Structural analyses of human ryanodine receptor type 2 channels reveal the mechanisms for sudden cardiac death and treatment

Abstract

Ryanodine receptor type 2 (RyR2) mutations have been linked to an inherited form of exercise-induced sudden cardiac death called catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT results from stress-induced sarcoplasmic reticular Ca²⁺ leak via the mutant RyR2 channels during diastole. We present atomic models of human wild-type (WT) RyR2 and the CPVT mutant RyR2-R2474S determined by cryo-electron microscopy with overall resolutions in the range of 2.6 to 3.6 Å, and reaching local resolutions of 2.25 Å, unprecedented for RyR2 channels. Under nonactivating conditions, the RyR2-R2474S channel is in a "primed" state between the closed and open states of WT RyR2, rendering it more sensitive to activation that results in stress-induced Ca²⁺ leak. The Rycal drug ARM210 binds to RyR2-R2474S, reverting the primed state toward the closed state. Together, these studies provide a mechanism for CPVT and for the therapeutic actions of ARM210.

Fig. 1.. Cryo-EM reconstructions of human RyR2 showing that the CPVT mutant RyR2-R2474S puts the channel into the primed state, and treatment with the Rycal ARM210 and CaM puts the channel back toward the closed state.

(A) Overlapped models of open PKA-phosphorylated RyR2 (PDB: 7U9R, yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray). The arrows show that the cytosolic shell of the PKA-phosphorylated RyR2 shifts downward and outward when going from the closed to the open state. To facilitate visualization, only the front protomer is shown in colors, while the other three protomers are shown as gray transparent volumes. The positions of the sarcoplasmic reticular membranes are shown as black discs. Conditions include 10 mM ATP, 150 nM free Ca²⁺, and 500 μM xanthine. (B) Overlapped models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X, magenta). The arrows show that the cytosolic shell of RyR2-R2474S shifts downward and outward compared to closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 going from the closed state to the open state. We define this intermediate between closed and open states as the primed state. (C) Overlapped models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X, magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1, cyan). The arrows show that the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 cytosolic domain shifts upward and inward compared to the RyR2-R2474S reversing the primed state back toward the closed state. (D) Overlapped models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X, magenta) and closed PKA-phosphorylated RyR2-R2474S + CaM (PDB: 7UA3, cyan). Similar to the effects of the Rycal ARM210, CaM reverses the primed state back toward the closed state.

Fig. 2.. Stabilization of RyR2-R2474S by the Rycal ARM210.

(A) Cryo-EM maps of closed PKA-phosphorylated RyR2 (gray) and primed PKA-phosphorylated RyR2-R2474S (magenta) from the side (left) and top (right) views. Conformation changes are shown with arrows. (B) Normalized differences in RMSD of the primed PKA-phosphorylated RyR2-R2474S. (C) Close-up view of the region around residue 2474 of closed PKA-phosphorylated RyR2 (left) and primed PKA-phosphorylated RyR2-R2474S (right). Distances are measured between Cβ atoms. (D) Aligned models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray), open PKA-phosphorylated RyR2 (PDB: 7U9R, yellow), and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X, magenta). Conformational changes are shown with arrows. Distances between closed PKA-phosphorylated RyR2 and primed PKA-phosphorylated RyR2-R2474S, and between closed and open PKA-phosphorylated RyR2 (in parentheses) are labeled. (E to G) Same as (A), (B), and (D) but including closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1, cyan). Distances between primed PKA-phosphorylated RyR2-R2474S and closed PKA-phosphorylated RyR2-R2474S + ARM210 are labeled (E). Changes introduced by the R2474S mutation are partially reversed by the addition of ARM210. The densities of ARM210 and BSol1-RY1&2 interface are highlighted (F, right). (H) Aligned models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray), primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X, magenta), and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1, cyan). Conformational changes of the RY1&2 and BSol domains are shown with arrows.

Fig. 3.. Stabilization of PKA-phosphorylated RyR2 and RyR2-R2474S by CaM.

(A) Aligned cryo-EM maps of closed PKA-phosphorylated RyR2 (gray) and closed PKA-phosphorylated RyR2 + CaM (cyan). (B) JSol and CSol models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray) and open PKA-phosphorylated RyR2 (PDB: 7U9R, yellow). Conformation changes are shown with arrows. (C) JSol and CSol models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q, gray) and closed PKA-phosphorylated RyR2 + CaM (PDB: 7U9T, cyan). Conformation changes are shown with arrows. (D) Aligned cryo-EM maps of primed PKA-phosphorylated RyR2-R2474S (magenta) and closed PKA-phosphorylated RyR2-R2474S + CaM (cyan). Conformation changes are shown with arrows. CaM reverses the changes in the BSol2 domain introduced by the mutation by stabilizing the BSol3 domain. (E) Model with cryo-EM map of closed PKA-phosphorylated RyR2-R2474S + CaM (PDB: 7UA3) centered on the BSol3 domain that is stabilized by CaM.

Fig. 4.. Structure-function relationship of the RY3&4 phosphorylation domain.

(A and B) Cryo-EM maps of the closed particles with destabilized (gray) and stabilized (magenta) RY3&4 domain. A downward shift in surrounding domains can be observed. Individual domains are labeled. (C and D) Same as (A) and (B) from a different point of view. (E) Aligned models of the closed state with destabilized (gray) and stabilized (magenta) RY3&4 domain, and open state (yellow). Models were aligned at the BSol1 domains to facilitate interpretation of conformational changes. When the RY3&4 domain is stabilized, the changes are in the same direction as the open state. Shown with arrows are the distance between the closed state with destabilized RY3&4 domain and the closed state with stabilized RY3&4 domain, and between the closed state with destabilized RY3&4 domain and the open state (in parentheses). (F) RMSD analysis of the closed state with stabilized RY3&4 domain. This analysis was performed on primed PKA-phosphorylated RyR2-R2474S because this dataset presents the most particles and best local refinement resolution.

Fig. 5.. Resolution of auxiliary intramembrane helices in RyR2.

Model with overlapped cryo-EM map of PKA-phosphorylated RyR2 (PDB: 7U9Q) highlighting the Sx helices from the side view (A) and bottom view (B). Auxiliary helices and pocket with extra densities are labeled.

Fig. 6.. Proposed mechanism of CPVT-related RyR2 variants, other gain-of-function mutants, and heart failure.

(A) Schematic representation of the normal function of RyR2. (B) Schematic representation of the CPVT-related Ca²⁺ leak during diastole under intense exercise or stress conditions. In the case of CPVT variants, the resting state is already in a primed state. This correlates with the higher open probability during exercise or stress conditions, which results in opening during diastole, afterdepolarizations, arrhythmias, and sudden cardiac death. This pathological state can be reversed by treatment with the Rycal ARM210 (R). This basal primed state scenario could be a shared mechanism among other RyR1 and RyR2 gain-of-function mutants. (C) Schematic representation of the heart failure–related primed state and Ca²⁺ leak. The heart failure–related primed state remains hypothetical as no structure has been solved yet.