Effects of Hydroxyl Radical and Sulfhydryl Reagents on the Open Probability of the Purified Cardiac Ryanodine Receptor Channel Incorporated into Planar Lipid Bilayers

1998 ◽  
Vol 249 (3) ◽  
pp. 938-942 ◽  
Author(s):  
Kazunori Anzai ◽  
Kunitaka Ogawa ◽  
Akihiko Kuniyasu ◽  
Toshihiko Ozawa ◽  
Haruhiko Yamamoto ◽  
...  
Keyword(s):  
Hydroxyl Radical ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Planar Lipid Bilayers ◽  
Sulfhydryl Reagents ◽  
Open Probability ◽  
Receptor Channel ◽  
Cardiac Ryanodine Receptor

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.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium

1997 ◽  
Vol 272 (6) ◽  
pp. C1908-C1918 ◽  
Author(s):  
K. R. Eager ◽  
L. D. Roden ◽  
A. F. Dulhunty
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Channel Activity ◽  
Irreversible Loss ◽  
Sulfhydryl Reagents ◽  
Open Probability ◽  
Open Time ◽  
Wide Range ◽  
The Mean ◽  
Channel Open Time

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1–10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

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 (&lt;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 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 &gt; 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 &lt;0.6 pA.

scholarly journals The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor

2020 ◽  
Vol 295 (22) ◽  
pp. 7620-7634
Author(s):  
Christian Holt ◽  
Louise Hamborg ◽  
Kelvin Lau ◽  
Malene Brohus ◽  
Anders Bundgaard Sørensen ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Hydrophobic Surface ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Open Probability ◽  
Calmodulin Binding ◽  
Terminal Domain ◽  
Genes Encoding ◽  
Cardiac Ryanodine Receptor

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia–causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84–2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

scholarly journals The Cytoplasmic Region of Inner Helix S6 Is an Important Determinant of Cardiac Ryanodine Receptor Channel Gating

2016 ◽  
Vol 291 (50) ◽  
pp. 26024-26034 ◽  
Author(s):  
Bo Sun ◽  
Wenting Guo ◽  
Xixi Tian ◽  
Jinjing Yao ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Important Determinant ◽  
Channel Gating ◽  
Cytoplasmic Region ◽  
Receptor Channel ◽  
Cardiac Ryanodine Receptor

scholarly journals A Model of the Putative Pore Region of the Cardiac Ryanodine Receptor Channel

2004 ◽  
Vol 87 (4) ◽  
pp. 2335-2351 ◽  
Author(s):  
William Welch ◽  
Shana Rheault ◽  
Duncan J. West ◽  
Alan J. Williams
Keyword(s):  
Ryanodine Receptor ◽  
Pore Region ◽  
Receptor Channel ◽  
Cardiac Ryanodine Receptor

scholarly journals Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2101
Author(s):  
Samantha C. Salvage ◽  
Esther M. Gallant ◽  
James A. Fraser ◽  
Christopher L.-H. Huang ◽  
Angela F. Dulhunty
Keyword(s):  
Atrial Fibrillation ◽  
Ryanodine Receptor ◽  
Polymorphic Ventricular Tachycardia ◽  
Sodium Currents ◽  
Wild Type ◽  
Open Probability ◽  
Active Channels ◽  
Separate Activation ◽  
Low Activity ◽  
Cardiac Ryanodine Receptor

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.

Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions

2003 ◽  
Vol 285 (5) ◽  
pp. C1059-C1070 ◽  
Author(s):  
A. Zahradníková ◽  
M. Dura ◽  
I. Györke ◽  
A. L. Escobar ◽  
I. Zahradník ◽  
...  
Keyword(s):  
Steady State ◽  
Dynamic Behavior ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Heart Cells ◽  
Local Dynamic ◽  
Open Probability ◽  
Ec Coupling ◽  
Activation Rate ◽  
Cardiac Excitation

Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+ release channels by competing with Ca2+ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained (≥10-ms) elevations in Ca2+ concentration. Paradoxically, Mg2+ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca2+ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+ did not affect the peak open probability or the probability of responses to brief Ca2+ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca2+ channels and RyRs in heart cells.

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.

Sign in / Sign up

Export Citation Format

Share Document