SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

2003 ◽  
Vol 285 (1) ◽  
pp. C119-C128 ◽  
Author(s):  
Ricardo Bull ◽  
Juan José Marengo ◽  
José Pablo Finkelstein ◽  
María Isabel Behrens ◽  
Osvaldo Alvarez
Keyword(s):  
Rat Brain ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Redox State ◽  
Conduction Mechanism ◽  
Brain Cortex ◽  
Single Channel ◽  
Noise Analysis ◽  
Maximal Activation ◽  
Subconductance States

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times ( Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 μM cytoplasmic [Ca2+]. At 10 μM [Ca2+], low- Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation ( KaATP) = 422 μM] than moderate- Po channels ( KaATP = 82 μM). Oxidation of low- Po channels with thimerosal or 2,2′-dithiodipyridine (DTDP) gave rise to moderate- Po channels and decreased KaATP from 422 to 82 μM. At 0.1 μM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low- Po channels. After oxidation to high- Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low- Po channels at 10 μM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode.

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

2007 ◽  
Vol 293 (1) ◽  
pp. C162-C171 ◽  
Author(s):  
Ricardo Bull ◽  
José Pablo Finkelstein ◽  
Alexis Humeres ◽  
María Isabel Behrens ◽  
Cecilia Hidalgo
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Brain ◽  
High Activity ◽  
Lipid Bilayers ◽  
Redox State ◽  
Brain Cortex ◽  
Moderate Activity ◽  
Rat Brain Cortex ◽  
Channel Inhibition ◽  
Living Neurons

Despite their relevance for neuronal Ca2+-induced Ca2+ release (CICR), activation by Ca2+ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg2+], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg2+], and RyR redox state on the Ca2+ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg2+] up to 1 mM inhibited vesicular [3H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg2+ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca2+ dependencies, defined by low, moderate, or high maximal fractional open time (Po), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca2+] for activation, increased maximal Po, and shifted channel inhibition to higher [Ca2+]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg2+] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg2+] induced a right shift in Ca2+ dependence for all channels so that [Ca2+] <30 μM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca2+ at physiological [ATP] and [Mg2+]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.

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.

Opening of large-conductance calcium-activated potassium channels by the substituted benzimidazolone NS004

1994 ◽  
Vol 71 (5) ◽  
pp. 1873-1882 ◽  
Author(s):  
M. C. McKay ◽  
S. I. Dworetzky ◽  
N. A. Meanwell ◽  
S. P. Olesen ◽  
P. H. Reinhart ◽  
...  
Keyword(s):  
Rat Brain ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Outward Current ◽  
Calcium Sensitivity ◽  
Bk Channels ◽  
Gh3 Cells ◽  
Xenopus Laevis Oocytes ◽  
Planar Lipid Bilayers ◽  
Whole Cell

1. We used electrophysiological techniques to examine the effects of 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidaz ole- 2-one (NS004) on large-conductance calcium-activated potassium (BK) channels. 2. We used recordings from excised membrane patches (cell-attached and inside-out single-channel configurations) and whole-cell patch-clamp recordings to examine the effects of NS004 on single BK channels and whole-cell outward currents, respectively, in rat GH3 clonal pituitary tumor cells. We also tested NS004 on voltage-clamped BK channels isolated from rat brain plasma membrane preparations and reconstituted into planar lipid bilayers. Finally, we used two-electrode voltage-clamp techniques to study the effects of NS004 on currents expressed in Xenopus laevis oocytes by the recently described Slo BK clone from Drosophila. 3. In GH3 cells and in Xenopus oocytes expressing the Slo gene product NS004 produced an increase in an iberiotoxin- or tetraethylammonium-sensitive whole-cell outward current, respectively. NS004 produced a significant increase in the activity of single GH3 cell BK channels and rat brain BK channels reconstituted into planar lipid bilayers. In both systems this was characterized by an increase in channel mean open time, a decrease in interburst interval, and an apparent increase in channel voltage/calcium sensitivity. 4. These data indicate that NS004 could be useful for investigating the biophysical and molecular properties of BK channels and for determining the functional consequences of the opening of BK channels.

scholarly journals Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

1998 ◽  
Vol 111 (5) ◽  
pp. 679-690 ◽  
Author(s):  
Ashutosh Tripathy ◽  
Wolfgang Resch ◽  
Le Xu ◽  
Hector H. Valdivia ◽  
Gerhard Meissner
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Binding Site ◽  
Lipid Bilayers ◽  
Rate Constant ◽  
Single Channel ◽  
Voltage Drop ◽  
Ryanodine Receptors ◽  
Subconductance States ◽  
Single Channel Currents

Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTxa binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.

