The cardiac ryanodine receptor (RyR2) governs the release of Ca ²⁺ from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca ²⁺ content reaches a critical level, spontaneous Ca ²⁺ release occurs, a process we refer to as store-overload-induced Ca ²⁺ release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca ²⁺ levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca ²⁺ and enhanced the basal level of [ ³ H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca ²⁺ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca ²⁺ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca ²⁺ overload-associated VT.

Gating a calcium channel: The type 2 ryanodine receptor (RyR2) controls the release of calcium ions from the sarcoplasmic reticulum in cardiac cells—the initiating step in cardiac muscle contraction. Mutations in RyR2 are associated with cardiac diseases. Penget al.used single-particle electron cryomicroscopy to determine the structure of RyR2 from porcine heart at 4.4-Å resolution with the calcium channel closed and at 4.2-Å resolution with the calcium channel open. The structures reveal how interdomain motions result in a conformational change in the cytoplasmic region of RyR2 that is transduced by a central domain to cause motions that open or close the channel.Science, this issue p.301

Hyperphosphorylation of the cardiac Ca ²⁺ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca ²⁺ dependence of [ ³ H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute β-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca²⁺ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca²⁺ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca²⁺. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca²⁺ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca²⁺ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca²⁺ revealed that caffeine reduced the luminal Ca²⁺ threshold at which spontaneous Ca²⁺ release occurs. Interestingly, spontaneous Ca²⁺ release in the form of Ca²⁺ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca²⁺ activation, but had little effect on the threshold for cytosolic Ca²⁺ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca²⁺ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca²⁺ activation of RyR2, and increased the propensity for spontaneous Ca²⁺ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca²⁺ release by reducing the threshold for luminal Ca²⁺ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.