scholarly journals Luminal Ca2+ controls activation of the cardiac ryanodine receptor by ATP

2012 ◽  
Vol 140 (2) ◽  
pp. 93-108 ◽  
Author(s):  
Barbora Tencerová ◽  
Alexandra Zahradníková ◽  
Jana Gaburjáková ◽  
Marta Gaburjáková
Keyword(s):  
Cardiac Myocytes ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Planar Lipid Bilayers ◽  
Synergic Effect ◽  
Allosteric Effect ◽  
Apparent Affinity ◽  
Ryr2 Channel ◽  
Fractional Inhibition ◽  
Cardiac Ryanodine Receptor

The synergic effect of luminal Ca2+, cytosolic Ca2+, and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose–response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca2+ concentration of 100 nM over a range of luminal Ca2+ concentrations and, vice versa, at a diastolic luminal Ca2+ concentration of 1 mM over a range of cytosolic Ca2+ concentrations. Low level of luminal Ca2+ (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca2+ (8–53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca2+ levels (<500 nM) greatly amplified the effects of luminal Ca2+. Fractional inhibition by cytosolic Mg2+ was not affected by luminal Ca2+. In models, the effects of luminal and cytosolic Ca2+ could be explained by modulation of the allosteric effect of ATP on the RYR2 channel. Our results suggest that luminal Ca2+ ions potentiate the RYR2 gating activity in the presence of ATP predominantly by binding to a luminal site with an apparent affinity in the millimolar range, over which local luminal Ca2+ likely varies in cardiac myocytes.

scholarly journals Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

2013 ◽  
Vol 142 (2) ◽  
pp. 127-136 ◽  
Author(s):  
Haiyan Chen ◽  
Giorgia Valle ◽  
Sandra Furlan ◽  
Alma Nani ◽  
Sandor Gyorke ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Contractile Function ◽  
Sudden Cardiac Arrest ◽  
Polymorphic Ventricular Tachycardia ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Model Studies ◽  
Pathological Conditions ◽  
Ryr2 Channel

Release of Ca2+ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca2+ regulation of SR Ca2+ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca2+ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca2+ regulates ryanodine receptor (RyR)2-mediated SR Ca2+ release through mechanisms localized inside the SR; one of these involves luminal Ca2+ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca2+] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca2+ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca2+ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca2+ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.

scholarly journals Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

1999 ◽  
Vol 113 (2) ◽  
pp. 177-186 ◽  
Author(s):  
Rafael Mejía-Alvarez ◽  
Claudia Kettlun ◽  
Eduardo Ríos ◽  
Michael Stern ◽  
Michael Fill
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Monovalent Cation ◽  
Planar Lipid Bilayers ◽  
Physiological Conditions ◽  
Single Channel Currents ◽  
The Relationship ◽  
Channel Currents ◽  
Cardiac Ryanodine Receptor

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2–30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

Ryanodine receptor dysfunction in porcine stunned myocardium

1997 ◽  
Vol 273 (2) ◽  
pp. H796-H804 ◽  
Author(s):  
C. Valdivia ◽  
J. O. Hegge ◽  
R. D. Lasley ◽  
H. H. Valdivia ◽  
R. Mentzer
Keyword(s):  
Coronary Artery ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Myocardial Stunning ◽  
Single Channel ◽  
Stunned Myocardium ◽  
Wall Thickening ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

scholarly journals Unitary Ca2+ Current through Mammalian Cardiac and Amphibian Skeletal Muscle Ryanodine Receptor Channels under Near-physiological Ionic Conditions

2003 ◽  
Vol 122 (4) ◽  
pp. 407-417 ◽  
Author(s):  
Claudia Kettlun ◽  
Adom González ◽  
Eduardo Ríos ◽  
Michael Fill
Keyword(s):  
Skeletal Muscle ◽  
Charge Carrier ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Skeletal Muscles ◽  
Planar Lipid Bilayers ◽  
High Concentrations

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1–30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (∼55%) in mammalian and amphibian channels. Two amplitudes, differing by ∼35%, were found in amphibian channel studies, probably corresponding to α and β RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (∼40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

scholarly journals Coupled Gating of Skeletal Ryanodine Receptor Channels - a Summary of Two Decades of Studies in Planar Lipid Bilayers

