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.

H2O2 and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

2000 ◽  
Vol 279 (5) ◽  
pp. C1366-C1374 ◽  
Author(s):  
Toshiharu Oba ◽  
Tatsuya Ishikawa ◽  
Takashi Murayama ◽  
Yasuo Ogawa ◽  
Mamoru Yamaguchi
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Physiological Role ◽  
Channel Activity ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Skeletal Muscle Function ◽  
Release Channel

We examined the effect of low concentrations of H2O2 on the Ca2+-release channel/ryanodine receptor (RyR) to determine if H2O2 plays a physiological role in skeletal muscle function. Sarcoplasmic reticulum vesicles from frog skeletal muscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle were incorporated into lipid bilayers. Channel activity of the frog RyR was not affected by application of 4.4 mM (0.02%) ethanol. Open probability ( P o) of such ethanol-treated RyR channels was markedly increased on subsequent addition of 10 μM H2O2. Increase of H2O2to 100 μM caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 μM H2O2-treated RyRs activated channel activity. Exposure to 10 or 100 μM H2O2 alone, however, failed to increase P o. Synergistic action of ethanol and H2O2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H2O2 at 100–500 μM had no effect on purified channel activity. Application of FKBP12 to the purified RyR1 drastically decreased channel activity but did not alter the effects of ethanol and H2O2. These results suggest that H2O2 may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.

Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 272 (2) ◽  
pp. C622-C627 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
M. Yamaguchi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Skeletal Muscles ◽  
Single Channel ◽  
Channel Activity ◽  
Electrical Parameters ◽  
Open Probability ◽  
Release Channel ◽  
Channel Current ◽  
Open Time

When sarcoplasmic reticulum (SR) vesicles prepared from frog skeletal muscles were actively loaded with Ca2+, pretreatment of the SR with 2.2 mM (0.01%) ethanol for 30 s significantly potentiated 5 mM caffeine-induced release of Ca2+ from 16.7 +/- 3.7 nmol/mg protein in control without ethanol to 28.0 +/- 2.6 nmol/mg (P < 0.05, n = 5). Ethanol alone caused no release of Ca2+ from the SR. Exposure of the Ca2+-release channel, incorporated into planar lipid bilayers, to 2 mM caffeine significantly increased open probability (Po) and mean open time, but unitary conductance was not affected. Ethanol (2.2 mM) enhanced caffeine-induced Ca2+-release channel activity, with Po reaching 3.02-fold and mean open time 2.85-fold the values in the absence of ethanol. However, ethanol alone did not affect electrical parameters of single-channel current, over a concentration range of 2.2 mM (0.01%) to 217 mM (1%). The synergistic action of ethanol and caffeine on the channel activity could be attributable to enhancement of caffeine-induced release of Ca2+ from the SR vesicles in the presence of ethanol.

scholarly journals Calsequestrin: more than ‘only’ a luminal Ca2+ buffer inside the sarcoplasmic reticulum

1998 ◽  
Vol 337 (1) ◽  
pp. 19-22 ◽  
Author(s):  
Csaba SZEGEDI ◽  
Sándor SÁRKÖZI ◽  
Anke HERZOG ◽  
István JÓNA ◽  
Magdolna VARSÁNYI
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Striated Muscle ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Open Probability ◽  
Physiological Event ◽  
Contraction Coupling ◽  
Open Time ◽  
The Mean

In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation–contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10-3–10-4 M in the SR lumen; in skeletal-muscle SR, it is mainly calsequestrin. Here we show that, depending on its phosphorylation state, calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated calsequestrin regulates Ca2+ release from the SR. Because calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of calsequestrin would appear to be a quintessential physiological event.

Single channel recordings of Nt- and L-type Ca2+ currents in rat neurohypophysial terminals

1993 ◽  
Vol 70 (4) ◽  
pp. 1617-1628 ◽  
Author(s):  
X. Wang ◽  
S. N. Treistman ◽  
J. R. Lemos
Keyword(s):  
Time Constant ◽  
Voltage Pulse ◽  
Single Channel ◽  
Single Channels ◽  
Open Probability ◽  
Time Constants ◽  
Channel Current ◽  
Open Time ◽  
The Mean ◽  
Channel Open Time

