Reduced threshold for store overload-induced Ca2+ release is a common defect of RyR1 mutations associated with malignant hyperthermia and central core disease.

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used ß-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain.

Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595-CFP and K4269-YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

Localization of the dantrolene-binding sequence near the FK506-binding protein-binding site in the three-dimensional structure of the ryanodine receptor.

Dantrolene is believed to stabilize interdomain interactions between the NH2-terminal and central regions of ryanodine receptors by binding to the NH2-terminal residues 590-609 in skeletal ryanodine receptor (RyR1) and residues 601-620 in cardiac ryanodine receptor (RyR2). To gain further insight into the structural basis of dantrolene action, we have attempted to localize the dantrolene-binding sequence in RyR1/RyR2 by using GFP as a structural marker and three-dimensional cryo-EM. We inserted GFP into RyR2 after residues Arg-626 and Tyr-846 to generate GFP-RyR2 fusion proteins, RyR2Arg-626-GFP and RyR2Tyr-846-GFP. Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST- or cyan fluorescent protein-tagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolene-binding sequence are located near the FKBP12.6-binding site. Using cryo-EM, we have mapped the three-dimensional location of Tyr-846-GFP to domain 9, which is also adjacent to the FKBP12.6-binding site. To further map the three-dimensional location of the dantrolene-binding sequence, we generated 10 FRET pairs based on four known three-dimensional locations (FKBP12.6, Ser-437-GFP, Tyr-846-GFP, and Ser-2367-GFP). Based on the FRET efficiencies of these FRET pairs and the corresponding distance relationships, we mapped the three-dimensional location of Arg-626-GFP or -cyan fluorescent protein, hence the dantrolene-binding sequence, to domain 9 near the FKBP12.6-binding site but distant to the central region around residue Ser-2367. An allosteric mechanism by which dantrolene stabilizes interdomain interactions between the NH2-terminal and central regions is proposed.

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor.

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca(2+) 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 Ca(2+) release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca(2+). We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or 'quantal' Ca(2+) release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca(2+) release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca(2+) revealed that caffeine reduced the luminal Ca(2+) threshold at which spontaneous Ca(2+) release occurs. Interestingly, spontaneous Ca(2+) release in the form of Ca(2+) 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 Ca(2+) activation, but had little effect on the threshold for cytosolic Ca(2+) activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca(2+) activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca(2+) activation of RyR2, and increased the propensity for spontaneous Ca(2+) release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca(2+) release by reducing the threshold for luminal Ca(2+) activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

ARVC-related mutations in divergent region 3 alter functional properties of the cardiac ryanodine receptor.

Two single-nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the nonsynonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with arrhythmogenic right ventricular cardiomyopathy, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser(1886)), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca(2+) release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly(1886) caused a significant increase in the cellular Ca(2+) oscillation activity compared with RyR2 wild-type-expressing HEK293 cells. It was even more pronounced if glycine 1885 or 1886 was replaced by the acidic amino acids glutamate (G1885E) or aspartate (G1886D). Surprisingly, when both substitutions were introduced in the same RyR2 subunit (RyR2-G1885E/G1886S), the store-overload-induced calcium release activity was nearly completely abolished, although the Ca(2+) loading of the intracellular stores was markedly enhanced, and the channel still displayed substantial Ca(2+) release on stimulation by 5 mM caffeine. These results suggest that the adjacent glycines 1885 and 1886, located in the divergent region 3, are critical for the function and regulation of RyR2.