From the Department of Biochemistry and Biophysics,
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 the
Department of Biochemistry, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, November 22, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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The skeletal muscle Ca2+
release channel/ryanodine receptor (RyR1) contains ~50 thiols per
subunit. These thiols have been grouped according to their
reactivity/responsiveness toward NO, O2, and glutathione,
but the molecular mechanism enabling redox active molecules to modulate
channel activity is poorly understood. In the case of NO, very low
concentrations (submicromolar) activate RyR1 by
S-nitrosylation of a single cysteine residue
(Cys-3635), which resides within a calmodulin binding domain.
S-Nitrosylation of Cys-3635 only takes place at
physiological tissue O2 tension (pO2;
i.e. ~10 mm Hg) but not at pO2 ~150 mm Hg.
Two explanations have been offered for the loss of RyR1 responsiveness
to NO at ambient pO2, i.e. Cys-3635 is oxidized
by O2 versus O2 subserves an
allosteric function (Eu, J. P., Sun, J. H., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509).
Here we report that the NO donors NOC-12 and
S-nitrosoglutathione both activate RyR1 by release of NO
but do so independently of pO2. Moreover, NOC-12 activates
the channel by S-nitrosylation of Cys-3635 and thereby
reverses channel inhibition by calmodulin. In contrast, S-nitrosoglutathione activates RyR1 by oxidation and
S-nitrosylation of thiols other than Cys-3635 (and
calmodulin is not involved). Our results suggest that the effect of
pO2 on RyR1 S-nitrosylation is exerted through
an allosteric mechanism.
The large, homotetrameric skeletal muscle Ca2+ release
channel/ryanodine receptor
(RyR1)1 contains several
classes of regulatory thiols. These classes are distinguished by
reactivity or responsiveness to O2 tension (pO2) (1, 2), redox active molecules such as glutathione (3) and nitric oxide (NO) (1), transmembrane glutathione redox
potential (4), and allosteric effector molecules (Ca2+,
Mg2+) (5). It has recently been shown that cysteine 3635, which is localized to the calmodulin (CaM) binding domain of RyR1
(6-8), confers responsiveness to NO. In contrast, the identities of
the remaining regulatory thiols are not known. NO forms a covalent bond
with the thiol group of Cys-3635 (i.e.
S-nitrosylation) in vivo and thereby reverses the
inhibitory effect of CaM on the channel (6). Full-length RyR1 channels
with an alanine residue substituted for Cys-3635 are not
S-nitrosylated by physiological concentrations of NO, and
channel activity is unaffected by NO. S-nitrosylation of
Cys-3635 only occurs at low O2 tension (pO2 ~10 mm Hg, comparable with that found in skeletal muscle in
vivo) (1, 6). At this pO2, 6-8 (of ~50) thiols per
RyR1 subunit are actively maintained in the reduced state (1). Thus,
one explanation for the failure of NO to S-nitrosylate RyR1
at ambient pO2 is that Cys-3635 is oxidized. An alternative
possibility is that the oxidation of pO2-sensitive thiols
leads to a change in channel conformation; in this state
S-nitrosylation of Cys-3635 is unfavorable. Alternatively
stated, O2 is either serving as an oxidant (of Cys-3635) or
as an allosteric effector (of Cys-3635 reactivity).
NO donors, compounds capable of donating NO and redox active forms
thereof, are widely used to mimic the effects of NO synthase (9). A
number of these compounds are capable of modulating RyR1 activity (1,
10-15). RyR1 contains a large number of reactive thiols (1, 2), and
the action of NO donors may differ widely depending on the mechanisms
and rates of NO release, the chemistry of NO group transfer, the base
structure of the NO donor compound, and the reactivity of substrate
thiol. In particular, members of the S-nitrosothiol (SNO)
class of NO donors can modulate protein function by transnitrosylation
as well as NO release (16, 17). In contrast, the NONOate class of NO
donors is thought to be less susceptible to transnitrosylation
chemistry (18). It is important to note, however, that NONOate
compounds may directly interact with proteins through polyamine
recognition sites and/or through ionic interactions.
