Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice.
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ABSTRACT:
SUBMITTER: Lehnart SE
PROVIDER: S-EPMC2381750 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.
| S-EPMC4216943 | biostudies-literature
Project description:Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias.
| S-EPMC3283708 | biostudies-literature
Project description:Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca(2+) and an increased propensity for spontaneous Ca(2+) release or store-overload-induced Ca(2+) release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca(2+), but had little effect on the sensitivity of the channel to activation by cytosolic Ca(2+). This specific impact of the A4860G mutation indicates that the luminal Ca(2+) activation of RyR2 is distinct from its cytosolic Ca(2+) activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca(2+) release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca(2+) activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.
| S-EPMC2084339 | biostudies-literature
Project description:Cardiorespiratory failure is the most common cause of sudden unexplained death in epilepsy (SUDEP). Genetic autopsies have detected "leaky" gain-of-function mutations in the ryanodine receptor-2 (RyR2) gene in both SUDEP and sudden cardiac death cases linked to catecholaminergic polymorphic ventricular tachycardia that feature lethal cardiac arrhythmias without structural abnormality. Here we find that a human leaky RyR2 mutation, R176Q (RQ), alters neurotransmitter release probability in mice and significantly lowers the threshold for spreading depolarization (SD) in dorsal medulla, leading to cardiorespiratory collapse. Rare episodes of sinus bradycardia, spontaneous seizure, and sudden death were detected in RQ/+ mutant mice in vivo; however, when provoked, cortical seizures frequently led to apneas, brainstem SD, cardiorespiratory failure, and death. In vitro studies revealed that the RQ mutation selectively strengthened excitatory, but not inhibitory, synapses and facilitated SD in both the neocortex as well as brainstem dorsal medulla autonomic microcircuits. These data link defects in neuronal intracellular calcium homeostasis to the vulnerability of central autonomic brainstem pathways to hypoxic stress and implicate brainstem SD as a previously unrecognized site and mechanism contributing to premature death in individuals with leaky RYR2 mutations.
| S-EPMC4995941 | biostudies-literature
Project description:Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia susceptibility genes occur in 5-10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels (KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current (I(Kr)) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1-I274V causes a gain-of-function in I(Ks) characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V-I(Ks) incorporated into the Luo-Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.
| S-EPMC2386856 | biostudies-literature
Project description:The ryanodine receptor/Ca(2+)-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca(2+) release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca(2+) channel CaV1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in "hot spot" regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca(2+) release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca(2+)-ATPase 1A and the ?1S subunit of the L-type Ca(2+) channel CaV1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca(2+) flux in skeletal muscle that mediates excitation-contraction coupling.
| S-EPMC3961684 | biostudies-literature
Project description:AimsThe cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer.Methods and resultsWe use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the β8-β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8-β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8-β9 domain in channel closing.ConclusionThese results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression.
| S-EPMC7898959 | biostudies-literature
Project description:Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction-oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3 knockout mouse, we discovered that deletion of CYB5R3 in male adult cardiomyocytes, but not female, causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3 KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3 KO hearts display decreased ATP, increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Lastly, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in African Americans with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a new genetic biomarker for individuals in the African American population that may potentially increase the risk of death from HFrEF.
2023-06-14 | GSE206121 | GEO
Project description:Modification of the ryanodine receptor (RyR)/Ca(2+) release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t(1/2)<30 s) at 25 degrees C with low concentrations of DNFB [C(50) (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding)=5 microM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca(2+) release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 degrees C); at pH values higher than 7.4 it reacted slowly (t(1/2)=20 min) with high concentrations of DNFB (C(50)=70 microM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca(2+)-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca(2+). The DNFB-modified ryanodine-activated RyR channel showed fast transitions between open, closed and several sub-conductance states, and was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified closed channel or, if present during modification, prevented the inhibition of RyR channel activity by DNFB. Neither the stimulation nor the inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3'-O-(4-benzoyl)benzoyl-[alpha-(32)P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.
| S-EPMC1220458 | biostudies-other
Project description:BackgroundRare variants in cardiac ion channel genes are associated with sudden cardiac death in rare primary arrhythmic syndromes; however, it is unknown whether common variation in these same genes may contribute to sudden cardiac death risk at the population level.Methods and resultsWe examined the association between 147 single nucleotide polymorphisms (SNPs) (137 tag, 5 noncoding SNPs associated with QT interval duration, and 5 nonsynonymous SNPs) in 5 cardiac ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2, and sudden and/or arrhythmic death in a combined nested case-control analysis among 516 cases and 1522 matched control subjects of European ancestry enrolled in 6 prospective cohort studies. After accounting for multiple testing, 2 SNPs (rs2283222 located in intron 11 in KCNQ1 and rs11720524 located in intron 1 in SCN5A) remained significantly associated with sudden/arrhythmic death (false discovery rate=0.01 and 0.03, respectively). Each increasing copy of the major T-allele of rs2283222 or the major C-allele of rs1172052 was associated with an odds ratio of 1.36 (95% confidence interval, 1.16 to 1.60; P=0.0002) and 1.30 (95% confidence interval, 1.12 to 1.51; P=0.0005), respectively. Control for cardiovascular risk factors and/or limiting the analysis to definite sudden cardiac death did not significantly alter these relationships.ConclusionIn this combined analysis of 6 prospective cohort studies, 2 common intronic variants in KCNQ1 and SCN5A were associated with sudden cardiac death in individuals of European ancestry. Further study in other populations and investigation into the functional abnormalities associated with noncoding variation in these genes may lead to important insights into predisposition to lethal arrhythmias.
| S-EPMC2891421 | biostudies-literature