From the Departments of Biochemistry and Biophysics
and Cell and Molecular Physiology, University of North Carolina, Chapel
Hill, North Carolina 27599-7260 and the § Howard Hughes
Medical Institute, Department of Medicine, Divisions of Pulmonary and
Cardiovascular Medicine, and Department of Biochemistry, Duke
University Medical Center, Durham, North Carolina 27710
Received for publication, January 4, 2001, and in revised form, February 13, 2001
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ABSTRACT |
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The skeletal muscle Ca2+
release channel/ryanodine receptor (RyR1) is a prototypic
redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1
subunit are kept in a reduced (free thiol) state under conditions
comparable with resting muscle. Here we assessed the effects of
physiological determinants of cellular redox state (oxygen tension,
reduced (GSH) or oxidized (GSSG) glutathione, and NO/O Ca2+ release channels/ryanodine receptors
(RyRs)1 are the largest known
ion channels, consisting of four ~565-kDa RyR subunits and four
associated 12-kDa FK506-binding protein subunits (1, 2).
Following an action potential, the cardiac and skeletal muscle RyR
isoforms release Ca2+ from an intracellular
Ca2+ storing membrane compartment, the sarcoplasmic
reticulum (SR), in a process known as excitation-contraction coupling.
Numerous endogenous effectors are known to regulate RyR function and
therefore muscle contractility. These effectors range from ions
(Ca2+ and Mg2+) to other small molecules
(adenine nucleotides) to polypeptides such as calmodulin (1, 2). Recent
work has also established RyRs as prototypic redox-sensitive ion
channels. The skeletal muscle isoform of RyR (RyR1) contains a large
number of free thiols: as many as 50 out of a total of 101 cysteine
residues/subunit (100 cysteines/RyR1 subunit (3) and 1 cysteine/FK506-binding protein subunit (4)) (5). RyR1 channel activity
is dramatically altered by redox modifications of critical thiols
(oxidation, S-nitrosylation, or alkylation) (5-14).
Conversely, RyR1 has thiols whose redox potential is dependent on
effectors that regulate RyR1 activity such as Ca2+ and
Mg2+ (15). In a physiological context, nitric oxide (NO)
and reactive oxygen species are produced in contracting muscle and have
been shown to modulate in vitro RyR redox state and channel
activity (5, 12, 16-21). Remarkably, RyR1 redox state and function are
dependent on O2 tension (5). Altering O2
tension alone dynamically reduced/oxidized as many as 6-8 thiols/RyR1
subunit and thereby modified channel responsiveness to physiological
concentrations of NO (5).
In this study, we varied reducing and oxidizing conditions to explore
in detail the effect of RyR1 redox state on channel function. At one
end of the redox spectrum, RyR1 was maintained in a highly reduced
state by GSH at low pO2 (~10 mm Hg). At the other end of the spectrum, we tested the effects of the strongly oxidizing conditions produced by high concentrations of NO/O Materials--
[3H]Ryanodine was obtained from
PerkinElmer Life Sciences; unlabeled ryanodine and monobromobimane
(mBB) from Calbiochem; SIN-1 from Molecular Probes, Inc. (Eugene, OR);
and CHAPS, leupeptin, and Pefabloc (a protease inhibitor) from Roche
Molecular Biochemicals. All other chemicals were of analytical grade.
Preparation of SR Vesicles--
"Heavy" rabbit skeletal
muscle SR membrane fractions enriched in [3H]ryanodine
binding, and Ca2+ release channel activities were prepared
in the presence of protease inhibitors (100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl
fluoride) as described (22).
Quantification of RyR1 Thiol and S-Nitrosothiol
Contents--
The number of free thiols in RyR1 was determined as
described previously (5). Briefly, skeletal SR vesicles, treated with or without SIN-1 in the absence or presence of GSH or GSSG at pO2 of ~10 or 150 mm Hg, were centrifuged
(100,000 × g) at 4 °C for 1 h. The pellets
were washed and resuspended and then probed with an excess (500 µM) of the lipophilic, thiol-specific agent mBB for
1 h in the dark at 24 °C. Following mBB treatment, SR vesicles
were solubilized with 1.5% CHAPS, and the bimane-labeled RyR1 was
isolated by sucrose density gradient centrifugation. The fluorescence
intensity of bimane (i.e. the thiol content) in the sucrose
gradient fraction most enriched in RyR1 (>95% purity) was determined
and normalized for protein concentration as described (5). The
S-nitrosothiol content of RyR1 was determined by isolating the receptor without prior mBB treatment and using a
photolysis/chemiluminescence method (5, 12).
