scholarly journals The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca 2+ and Mg 2+

2021 ◽  
Author(s):  
Abigail D. Wilson ◽  
Jianshu Hu ◽  
Charalampos Sigalas ◽  
Elisa Venturi ◽  
Héctor H. Valdivia ◽  
...  
Keyword(s):  
Ryr2 Channel

scholarly journals Control of Diastolic Activity of the RyR2 Channel by Luminal Calcium and ATP

2012 ◽  
Vol 102 (3) ◽  
pp. 316a
Author(s):  
Barbora Tencerova ◽  
Jana Gaburjakova ◽  
Marta Gaburjakova ◽  
Alexandra Zahradnikova
Keyword(s):  
Ryr2 Channel ◽  
Luminal Calcium

scholarly journals Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response

2018 ◽  
Vol 151 (2) ◽  
pp. 131-145 ◽  
Author(s):  
Duilio M. Potenza ◽  
Radoslav Janicek ◽  
Miguel Fernandez-Tenorio ◽  
Emmanuel Camors ◽  
Roberto Ramos-Mondragón ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Cardiac Contractility ◽  
Phosphorylation Site ◽  
Adrenergic Stimulation ◽  
Permeabilized Cells ◽  
Whole Cell Patch Clamp ◽  
Adrenergic Response ◽  
Pump Activity ◽  
Ryr2 Channel ◽  
A Site

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

scholarly journals RYR2 Channel Inhibition Is the Principal Mechanism 0f Flecainide Action in CPVT

2020 ◽  
Author(s):  
Dmytro O Kryshtal ◽  
Daniel Blackwell ◽  
Christian Egly ◽  
Abigail N Smith ◽  
Suzanne M Batiste ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Antiarrhythmic Action ◽  
Channel Block ◽  
Principal Mechanism ◽  
Channel Inhibition ◽  
Ryr2 Channel ◽  
Cardiac Sodium Channels

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-FL and NM-FL) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-Methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

scholarly journals Stereoselectivity of Propafenone RyR2 Channel Block and Prevention of Ventricular Arrhythmia In Vivo

2011 ◽  
Vol 100 (3) ◽  
pp. 180a
Author(s):  
Michela Faggione ◽  
Hyun Seok Hwang ◽  
Bjorn C. Knollmann
Keyword(s):  
Ventricular Arrhythmia ◽  
Channel Block ◽  
Ryr2 Channel

Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum

2008 ◽  
Vol 294 (1) ◽  
pp. H121-H133 ◽  
Author(s):  
Toshiharu Oba ◽  
Yoshitaka Maeno ◽  
Masataka Nagao ◽  
Nagahiko Sakuma ◽  
Takashi Murayama
Keyword(s):  
Redox Potential ◽  
Redox State ◽  
Ventricular Myocytes ◽  
Imaging System ◽  
Reduced Glutathione ◽  
Confocal Imaging ◽  
Minor Effect ◽  
Channel Activity ◽  
Ryr2 Channel ◽  
Cardiac Excitation

Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca2+ handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species, acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca2+ handling by measuring Ca2+ transient using a confocal imaging system and single ryanodine receptor type 2 (RyR2) channel activity using the planar lipid bilayer method. Ca2+ transient was recorded in isolated rat ventricular myocytes with incorporated fluo 3. Intracellular reduced glutathione level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 μM increased Ca2+ transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 μM decreased Ca2+ transient area significantly. Acetaldehyde showed a minor effect on Ca2+ transient in myocytes in which intracellular reduced glutathione content had been decreased against challenge of diethylmaleate to a level comparable to that induced by exposure to ∼50 μM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (−213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1–100 μM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (−230 mV) or markedly oxidized (−180 mV) state. This result was similar to effects of acetaldehyde on Ca2+ transient in diethylmaleate-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.

Mechanism of Destabilized RyR2 Channel in Catecholaminergic Polymorphic Ventricular Tachycardia

2010 ◽  
Vol 16 (9) ◽  
pp. S151
Author(s):  
Takeshi Suetomi ◽  
Masafumi Yano ◽  
Masakazu Fukuda ◽  
Akihiro Hino ◽  
Xiaojuan Xu ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Ryr2 Channel

scholarly journals Slow Diffusion Across Cell Membrane Delays Onset of RyR2-Channel Block and Ca Wave Suppression by Flecainide in Intact Myocytes

2011 ◽  
Vol 100 (3) ◽  
pp. 416a
Author(s):  
Hyun Seok Hwang ◽  
Eleonora S. Galimberti ◽  
Bjorn C. Knollmann
Keyword(s):  
Cell Membrane ◽  
Channel Block ◽  
Slow Diffusion ◽  
Ryr2 Channel

scholarly journals Flux regulation of cardiac ryanodine receptor channels

2009 ◽  
Vol 135 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Yiwei Liu ◽  
Maura Porta ◽  
Jia Qin ◽  
Jorge Ramos ◽  
Alma Nani ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Positive Feedback ◽  
Open Channel ◽  
Single Channel ◽  
Single Channels ◽  
Ryr2 Channel ◽  
Regulatory Sites ◽  
Activation Site ◽  
In Cells

The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel.