Ryanodine receptor function in newborn rat heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2527-H2540 ◽  
Author(s):  
Claudia G. Pérez ◽  
Julio A. Copello ◽  
Yanxia Li ◽  
Kimberly L. Karko ◽  
Leticia Gómez ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Functional Properties ◽  
Rat Heart ◽  
Single Channel ◽  
Binding Assays ◽  
Adult Rats ◽  
Intracellular Ca ◽  
Cardiac Excitation

The role of ryanodine receptor (RyR) in cardiac excitation-contraction (E-C) coupling in newborns (NB) is not completely understood. To determine whether RyR functional properties change during development, we evaluated cellular distribution and functionality of sarcoplasmic reticulum (SR) in NB rats. Sarcomeric arrangement of immunostained SR Ca2+-ATPase (SERCA2a) and the presence of sizeable caffeine-induced Ca2+ transients demonstrated that functional SR exists in NB. E-C coupling properties were then defined in NB and compared with those in adult rats (AD). Ca2+ transients in NB reflected predominantly sarcolemmal Ca2+ entry, whereas the RyR-mediated component was ∼13%. Finally, the RyR density and functional properties at the single-channel level in NB were compared with those in AD. Ligand binding assays revealed that in NB, RyR density can be up to 36% of that found in AD, suggesting that some RyRs do not contribute to the Ca2+ transient. To test the hypothesis that RyR functional properties change during development, we incorporated single RyRs into lipid bilayers. Our results show that permeation and gating kinetics of NB RyRs are identical to those of AD. Also, endogenous ligands had similar effects on NB and AD RyRs: sigmoidal Ca2+ dependence, stronger Mg2+-induced inhibition at low cytoplasmic Ca2+ concentrations, comparable ATP-activating potency, and caffeine sensitivity. These observations indicate that NB rat heart contains fully functional RyRs and that the smaller contribution of RyR-mediated Ca2+ release to the intracellular Ca2+ transient in NB is not due to different single RyR channel properties or to the absence of functional intracellular Ca2+ stores.

scholarly journals Molecular Basis of Ca2+ Activation of the Mouse Cardiac Ca2+ Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 118 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pin Li ◽  
S.R. Wayne Chen
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Single Channel ◽  
Single Point ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Single Channels ◽  
Wild Type ◽  
Structural And Functional Properties

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor

2000 ◽  
Vol 351 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Salim M. HAYEK ◽  
Xinsheng ZHU ◽  
Manjunatha B. BHAT ◽  
Jiying ZHAO ◽  
Hiroshi TAKESHIMA ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Expression System ◽  
Binding Assays ◽  
Release Channel ◽  
Important Region ◽  
Functional Consequences ◽  
Increased Sensitivity ◽  
Regulatory Functions

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872–1923, is involved in Ca 2+-dependent regulation of the Ca2+-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca2+ exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentrations respectively. To characterize the role of D3 in the Ca2+-dependent regulation of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (∆D3-RyR1) using a heterologous expression system, [3H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-dependent regulation of RyR1, but did not alter [3H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca2+-dependence curve of ∆D3-RyR1 is broader, reflecting increased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inactivation. In addition, ∆D3-RyR1 was more resistant to inhibition by Mg2+. Comparison of the effect of caffeine on wt-RyR1 and ∆D3-RyR1 suggested that D3 is an important region of RyR that participates in Ca2+-dependent activation and inactivation of the Ca2+-release channel.

scholarly journals Ischemia Enhances Activation by Ca2+ and Redox Modification of Ryanodine Receptor Channels from Rat Brain Cortex

2008 ◽  
Vol 28 (38) ◽  
pp. 9463-9472 ◽  
Author(s):  
R. Bull ◽  
J. P. Finkelstein ◽  
J. Galvez ◽  
G. Sanchez ◽  
P. Donoso ◽  
...  
Keyword(s):  
Rat Brain ◽  
Ryanodine Receptor ◽  
Brain Cortex ◽  
Rat Brain Cortex

scholarly journals Reversible Block of the Calcium Release Channel/Ryanodine Receptor by Protamine, a Heparin Antidote

2000 ◽  
Vol 11 (7) ◽  
pp. 2213-2219 ◽  
Author(s):  
Peter Koulen ◽  
Barbara E. Ehrlich
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Single Channel ◽  
Reversible Inhibitor ◽  
Channel Activity ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Mouse Muscle ◽  
Cytoplasmic Side

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C2C12 mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.

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