Caffeine has long been used as a pharmacological probe for studying ryanodine receptor (RyR)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. Here we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single cardiac RyR (RyR2) channels to luminal or cytosolic Ca2+. We found that HEK293 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 diseased-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.
Dantrolene is believed to stabilize interdomain interactions between the NH 2 -terminal and central regions of ryanodine receptors by binding to the NH 2 -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, RyR2 Arg-626-GFP and RyR2 Tyr-846-GFP . Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST-or cyan fluorescent proteintagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolenebinding 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 NH 2 -terminal and central regions is proposed.Ryanodine receptor (RyR) 6 Ca 2ϩ release channels are located in the sarco(endo)plasmic reticulum of muscle and some nonmuscle cells and play an essential role in muscle contraction and Ca 2ϩ signaling (1, 2). There are three mammalian RyR isoforms, RyR1, RyR2, and RyR3, with distinct patterns of expression. RyR1 is predominantly expressed in skeletal muscle, whereas RyR2 is mainly expressed in cardiac muscle and the brain. RyR3 expression is widespread but at relatively low levels (3). RyRs also play a critical role in the pathogenesis of muscle disorders. Naturally occurring mutations in RyR1 are linked to malignant hyperthermia and central core disease (4, 5), whereas mutations in RyR2 are associated with ventricular arrhythmias and sudden cardiac death (6, 7). Interestingly, most of the disease-causing RyR1 and RyR2 mutations are located within the NH 2 -terminal, central, and COOH-terminal "hot spot" regions of the two channel isoforms (6, 7). This similar pattern of mutation distribution suggests that disease-causing RyR1 and RyR2 mutations are likely to affect the same aspects of channel structure and function. It has been proposed that interdomain interactions between the NH 2 -terminal and central regions of RyR1 or RyR2 are critical for stabilizing the closed state of the channel and that disease-cau...
SummaryCalmodulin (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 (RyR2 R3595-CFP/K4269-YFP ) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2 R3595-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 CaMbinding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2 R3595-CFP ) and single YFP (RyR2 K4269-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.
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 β-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 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 Ser1886), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca2+ release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly1886 caused a significant increase in the cellular Ca2+ 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 Ca2+ loading of the intracellular stores was markedly enhanced, and the channel still displayed substantial Ca2+ 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.
amino acids of RyR1 are predicted to fold into a second b-trefoil and an alpha helical domain. In this report, the relative orientation of these three domains (termed here as b1, b2 and a1) within the full-length RyR1 protein has been investigated using a novel FRET-based technique. This method monitors the relative proximity of a GFP fluorescent donor fused into RyR1 and a fluorescent acceptor, Cy3NTA, targeted to poly-histidine (His) tags inserted into the primary sequence of RyR1. In this study, GFP was fused to single positions within b1, b2 and a1 and FRET was then measured to Cy3NTA targeted to 6 His tags singly introduced into these three domains. The results of these measurements provide a detailed picture of the relative orientation of these three sub-domains within full-length RyR1 expressed in a cellular context. Further structural studies using this technique can now be undertaken to determine how these domains move during channel gating. (Supported by NIH grant R21ARO56406). 1030-Plat3D Mapping of the SPRY2 Domain of RyR1 by Antibody Labeling and Single-Particle cryo-EM
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