2012 ◽  
Vol 102 (3) ◽  
pp. 307a
Author(s):  
Paula L. Diaz-Sylvester ◽  
Jacob T. Neumann ◽  
Maura Porta ◽  
Alma Nani ◽  
Ariel L. Escobar ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Planar Lipid Bilayers ◽  
Coupled Gating

scholarly journals Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

2008 ◽  
Vol 294 (4) ◽  
pp. C1103-C1112 ◽  
Author(s):  
Paula L. Diaz-Sylvester ◽  
Maura Porta ◽  
Julio A. Copello
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Genetic Disorder ◽  
Ryanodine Receptors ◽  
Volatile Anesthetic ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Highly Sensitive

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca2+ release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs+ solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca2+ release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca2+) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca2+ (channel agonist) and counteracted by Mg2+ (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca2+ release is enhanced by high ATP-low Mg2+ in the cytosol and by increased SR Ca2+ load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

scholarly journals Imaging Single Cardiac Ryanodine Receptor Ca2+ Fluxes in Lipid Bilayers

2004 ◽  
Vol 86 (1) ◽  
pp. 134-144 ◽  
Author(s):  
S. Peng ◽  
N.G. Publicover ◽  
G.J. Kargacin ◽  
D. Duan ◽  
J.A. Airey ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Cardiac Ryanodine Receptor

Abstract 795: Enhanced Sensitivity Of The Cardiac Ryanodine Receptor To Activation By Luminal Ca 2+ As A Primary Cause Of Catecholaminergic Polymorphic Ventricular Tachycardia

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hitoshi Uchinoumi ◽  
Masafumi Yano ◽  
Makoto Ohno ◽  
Xiaojuan Xu ◽  
Hiroki Tateishi ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Underlying Mechanism ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Enhanced Sensitivity ◽  
Histological Abnormality ◽  
Ryr2 Channel ◽  
The Relationship ◽  
Cardiac Ryanodine Receptor

Mutations in cardiac ryanodine receptor (RyR2) was found to be linked with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of CPVT, we developed knock-in mice harboring the Arg-to-Ser (R2474S) mutation. The RyR2 R2474S/+ knock-in (KI) mice revealed no structural or histological abnormality in hearts. Echocardiography showed no contractile or relaxation dysfunction at rest. In all KI mice (n=6), bidirectional ventricular tachycardia (VT) was observed during or after exercise with treadmill, but never observed in wild-type (WT) mice (n=6). In intact cardiomyocytes, the frequency of Ca 2+ sparks (SpF; s −1 ·100μm −1 ) was significantly increased in KI mice, but not in WT mice (at 2 mM [Ca 2+ ]; KI:6.4±0.7, WT:0.9±0.08, p<0.01). To investigate the sensitivity of the RyR2 channel to activation by luminal Ca 2+ {[Ca 2+ ] in sarcoplasmic reticulum (SR)}, we measured cytoplasmic [Ca 2+ ] ([Ca 2+ ] C ) and luminal [Ca 2+ ] ([Ca 2+ ] L ) simultaneously in saponin-permeabilized cardiomyocytes, using Rhod-2 and Fluo-5N AM as Ca 2+ indicators, respectively. When [Ca 2+ ] C was buffered at 100 nM (by 1 mM EGTA), the spontaneous Ca 2+ sparks were frequently observed both in KI and WT cardiomyocyts (SpF: KI:22.1±0.9, WT:22.0±0.8, p=ns). When we added thapsigargin (1 μM) to the cardiomyocytes under this condition ([Ca 2+ ] C =100 nM), both SpF and [Ca 2+ ] L gradually decreased due to a decrease in SR Ca 2+ content caused by an inhibition of SR Ca 2+ ATPase. The relationship curve between SpF and [Ca 2+ ] L (SpF -[Ca 2+ ] L ) during the addition of thapsigargin was markedly shifted to the left in KI cardiomyocytes compared to WT cardiomyocytes, thereby lowering the threshold of [Ca 2+ ] L to induce Ca 2+ sparks to approximately one-fifth in KI cardiomyocytes. In conclusion, the enhanced sensitivity of the RyR2 channel to activation by [Ca 2+ ] L : i.e. decreased threshold [Ca 2+ ] L to induce spontaneous Ca 2+ release, may be a primary cause of CPVT.

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