[3H]Ryanodine Binding--
Unless otherwise
indicated, skeletal SR vesicles, treated with various concentrations of
SIN-1 in the absence or presence of GSH or GSSG at
pO2 of ~10 or 150 mm Hg, were incubated with 5 nM [3H]ryanodine at 24 °C in media
containing 0.125 M KCl, 20 mM imidazole, pH
7.0, 0.3 mM Pefabloc, 30 µM leupeptin, and
the indicated concentrations of free Ca2+. Nonspecific
binding was determined using a 1000-fold excess of unlabeled ryanodine.
After 5 h, aliquots of the samples were diluted with 20 volumes of
ice-cold water and placed on Whatman GF/B filters soaked with 2% (w/w)
polyethyleneimine. Filters were washed with three 5-ml volumes of
ice-cold buffer, and the radioactivity remaining on the filters was
determined by liquid scintillation counting to obtain bound
[3H]ryanodine.
Single Channel Recordings--
Single channel measurements were
performed by fusing skeletal SR vesicles with Mueller-Rudin type
bilayers containing phosphatidylethanolamine, phosphatidylserine, and
phosphatidylcholine in the ratio 5:3:2 (25 mg of total phospholipid/ml
n-decane) (5). The side of the bilayer to which the SR
vesicles were added was defined as the cis (cytoplasmic) side. The
trans (SR luminal) side of the bilayer was defined as ground. Single
channels were recorded in a symmetric CsCH3SO3
buffer solution (0.25 M CsCH3SO3,
10 mM Cs-HEPES, pH 7.3) containing the additions indicated
(see Fig. 4). Measurement of the sensitivity of the channels to
cytosolic Ca2+ indicated that in a majority of recordings
(>98%) the cytosolic side of the RyRs faced the cis side, and the
luminal side faced the trans side of the bilayer. Electrical signals
were filtered at 2 kHz, digitized at 10 kHz, and analyzed. Data
acquisition and analysis were performed with a commercially available
software package (pClamp 6.0.4; Axon Instruments, Burlingame, CA) with an IBM-compatible Pentium II computer and 12-bit A/D to D/A converter (Digidata 1200, Axon Instruments).
Immunoblotting--
RyR1 and nitrotyrosine contents of SR
vesicles were determined by immunoblot analysis. Samples were
solubilized in nonreducing sample buffer, containing 62.5 mM Tris-HCl, pH 6.8, 20% glycerol (w/v), 1.2% SDS, and
0.05% bromphenol blue, and loaded onto 3-15% SDS-PAGE gradient gels.
After electrophoresis, the proteins were transferred overnight to a
nitrocellulose membrane (Schleicher and Schuell). The transferred
membrane was blocked in Tris-buffered saline buffer, containing 0.05%
Tween 20, 20 mM Tris-HCl, pH 7.4, 250 mM NaCl,
and 5% nonfat milk, with agitation for 2 h at room temperature.
RyR1 and nitrotyrosine proteins were identified using D110 anti-RyR1
monoclonal antibody (23) and anti-nitrotyrosine polyclonal antibody
(Upstate Biotechnology, Inc., Lake Placid, NY), respectively. Secondary
goat anti-mouse and anti-rabbit IgG-horseradish peroxidase-linked
antibodies were used at 1:2000 dilution. Color development was
accomplished using the DAB substrate kit (Roche Molecular Biochemicals).
Other Biochemical Assays--
Free Ca2+
concentrations were obtained by including in the solutions the
appropriate amounts of Ca2+ and EGTA as determined using
the stability constants and computer program published by Shoenmakers
et al. (24). Free Ca2+ concentrations >1
µM were verified with the use of a
Ca2+-selective electrode (World Precision Instruments,
Inc.). The protein concentrations were determined by the Amido Black
method (25).