In the present study, we examined the activation of the skeletal muscle
Ca2+ release channel by NOC-12 and GSNO, an endogenous
S-nitrosothiol, and compared their effects to solutions of NO. We found
that both NOC-12 and GSNO activated RyR1 independently of
O2 tension and that the NO scavenger, C-PTIO, blocked the
effects of both. But whereas NOC-12 mediated its effects by
S-nitrosylation of a single cysteine (Cys-3635), GSNO
activation involved the S-nitrosylation and oxidation of
multiple thiols. Moreover, Cys-3635 was not required for activation by
GSNO. Thus, NO, NOC-12, and GSNO activate the prototypic
redox-sensitive RyR1 channel by different mechanisms, and the effect of
O2 tension on S-nitrosylation by NO is best rationalized by an allosteric mechanism.
Materials--
[3H]Ryanodine was a product of
PerkinElmer Life Sciences. CaM was obtained from Sigma. NO
donors, monobromobimane, myosin light chain kinase-derived CaM binding
peptide and anti-S-nitrosocysteine polyclonal antibody were
from Calbiochem, and leupeptin and Pefabloc (protease inhibitors) were
from Roche Molecular Biochemicals. An ECL detection reagent kit was
from Amersham Biosciences. NO gas (purity >99%, National Welders) was
scrubbed to remove O2 and nitrite by passing through an
argon-purged column filled with KOH pellets and then a solution of
NaOH. The concentration of NO was determined by a hemoglobin titration
assay and an NO electrode (WPI Instruments) as described (1). All other
chemicals were of analytical grade.
Sample Preparations--
Skeletal muscle sarcoplasmic reticulum
(SR) vesicles enriched in RyR1 were prepared from rabbit skeletal
muscle in the presence of protease inhibitors (19). The construction
and expression of wild type (WT) and C3635A mutant RyR1s have been
described (6). WT and C3635A RyR1s were expressed in HEK293 cells, and crude membrane fractions were prepared as described (6).
Quantification of RyR1 Free Thiols and S-Nitrosothio1s--
RyR1
free thiol (SH) and SNO contents were determined by the monobromobimane
fluorescence method and a photolysis/chemiluminescence-based NO
detection assay, respectively (1).
Electrophoresis and Detection of S-Nitrosocysteine on Western
Blots--
All procedures were performed under non-reducing conditions
(6). Membranes were incubated in 0.125 M KCl, 20 mM imidazole, pH7.0, and 8 µM free
Ca2+ for 1 h at 24 °C in room air in the absence
and presence of NOC-12 or GSNO. Protein samples were separated by
3-20% SDS-PAGE under non-reducing conditions and transferred to
polyvinylidene difluoride membranes. The membranes were blotted
with 5% nonfat milk in 0.05% Tween 20 phosphate-buffered 0.1 M saline solution at 24 °C for 2 h and probed with
anti-S-nitrosocysteine polyclonal antibody (Calbiochem;
1:500) and secondary peroxidase-conjugated anti-rabbit IgG antibody
(Calbiochem; 1:2000). Anti-S-nitrosocysteine signals were
detected with an ECL kit (Amersham Biosciences). After that, the
membranes were re-probed with anti-RyR1 monoclonal antibody D110 (1:10)
and peroxidase-conjugated anti-mouse IgG (Calbiochem, 1:2000) using the
ECL detection method.
[3H]Ryanodine Binding--
Functional effects of
NO donors were determined in [3H]ryanodine binding
measurements as described (1). The assay conditions of
[3H]ryanodine binding are indicated in the legends to
Figs. 1, 2, and 4.
Single Channel Recordings--
Single channel measurements were
performed at room air by fusing RyR1-containing membrane fractions with
Mueller-Rudin-type bilayers containing phosphatidylethanolamine,
phosphatidylserine, and phosphatidylcholine in the ratio 5:3:2 (25 mg
of total phospholipid per milliliter of n-decane) (1, 20).
The side of the bilayer to which the RyR1-containing membrane fractions
were added was defined as the cis (cytoplasmic) side. The
trans (lumenal) side of the bilayer was defined as ground.