1. Ca2+ currents through single channels in acutely dissociated nerve terminals from rat neurohypophyses were recorded using cell-attached patch recordings with 110 mM Ba2+ as the charge carrier. 2. One type (Nt, where the t denotes terminal) of single Ca2+ channel current was evoked only by depolarizing steps from holding potentials less negative than -50 mV. Because this channel opened primarily at the beginning of a 180-ms-long voltage pulse, the averaged ensemble current decayed rapidly (approximately 30 ms). Infrequently, the channel opened throughout such a long pulse, resulting in a long-lasting averaged ensemble current. The averaged channel open time constant (tau) was 0.34 ms and the two averaged closed time constants were 1.78 (tau 1) and 86.57 (tau 2) ms. The mean unitary slope conductance for this channel was 11 pS and its threshold for activation was approximately -10 mV. 3. The other type (L) of single Ca2+ channel current could be evoked in isolation by depolarizations from holding potentials more positive than or equal to -50 mV. This channel opened throughout an entire 180-ms-long voltage pulse. The averaged ensemble current, therefore, showed little inactivation. The averaged channel open-time constant was 0.49 ms and the two average closed time constants were 2.02 (tau 1) and 79.91 (tau 2) ms. The mean unitary slope conductance for this channel was 25 pS. 4. Bay K 8644 (5 microM), a dihydropyridine (DHP) Ca2+ channel agonist, increased the open probability of the larger-conductance L-type Ca2+ channel by prolonging the average duration (to 2.79 ms) of channel openings, but did not alter the single channel slope conductance. In contrast, the same concentration of Bay K 8644 did not affect the smaller-conductance Nt-type Ca2+ channel. The DHP Ca2+ channel antagonist nicardipine (5 microM), but not nifedipine (5 microM), reduced the open probability of the large-conductance L-type Ca2+ channel by shortening the duration (to 0.36 ms) of channel openings. 5. The voltage- and time-dependent properties of these two types of single Ca2+ channel currents are in close agreement with those of the two components of macroscopic Ca2+ currents previously reported using the "whole-terminal" recording method. Therefore these two types of single channels appear to underlie the macroscopic current. 6. Our studies suggest that the terminal Nt-type Ca2+ channel differs from the conventional somatic N- and T-type Ca2+ channels in some respects, and that the terminal L-type Ca2+ channel is similar to the conventional somatic L-type Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)

U46619, a thromboxane A2 agonist, inhibits KCa channel activity from pig coronary artery

1992 ◽  
Vol 262 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
F. S. Scornik ◽  
L. Toro
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Lipid Bilayers ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Open Probability ◽  
Control Value ◽  
Kca Channels ◽  
Internal Side

Thromboxane A2 (TxA2) is a potent vasoconstrictor derived from the metabolism of arachidonic acid. Because potassium channels are involved in the contraction of vascular smooth muscle, their blockade could contribute to the TxA2-induced contraction. To test this possibility, we studied the effect of the TxA2 stable analogue U46619 on calcium-activated potassium (KCa) channels from coronary artery reconstituted into lipid bilayers. Addition of U46619 (50-150 nM) to the external but not to the internal side of the channel decreased the channel open probability (Po) between 15 and 80% of the control value. The inhibitory effect of U46619 affected both the open and closed states of the channel and could be reversed by internal calcium. Thromboxane B2, the inactive hydrolysis derivative of TxA2, did not affect channel activity. SQ 29548, a TxA2 receptor antagonist, was able to prevent the inhibition by U46619. Furthermore, SQ 29548 added after U46619 could restore channel activity to near control values. These results suggest that TxA2 could be a regulatory factor of KCa channels from coronary smooth muscle and that this regulation could be related to its action as a vasoconstrictor.

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 Interdomain Interactions within Ryanodine Receptors Regulate Ca2+ Spark Frequency in Skeletal Muscle

2001 ◽  
Vol 119 (1) ◽  
pp. 15-32 ◽  
Author(s):  
Alexander Shtifman ◽  
Christopher W. Ward ◽  
Takeshi Yamamoto ◽  
Jianli Wang ◽  
Beth Olbinski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Laser Scanning ◽  
Muscle Fibers ◽  
Mammalian Skeletal Muscle ◽  
Open Probability ◽  
Release Channel ◽  
Pronounced Increase ◽  
Open Time ◽  
Interdomain Interactions

DP4 is a 36-residue synthetic peptide that corresponds to the Leu2442-Pro2477 region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca2+ release events (Ca2+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca2+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca2+ release channel(s) generating the sparks. DP4 also increased [3H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca2+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg17 to Cys17 replacement (DP4mut), which corresponds to the Arg2458-to-Cys2458 mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks.

Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Niels Voigt ◽  
Ange Maguy ◽  
Yung-Hsin Yeh ◽  
Xiao-Yan Qi ◽  
Ursula Ravens ◽  
...  
Keyword(s):  
Atrial Tachycardia ◽  
Single Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Direct Demonstration ◽  
Open Time ◽  
Therapy Target ◽  
Channel Properties ◽  
Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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