Ryanodine receptors (RyRs) are intracellular Ca²⁺ channels that control the release of Ca²⁺ from the sarco(endo)plasmic reticulum. Among the three mammalian RyR isoforms, RyR2 is primarily expressed in the heart and brain and is activated by Ca²⁺ influx by a mechanism known as calcium-induced calcium release. This Ca²⁺ release is fundamental to cellular processes ranging from muscle contraction to learning and memory.

RyRs are the largest known ion channels with a molecular mass of > 2 megadalton for a homotetramer. Each RyR protomer consists of a cytoplasmic region of over 4500 residues and a carboxyl terminal transmembrane (TM) domain. The four identical TM segments enclose a central ion-conducting pore, whereas the cytoplasmic regions serve as a scaffold for interactions with diverse ligands and protein modulators.

RyR channel gating involves a long-range allosteric mechanism. The structures of rabbit skeletal muscle RyR1 were determined at 3.8-Å resolution for the closed state and various lower resolutions for the potentially open states. Elucidating the gating mechanism of RyR requires structural determination of the open states at high resolution.

RyR2 harbors more than 150 mutations associated with cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia type 1, idiopathic ventricular fibrillation, and sudden cardiac death. Structural elucidation of RyR2 may provide the molecular basis for understanding disease mechanisms and for the development of potential novel therapeutics. We purified endogenous RyR2 from porcine heart using glutathione S-transferase–fused FKBP12. To obtain the structure of RyR2 in the closed state, 5 mM EDTA was included throughout purification. To capture an open RyR2, the protein was purified in the presence of 20 μM Ca²⁺ and the compound 2,2′,3,5′,6-pentachlorobiphenyl (PCB95) that can stabilize RyR1 in the open state.

The electron microscopy (EM) maps for RyR2 were reconstructed to 4.4- and 4.2-Å resolutions for the closed and open states, respectively. Compared to the structure of RyR1, a number of armadillo repeats in the C terminus of the helical domain 2 were invisible in RyR2, likely due to intrinsic flexibility. At 20 μM Ca²⁺, the cytoplasmic gate in the closed structure is dilated by approximately 8 Å, resulting in the shift of the constriction site from Ile4868 to Gln4864. The four Gln4864 residues enclose a gate with a diameter of approximately 4 Å, allowing Ca²⁺ passage in a single file.

Structure comparison of the open and closed RyR2 shows little intradomain rearrangement of the armadillo-containing cytoplasmic domains including the amino terminal domain, the Handle domain, and the Helical domain. Relative shifts between these domains result in the breathing motion of the periphery of the cytoplasmic canopy and the rotation of the Central domain. The horseshoe-shaped Central domain, with its convex side interacting with the three armadillo domains and its concave side wrapping around the cytoplasmic O-ring of the channel domain, serves as the primary transducer that integrates and translates the conformational changes of the cytoplasmic domains to channel gating. However, the mechanism of Ca²⁺ sensing and activation of RyR2 remains to be elucidated.

(Top) Representative two-dimensional class averages of electron micrographs of the closed and open RyR2. (Bottom) The two structures are superimposed relative to the transmembrane domain. The blue …