Ca2+-ATPase activity in SR vesicles was assayed by
malachite green ATPase method (26) in the presence of a
Ca2+ ionophore (1 µM ionomycin) and the
absence or presence of SIN-1. Mg2+-ATPase remaining in the
SR preparations was subtracted from the total ATPase by adding 1 mM EGTA to assay media.
Data Analysis--
Results are given as means ± S.D. with
the number of experiments in parentheses. Significance of differences
of data was analyzed with Student's t test. Differences
were regarded to be statistically significant at p < 0.05 (*) and p < 0.01 (**).
Correlation of RyR1 Redox State and Activity--
The highly
specific plant alkaloid ryanodine is widely used as a probe of RyR
channel activity because of its preferential binding to open channel
states (1, 2). In Fig. 1,
[3H]ryanodine binding to rabbit skeletal muscle SR
vesicles, and therefore RyR1 channel activity, was determined as a
function of three different redox modifiers: glutathione (5 mM GSH versus 5 mM GSSG), SIN-1
(which releases NO/O
The effects of SIN-1 on RyR1 activity were dependent on oxygen tension
and the presence of GSH or GSSG. In ambient O2 tension and
the absence of glutathione, SIN-1 activated RyR1 channel activity maximally at 0.2 mM. Higher concentrations of SIN-1 reduced
[3H]ryanodine binding ultimately back to base line (Fig.
1B). The biphasic concentration dependence indicates that
moderate amounts of NO/O
Recent studies have indicated that oxidation and
S-nitrosylation affect channel activity by altering RyR1's
interaction with calmodulin (5, 13). SR vesicles contain 0.10-0.15
calmodulin/RyR1 subunit (5). To eliminate the effects of endogenous
calmodulin, SR vesicles were pretreated with 2 µM myosin
light chain kinase-derived calmodulin binding peptide, followed by
centrifugation through a layer of 0.3 M sucrose to remove
complexed calmodulin and calmodulin binding peptide. After
centrifugation, the endogenous SR-associated calmodulin content was
reduced to ~5% of the control value, as determined by a
phosphodiesterase activation assay (5). The effects of SIN-1 on RyR1
activity, as measured by [3H]ryanodine binding under the
conditions in Fig. 1, were then studied by exposing control and
pretreated vesicles to 0, 0.2, and 1.0 mM SIN-1 at
pO2 ~150 mm Hg. An essentially identical
activation by 0.2 mM SIN-1 and inactivation by 1.0 mM SIN-1 for vesicles pretreated and not pretreated with
the calmodulin binding peptide (data not shown) indicated that SIN-1
did not transduce its effects in Fig. 1 via the small amounts of
calmodulin associated with the SR vesicles.
The oxidation or reduction of a large number of thiols is the principle
mechanism by which O2 tension and reducing agents such as
glutathione modulate RyR1 channel activity in SR vesicles. A specific
free thiol-labeling agent, mBB, was used to correlate RyR1 free thiol
content and activities in the presence of redox active molecules. As
reported previously (5), RyR1 in SR vesicles exposed to 5 mM GSH had ~48 and ~40 free thiols/subunit at
pO2 ~10 or ~150 mm Hg, respectively.
Exposure of the vesicles to variable glutathione, O2,
and/or NO/O Ca2+ Dependence of Redox Modulation of RyR1 by
SIN-1/(NO/O
The effects of SIN-1 on RyR1 activity were also explored in single
channel measurements using maximally activating concentrations of
Ca2+ (10 µM; Fig. 3) and SIN-1 (0.2 mM; Fig. 1). Skeletal SR vesicles were incorporated into
planar lipid bilayers, and RyR1 single channel activity was recorded at
ambient oxygen tension with Cs+ as the current carrier to
eliminate other ion currents also present in SR vesicles (32). In the
presence of an optimally activating Ca2+ concentration of
10 µM, channel open probability (Po) increased 2-3-fold (n = 6) after the addition of 0.2 mM SIN-1 to the cytosolic (cis) chamber of the bilayer
apparatus (Fig. 4). Thus, in agreement
with the [3H]ryanodine binding measurements, single
channel recordings show that a moderately oxidizing concentration of
SIN-1 activates the RyR1.