Differential Ca2+ sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

2000 ◽  
Vol 279 (3) ◽  
pp. C724-C733 ◽  
Author(s):  
Bradley R. Fruen ◽  
Jennifer M. Bardy ◽  
Todd M. Byrem ◽  
Gale M. Strasburg ◽  
Charles F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Ryanodine Receptors ◽  
Cross Linking ◽  
Release Channel ◽  
Channel Activation ◽  
Functional Interactions ◽  
Ryr2 Channel

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) in the presence of nanomolar Ca2+ concentrations. However, the role of CaM activation in the mechanisms that control Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle and in the heart remains unclear. In media that contained 100 nM Ca2+, the rate of45Ca2+ release from porcine skeletal muscle SR vesicles was increased approximately threefold in the presence of CaM (1 μM). In contrast, cardiac SR vesicle45Ca2+ release was unaffected by CaM, suggesting that CaM activated the skeletal RyR1 but not the cardiac RyR2 channel isoform. The activation of RyR1 by CaM was associated with an approximately sixfold increase in the Ca2+ sensitivity of [3H]ryanodine binding to skeletal muscle SR, whereas the Ca2+ sensitivity of cardiac SR [3H]ryanodine binding was similar in the absence and presence of CaM. Cross-linking experiments identified both RyR1 and RyR2 as predominant CaM binding proteins in skeletal and cardiac SR, respectively, and [35S]CaM binding determinations further indicated comparable CaM binding to the two isoforms in the presence of micromolar Ca2+. In nanomolar Ca2+, however, the affinity and stoichiometry of RyR2 [35S]CaM binding was reduced compared with that of RyR1. Together, our results indicate that CaM activates RyR1 by increasing the Ca2+ sensitivity of the channel, and further suggest differences in CaM's functional interactions with the RyR1 and RyR2 isoforms that may potentially contribute to differences in the Ca2+ dependence of channel activation in skeletal and cardiac muscle.

P5025Structural insights into catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant channels using a three-dimensional in silico model

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Gao ◽  
T Makiyama ◽  
S Ohno ◽  
Y Yamamoto ◽  
Y Wuriyanghai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ventricular Tachycardia ◽  
In Silico ◽  
Structural Model ◽  
Three Dimensional ◽  
Polymorphic Ventricular Tachycardia ◽  
In Silico Modeling ◽  
Ryr2 Channel ◽  
The Relationship ◽  
Inherited Arrhythmias

Abstract Background The cardiac ryanodine receptors (RyR2) are large tetrameric calcium-permeant ion channels found in cardiac muscle sarcoplasmic reticulum, which play an important role in the control of intracellular Ca2+ release and cardiac contraction. Mutations in the RYR2 gene are associated with lethal arrhythmia diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) resulting from increased diastolic Ca2+ leak from mutant channels. RyR2 is a huge protein that each subunit of tetramer is comprised of 4967 amino acids, which hampers the detailed in vitro analysis of RyR2 mutant channels. Purpose We aimed to analyze the structural features of RyR2 mutant channels identified in our cohort with inherited arrhythmias using RyR2 three-dimensional (3D) in silico model to reveal the arrhythmogenic mechanisms. Methods A targeted next-generation sequencing panel for inherited arrhythmias was employed for genetic diagnosis of the patients. Then, we mapped the identified mutations on RyR2 3D structural model developed by cryo-EM images (PDB: 5go9, 5goa, Peng Science 2016) and investigated the relationship between the location of the mutations and specific functional sites. Results As a result of genetic analysis, we identified 93 RYR2 mutations from 112 probands with CPVT (n=93) or long-QT syndrome (LQTS) (n=19).64 of 93 (69%) RYR2 mutations are located in three “hot-spot” area (N-terminal (residues 77–466), central (2246–2534), and channel (3778–4959) hotspot. RyR2 3D in silico modeling revealed that the mutations are regionally distributed mainly in three parts: N-terminal, periphery, and channel part (Figure A). In N-terminal part (1–642 amino acid), 9 of 13 mutations alter the charges of the amino acids (Figure B). Especially, R169L, R169Q, and G172E are close to the interface between two neighboring subunits (∼20Å). These mutations which change the amino acid charge may cause a complete disruption of the ionic pair network and result in largest structural changes, which facilitates RyR2 channel opening. In periphery part (643–3528aa), 22 of 33 mutations are close to the two predicted binding sites of FKBP12.6, a stabilizer of RyR2 (∼5–40Å, Figure C). The mutations are supposed to disturb the binding affinity to the FKBP12.6 resulting in RyR2 channel instability. In channel part (3613–4968aa), 16 of 40 mutations are located near two interface. (FigureD) 12 mutations are close to the Ca2+ sensor and the other 4 mutations are adjacent to the pore-forming segment. Especially, V4821I is just located on this segment and strongly expected to impair the channel function. Above all, RyR2 3D in silico modeling revealed that 63 of all 93 (68%) identified mutations are supposed to be pathogenic. Location of RYR2 mutations in 3D model Conclusion 3D structural model of RyR2 is useful for the investigation of the pathogenic mechanisms of CPVT-related mutations. Further studies are needed to elucidate the relationship between the location of the mutations and clinical phenotypes.

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