Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo–electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major “hyperactive” feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain. Because the more flexible conformation may correspond to RyR2 with a phosphorylated P2 domain, we suggest that phosphorylation may set RyR2 in a conformation that needs less energy to transition to the open state. Comparison of RyR2 from cardiac muscle and RyR1 from skeletal muscle showed substantial structural differences between the two, especially in the helical domain 2 (HD2) structure forming the Clamp domain, which participates in quaternary interactions with the dihydropyridine receptor and neighboring RyRs in RyR1 but not in RyR2. Rigidity of the HD2 domain of RyR2 was enhanced by binding of FKBP12.6, a ligand that stabilizes RyR2 in the closed state. These results help to decipher the molecular basis of the different mechanisms of activation and oligomerization of the RyR isoforms and could be extended to RyR complexes in other tissues.

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 of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly-knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for seamless transition from inactivated to closed conformations.

No abstract

Ca 2þ sparks constitute the fundamental units of sarcoplasmic reticulum (SR) Ca 2þ release in cardiomyocytes. However, despite more than 25 years of investigation, the precise nature by which ryanodine receptors (RyRs) collaborate to generate these release events remains unclear. This challenge is related to both technical limitations in imaging RyRs and the rapid time frame in which sparks occur. Unfortunately, various imaging techniques capable of resolving RyRs, including super-resolution microscopy (dSTORM, DNA-PAINT) and electron microscopy, require fixed samples. To circumvent this limitation, we developed a transgenic mouse with photo-activated (PA) tagRFP targeted to RyR2. This approach allows correlative pairing of RyR localization, determined by PA Localization Microscopy (PALM), and Ca 2þ sparks detected by high-speed imaging with a highly inclined light sheet (HILO). Ca 2þ spark recordings showed that a subset of events exhibited slow kinetics, with protracted rise times and durations. Subtracting estimated Ca 2þ diffusion revealed that prolonged Ca 2þ sparks exhibited multiple distinct releases, numbering between 2 and 8 events. Notably, consecutive releases were associated with displacement of the spark centroid. Paired imaging of RyRs confirmed that these ''travelling sparks'' moved between nearby RyR clusters, with some sparks exhibiting displacement as far as 500 nm along z-lines. Importantly, spark propagation often proceeded between clusters that were not within the closest proximity. Treatment with isoproterenol exaggerated this phenomenon, as a larger fraction of travelling sparks was observed which included as many as 12 distinct release sites. These data suggest that participation of discrete RyR clusters in Ca 2þ spark generation is dependent not only on RyR cluster position, but also other factors such as local post-translational modifications which are critically altered during badrenergic stimulation.