Single channels were recorded in the buffer solutions given in the
legends to Figs. 3 and 5. Measurement of the sensitivity of the
channels to cytosolic Ca2+ indicated that in a majority of
recordings (>98%) the cytosolic side of RyR1 faced the cis
side and the lumenal side faced the trans side of the
bilayer. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed with a commercially available software package
(pClamp 8.2, Axon Instruments, Foster City, CA). Po values in multichannel recordings were
calculated using the equation Po = 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 Schoenmakers
et al. (21). Free Ca2+ concentrations were
verified with the use of a Ca2+ selective electrode. The
protein concentrations were determined by the Amido Black method
(22).
Data Analysis--
Results are given as means ± S.D.
unless otherwise indicated. 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.
Release of NO by NOC-12 and GSNO--
NOC-12 releases two NO
molecules per donor (23), whereas GSNO releases only one (24). An NO
electrode was used to characterize the peak concentrations and the
durations of NO release under conditions employed in the
[3H]ryanodine binding measurements. NOC-12 (0.1 mM) and GSNO (0.1 mM) attained peak
concentrations of 2.6 ± 0.4 µM and 1.4 ± 0.3 µM (n = 3 each) with half-life times of
~6.5 and 2.8 h, respectively (Table
I). In a majority of the experiments, we
matched the NO peak concentrations by comparing the groups treated with
0.1 mM NOC-12 with those treated with 0.2 mM
GSNO. There was no difference in peak concentrations of NO released by
either donor as a function of pO2 (pO2 ~10 mm
Hg versus ~150 mm Hg) (data not shown). The half-life time
of NO was ~10 min.
O2 Tension-independent Modulation of RyR1 by NOC-12 and
GSNO--
Modulation of RyR1 by NO is O2 tension
dependent; only at a pO2 comparable with that found in
skeletal muscle in vivo (pO2 ~10 mm Hg) can
physiological amounts of NO (submicromolar) S-nitrosylate and activate RyR1 (1). In Fig. 1, SR
vesicles were treated with increasing concentrations of NO, NOC-12, or
GSNO, and RyR1 activities were determined by
[3H]ryanodine binding at pO2 ~10 mm Hg
(Fig. 1A) or at pO2 ~150 mm Hg (Fig.
1B). Ryanodine is a highly specific plant alkaloid that is
widely used as a probe of channel activity because of its preferential
binding to the open channel states (25, 26). As shown previously (1),
only at pO2 ~10 mm Hg did NO (1-10 µM)
cause a significant increase in [3H]ryanodine binding
(Fig. 1A). Elevated levels of NO were inhibitory at
pO2 ~10 mm Hg. In striking contrast, NOC-12 and GSNO
activated RyR1 channel activity at either O2 tension.
Control experiments showed that NOC-12 and GSNO left to incubate for
48 h at room air (i.e. spent compounds) were without
effect on RyR1 channel activity (data not shown). Under both
O2 tensions, NOC-12 concentrations higher than 0.1 mM caused a slight decrease in [3H]ryanodine
binding, whereas GSNO concentrations higher than 0.2 mM
further increased [3H]ryanodine binding. Thus, in
contrast to NO, both NOC-12- and GSNO-activation of RyR1 is independent
of pO2 (over a wide range of NO donor concentrations).
Modulation of RyR1 by NOC-12 Is Dependent on CaM, whereas GSNO
Modulation Is Not--
The functional effects of
S-nitrosylation of RyR1 Cys-3635 by NO are
CaM-dependent (1, 6). At [Ca2+] >1
µM, the Ca2+-bound form of CaM (CaCaM)
inhibits RyR1, whereas at [Ca2+] <1 µM the
Ca2+-free form of CaM (apoCam) activates the receptor (27).
We therefore assessed the effects of the NO donors on
[3H]ryanodine binding in the presence or absence of both
the Ca2+-bound form of CaM and the Ca2+-free
form of CaM. Sequestration of endogenous CaM with a CaM binding peptide
(28) caused an increase in RyR1 channel activity over control at 8 µM free Ca2+ (Fig.