Mechanism of Redox Modification of RyR1 by
SIN-1/ (NO/O
Peroxynitrite oxidizes thiols reversibly to disulfide bonds or sulfenic
(SOH) acids or irreversibly to sulfinic (SO2H) or sulfonic
acids (SO3H) (33-35). The reversibility of RyR1 oxidation was determined at ambient oxygen tension from the thiol content of SR
vesicles that were first treated with 0, 0.2, or 1.0 mM SIN-1 at 24 °C for 5 h and then exposed to 5 mM GSH
(experimental group) or no reducing equivalent (control) for another
5 h. [3H]Ryanodine binding and free RyR1 thiol
content were determined after the final 5 h of incubation. The
results are summarized in Fig. 5, with
the number of free thiols per RyR1 subunit (mean ± S.D.,
n
Peroxynitrite may also S-nitrosylate free thiols in proteins
(33, 34). We have shown previously that S-nitrosylation of a
single thiol per RyR1 can activate the channel (5). To see if RyR1 is
S-nitrosylated by SIN-1, control and SIN-1 (0.2 mM)-treated samples were assayed for
S-nitrosothiol content using a photolysis/chemiluminescence method (5, 12). In the control group, there were ~0.4
S-nitrosothiol/RyR1 subunits, a level comparable with the
endogenous amount of S-nitrosylation found in our previous
study (5). SIN-1/peroxynitrite did not S-nitrosylate
any additional RyR1 thiols (Table I).
Therefore, unlike NO (5), SIN-1/peroxynitrite did not activate RyR1 by S-nitrosylation.
Peroxynitrite can also modify proteins by the addition of a nitro group
to the ortho position of tyrosine to form nitrotyrosine (33, 34). In
the case of the SR Ca2+-ATPase, in vitro
exposure of skeletal SR vesicles to peroxynitrite resulted in both
S-nitrosylation (36) and nitrotyrosine formation (37). Both
modifications contributed to inhibition of SR Ca2+-ATPase
activity. Nitrotyrosine formation could therefore represent an
additional mechanism by which SIN-1 modulates RyR1, and this possibility was examined in immunoblots. A polyclonal antibody recognizing nitrotyrosine (Fig.
6A, lanes 4-6) did
not detect any nitrotyrosine formation in the region of the blots
corresponding to RyR1 (Fig. 6A, lanes
1-3) in controls and SR vesicles exposed to 0.2 or 1.0 mM SIN-1. In contrast, the anti-nitrotyrosine antibody revealed a weak band in the control sample (lane
4) and two stronger bands in the samples treated with 0.2 mM SIN-1 (lane 5) and 1.0 mM SIN-1
(lane 6), corresponding to a protein with an apparent molecular mass of ~100 kDa. The results suggested that a 100-kDa protein such as the skeletal muscle SR Ca2+-ATPase has
endogenous nitrotyrosine(s) and that its level is increased by
exogenous SIN-1/peroxynitrite (37). In favor of this interpretation,
SIN-1 (0.2 or 1.0 mM) inhibited SR Ca2+-ATPase
activity by about ~30 and 50%, respectively (Fig.
6B).
Taken together, the results suggest that SIN-1 acts in our assay
conditions as an oxidant rather than a NO donor. In the absence of
glutathione, NO/O Contracting muscle produces reactive oxygen and nitrogen species
(16, 17, 38, 39). A functional role of these molecules is indicated by
the finding that force development in muscle is affected using
nitric-oxide synthase inhibitors and scavengers of superoxide. The data
we have presented here imply that the effects of NO/O The RyR1 is exquisitely sensitive to redox modulation. Ample evidence
indicates that RyRs are activated or inactivated or both by
sulfhydryl-modifying molecules (5-15, 18-21, 40, 41). We previously
uncovered a striking plasticity of redox state in RyR1 channel activity
(5). RyR1 free thiol content and channel activity were dynamically
controlled by GSH/GSSG, oxygen tension, and NO. Other recent studies
have shown that RyR1 responds to changes in transmembrane
glutathione redox potential (14) and contains thiols whose redox
potential is dependent on ligands (Ca2+, Mg2+)
that control RyR1 activity (15).
