates the S6 four-helix bundle and opens the channel. Further out-of-plane rotation of the quasi-rigid unit pushes S6 towards the central axis, closing (inactivating) the channel. The inactivated conformation is characterized by a downward conformation of the cytoplasmic assembly, a tightly-knit subunit interface contributed by a fully occupied and partially remodeled Ca²⁺activation site, and two salt bridges between the EF hand domain and the S2-S3 loop of the neighboring subunit validated by naturally-occurring diseasecausing mutations. Ca²⁺also bound to ATP, mediating a tighter interaction between S6 and CTD. Our study suggests that theclosed-inactivatedis a distinctive state of the RyR1 and its transition to theclosed-activablestate is not a simple reverse of the Ca²⁺mediated activation pathway.