2A) and a decrease at 0.3 µM free Ca2+ (Fig. 2B). NOC-12
caused an increase in [3H]ryanodine binding in the
presence of CaM but not after CaM had been sequestered (Fig. 2,
A and B). In contrast, GSNO caused an additional
enhancement of RyR1 channel activity even after endogenous CaM
sequestration. These results support the idea that NOC-12 controls RyR1
via the S-nitrosylation of Cys-3635, which is found in the
CaM binding region of RyR1. On the other hand, redox modulation by GSNO
does not appear to be dependent on S-nitrosylation or oxidation of
Cys-3635. More definitive evidence for the role of Cys-3635 in the
redox modulation of RyR1 is given below using a RyR1 construct with a
Cys-3635 to Ala substitution.
Modulation of RyR1 Single Channel Activities by NOC-12 and
GSNO--
The ability of the two NO donors to activate RyR1 under
ambient oxygen tension was confirmed in single channel recordings. Skeletal SR vesicles were incorporated into planar lipid bilayers, and
single RyR1 channels were recorded with Cs+ as the current
carrier. As shown in Fig. 3A,
0.1 mM NOC-12 significantly activated RyR1 channel in the
presence of 2 µM free Ca2+ and 1 µM CaM. Similarly, 0.2 mM GSNO activated the
channel (Fig. 3B). Fig. 3C shows that the
averaged channel open probability (Po) of RyR1
tripled after the addition of 0.1 mM NOC-12 or 0.2 mM GSNO. Thus, both [3H]ryanodine binding and
single channel measurements show that under comparable conditions these
two NO donors activate the RyR1 to the same extent.
Redox-related Basis of RyR1 Modulation by NOC-12 and GSNO--
We
next determined whether modulation of RyR1 by NO donors involved the
formation of a single SNO per RyR1 subunit, as was shown previously for
NO at pO2 ~10 mm Hg (1). We thus determined both the free
thiol and SNO content of RyR1s treated with NOC-12 or GSNO at
pO2 ~150 mm Hg. Exposure of SR vesicles to 0.1 or 1.0 mM NOC-12 increased [3H]ryanodine binding to
a similar extent and reduced the RyR1 thiol content by ~1 per RyR1
subunit, which was accounted for by the formation of ~1 SNO per RyR1
subunit (Table II). The stoichiometry of
1 SNO/RyR1 subunit agreed with that obtained by exposure to 0.75 µM NO at pO2 ~10 mm Hg (1). 0.1 mM NOC-12 optimally activated RyR1 in single channel
recordings in less than 1 min (Fig. 3A).
In contrast to NOC-12, 0.2 mM GSNO activated RyR1 at
ambient O2 tension via the S-nitrosylation or
oxidation of multiple thiols or a combination of both redox-based
modifications. As shown in Table II, 0.2 mM GSNO
S-nitrosylated approximately two RyR1 thiols in addition to
oxidizing approximately two thiols per RyR1 subunit (loss of
approximately four thiols per RyR1 subunit). At an elevated concentration (1.0 mM), GSNO further increased the level of
[3H]ryanodine binding and S-nitrosylated
approximately three thiols and oxidized approximately four thiols (loss
of approximately seven thiols/RyR1 subunit). Both oxidation and
S-nitrosylation of RyR1 by GSNO (and
S-nitrosylation by NOC-12) were prevented in the presence of
5 mM reduced glutathione (not shown).
We considered the possibility that GSNO S-nitrosylates RyR1
via transnitrosylation using C-PTIO, a NO scavenger, and NOC-12 as a
control. NOC-12 (0.1 mM) no longer had any effect on RyR1 in the presence of 0.1 mM C-PTIO, neither S-nitrosylating
nor activating RyR1 (not shown). Similarly, 0.1 mM C-PTIO
eliminated RyR1 S-nitrosylation and activation by 0.2 mM GSNO (not shown). These results suggest that
S-nitrosylation of RyR1 by GSNO is dependent on release of
NO, as is the release of NO from NOC-12. We caution, nevertheless, that
C-PTIO may have other effects, including scavenging and generating
additional reactive radicals.