In this study, we used SIN-1 to probe the effect of redox state on RyR1
activity and have done so under a spectrum of redox conditions that are
encountered in muscle, by further varying GSH/GSSG and
pO2. SIN-1 spontaneously generates NO and
O Exposure to the three redox variables O2, glutathione, and
SIN-1 revealed three distinct redox states of RyR1. The free thiol content ranged from as low as 13 to as high as 48 thiols/RyR1 subunit
(SH/RyR1s) (see scheme below).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
38 free thiols/RyR1 subunit)
or oxidizing (
15 free thiols/RyR1 subunit) conditions, and 4) NO and
O
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dose-dependent effects of SIN-1
on RyR1 channel activity in low and high O2 tension.
Effects of SIN-1 on specific [3H]ryanodine binding to
skeletal SR vesicles were determined either in the absence
(circles) or presence of 5 mM GSH
(triangles) or GSSG (squares) as described under
"Experimental Procedures." A, pO2
~10 mm Hg; B, pO2 ~150 mm Hg.
Compared with each control, asterisks represent
p < 0.05, and double asterisks
represent p < 0.01.
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Fig. 2.
RyR1 channel activity is controlled by redox
state. [3H]Ryanodine binding and free thiol content
were determined as described under "Experimental Procedures" either
in the absence (circles) or presence of 5 mM GSH
(squares) or GSSG (triangles) in either
pO2 ~10 mm Hg (symbols without
cross) or ~150 mm Hg (symbols with cross) and in the
absence (open symbols) or presence of 0.2 mM
(gray symbols) or 1 mM (dark symbols)
SIN-1. [3H]Ryanodine binding was determined by incubating
SR vesicles for 1 h at 24 °C with 25 nM
[3H]ryanodine in medium containing 0.125 M KCl, 20 mM imidazole, pH 7.0, 0.3 mM Pefabloc, 30 µM leupeptin, and 10 µM free Ca2+. The free thiol content of RyR1
was determined by the mBB fluorescence method in the same conditions.
Data are the averages of at least three experiments done in duplicate
with the S.D. being less than 20% for the [3H]ryanodine
binding and less than 15% for the free thiol content
determinations.
7). Extensive oxidation by 1.0 mM
SIN-1 resulted in reduced [3H]ryanodine binding and a
broadened Ca2+ activation/inactivation profile. These
results suggest that SIN-1 activates and inactivates the RyR1, altering
the Ca2+ dependence of channel activity.
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Fig. 3.
Effects of SIN-1 on Ca2+
dependence of [3H]ryanodine binding to SR vesicles.
Vesicles were incubated for 30 min at 24 °C with either 0, 0.2, or
1.0 mM SIN-1 and then incubated for 5 h with 5 nM [3H]ryanodine at the indicated
Ca2+ concentrations. Data are the means ± S.D. of
three experiments.
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Fig. 4.
Effect of SIN-1 on RyR1 single channel
activities. Skeletal SR vesicles were fused with lipid bilayers at
pO2 ~ 150 mm Hg. A, single RyR1
channel currents, shown as downward deflections from closed
(c) levels, were recorded in symmetrical 0.25 M
CsCH3SO3, pH 7.3 buffer at a holding potential
of 35 mV. Top trace, control with 10 µM free Ca2+, Po = 0.08;
second trace, immediately after the addition of
0.2 mM SIN-1 to the cytosolic side of the bilayer,
Po = 0.16. B, quantitative
presentation of changes in Po. Values of controls
were normalized as 100%, and the changes were expressed as percentage
of the controls. Normalized Po before (open
columns) and after the addition (filled columns) of 0.2 mM SIN-1 (n = 6) is shown. Data are the
means ± S.D. of six experiments. Compared with control,
asterisks represent p < 0.05.
1 s
1)
to form peroxynitrite (27, 28). Therefore, peroxynitrite is probably a
dominant oxidative species in experiments involving SIN-1. Rapid
formation of peroxynitrite was supported by the finding that a NO
electrode with a high sensitivity (5) failed to detect any NO release
from SIN-1 in our assay conditions at either oxygen tension (data not shown).
3) labeled at the top of each
column. Channel activation and thiol oxidation by 0.2 mM SIN-1 were nearly completely reversed by 5 mM GSH. In contrast, 5 mM GSH could not reverse
the effects of 1.0 mM SIN-1 on RyR1 channel activity or
redox state (Fig. 5, third pair of
columns). These results suggest, but do not prove, that at
high concentrations of peroxynitrite, numerous RyR1 thiols (~10/RyR1
subunit) proceeded to high degrees of oxidation.
