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.

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Ryanodine receptors (RyRs) are main regulators of intracellular Ca 2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a “spacer” within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca 2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca 2+ , increasing by ∼20-fold the potency of Ca 2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

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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.

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