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Fig. 5.
Reversibility of SIN-1 induced changes in
RyR1 channel activity and free thiol content. SR vesicles were
treated for 5 h at 24 °C at pO2 ~150
mm Hg with 0, 0.2, or 1.0 mM SIN-1 and then incubated for
another 5 h in the absence (open columns) or presence
of 5 mM GSH (filled columns) either in the
absence (free thiol quantification) or presence of 5 nM
[3H]ryanodine ([3H]ryanodine binding). The
free thiol content (thiols per RyR1 subunit) is given above
the columns. Data are the means ± S.D. of at least
three experiments.
SNO/RyR1 and [3H]ryanodine binding with or without SIN-1
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Fig. 6.
Immunoblots and Ca2+-ATPase
activity of SR vesicles treated with SIN-1. A, SR
vesicles were incubated for 1 h at 24 °C either in the absence
(lanes 1 and 4) or presence of 0.2 mM (lanes 2 and 5) or 1.0 mM (lanes 3 and 6) SIN-1.
Proteins separated by 3-15% gradient SDS-PAGE were transferred to
nitrocellulose membranes and probed with an anti-RyR1 (lanes
1-3) or anti-nitrotyrosine (lanes
4-6) antibody. The anti-nitrotyrosine antibody did not
detect nitrotyrosines in RyR1. However, a protein with a molecular mass
of ~100 kDa was recognized by the antibody, and SIN-1 significantly
increased the level of nitrotyrosines. B, SIN-1 (0.2 or 1.0 mM) significantly decreased SR Ca2+-ATPase
activity. Data are the means ± S.D. of three experiments.
Compared with control, double asterisks represent
p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RyR1 free thiol content was high and channel activity was low in
the presence of 5 mM GSH at pO2
~10 mm Hg; i.e. there are ~48 free thiols/RyR1 subunit
under conditions encountered in normally functioning skeletal muscle.
Remarkably, oxidation of ~10 free thiols/RyR1 subunit (SIN-1 in the
presence of 5 mM GSH) had virtually no effect on RyR1
activity, suggesting that RyR1 has a large buffer capacity against
oxidants such as O
This analysis of RyR1 is reminiscent of our previous studies of RyR2
(12). In the case of RyR1, however, the oxidation of up to 25 thiols
was reversible (suggesting the formation of disulfides and/or sulfenic
acids), whereas only 5-6 thiols could be oxidized in the cardiac
channel before irreversible changes were encountered. Irreversible
thiol oxidation (suggesting oxidation to sulfinic or sulfonic acids)
was dependent on the concentration of the oxidant (1.0 mM
SIN-1) and required the absence of GSH. Although the
(patho)physiological correlative of thiol oxidation remains to be
elucidated, the finding of endogenous nitration in the SR (Ref. 37 and
this study) indicates that such oxidative modifications are likely
(i.e. thiols are generally more reactive toward
NO/O
Intracellular Ca2+ concentration, the main determinant of
skeletal muscle contractile function, is controlled by the RyR1 and an
ATP-driven Ca2+ pump, with the former releasing the stored
Ca2+ from SR to initiate contraction and the later
sequestering Ca2+ back in SR to initiate relaxation. We
previously showed that O2 tension dynamically
reduced/oxidized 6-8 thiols/RyR1 subunit. The alteration of channel
redox state determined its responsiveness to S-nitrosylation
by NO of one cysteine per RyR1 subunit, and the effect of
S-nitrosylation on channel activity was transduced via
calmodulin (5). We believe that such regulation may impact on
excitation-contraction coupling. On the other hand, we now show that
NO/O
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This
work was supported by National Institutes of Health Grants HL04053 (to J. P. E.); HL52529, ES-09206, and HL59130 (to J. J. S.); and AR18687 and HL27430 (to G. M.).
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260. Tel.: 919-966-5021; Fax: 919-966-2852; E-mail: meissner@med.unc.edu.
Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M100083200
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ABBREVIATIONS |
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The abbreviations used are:
RyR, ryanodine receptor;
RyR1, skeletal muscle isoform of RyR;
SR, sarcoplasmic reticulum;
mBB, monobromobimane;
NO, nitric oxide;
O
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