Cysteine 3635 Is Critical for RyR1 Modulation by NOC-12 but Not by
GSNO--
The aforementioned data using SR vesicles suggest that at
ambient pO2 NOC-12 S-nitrosylates Cys-3635 and activates
RyR1 by antagonizing the inhibitory effect of CaM. In contrast, GSNO
works by a different mechanism. We tested this hypothesis using a
strategy that was previously employed to demonstrate selective
modification of Cys-3635 by NO (6). Full-length WT or single-site
C3635A RyR1 mutant channels were expressed in HEK293 cells. Membranous fractions containing WT and C3635A mutant RyR1s were isolated from the
HEK293 cells, and the effects of the two NO donors were assessed at
pO2 ~150 mm Hg in
[3H]ryanodine binding (Fig.
4) and in single channel measurements (Fig.
5). NOC-12 had no effect on the mutant
RyR1 (Figs. 4A and 5, A and C),
whereas the GSNO effect was preserved (Figs. 4B and 5,
B and C). The failure of NOC-12 to activate RyR1
C3635A was not due to a lack of CaM binding, because the C3635A
mutation does not eliminate modulation of RyR1 activity by CaM (6, 8).
We used an anti-nitrosocysteine polyclonal antibody to determine
whether NOC-12 and GSNO S-nitrosylated the RyR1 C3635A
mutant channel. We first confirmed that NO increased the
immunoreactivity of the native and WT RyR1s in pO2 ~10 mm
Hg but not pO2 ~150 mm Hg (6) (not shown). NO did not,
however, increase immunoreactivity of the C3635A mutant RyR1 at either
oxygen tension. A weak signal was detected by the antibody in the
control samples (without NO donor) in a region of the immunoblots
containing the RyR1 (Fig. 6,
left panel), as determined by an anti-RyR1
antibody (Fig. 6, right panel). NOC-12 (0.1 and
1.0 mM) produced virtually the same signal as NO (6) but in
ambient pO2, thus increasing the level of
S-nitrosylation of native and WT RyR1s but not of C3535A
RyR1. Specificity of S-nitrosylation was proven by showing that prior treatment with HgCl2 nearly eliminated the signal (not
shown). In contrast, 0.2 and 1.0 mM GSNO did not noticeably
increase the low levels of endogenous immunoreactivity. Taken together,
the data of Figs. 4-6 suggest that NOC-12 and GSNO affect the RyR1 by two different mechanisms, i.e. NOC-12 by
S-nitrosylation of Cys-3635 and GSNO by
S-nitrosylation and/or oxidation of an
additional/alternative class of RyR1 thiols.
The massive (~2,200 kDa) ryanodine receptors contain numerous
allosteric sites subserving multiple levels of control (25). It has
been firmly established that all three mammalian ryanodine receptor
isoforms are redox sensitive, i.e. the channels contain regulatory thiols whose oxidation or covalent modification alters their
activities (1, 2, 29-32). These thiols (~50/subunit) have been
grouped according to their differential reactivities toward NO,
O2, and glutathione, which in turn may be linked to binding
of allosteric effectors (1, 2). We have recently shown that NO, at low
pO2, selectively modifies Cys-3635 (6). PO2 is
dynamically linked to the redox state of a class of 6-8 thiols.
However, the identities of these regulatory thiols and the mechanistic
basis of the pO2 regulation of NO binding (homotropic versus heterotropic) remain to be determined. Here we have
probed this question by taking advantage of the different reactivities and properties of alternative classes of NO donors.
Cysteine 3635 is part of a predicted hydrophobic motif for
S-nitrosylation (33) located within the RyR1 CaM binding
domain (7, 8); NO regulation of RyR1 activity is thus CaM dependent (1,
6). We posited that the inability of NO to S-nitrosylate Cys-3635 at ambient O2 tension is either due to Cys-3635
being oxidized (i.e. Cys-3635 is one of the 6-8 thiols) or
to a change in channel conformation brought about by the oxidative
posttranslational modification. As a first step to address this
question, we determined the dependence of NOC-12 and GSNO on Cys-3635
and pO2. NOC-12 and GSNO had very similar effects on RyR1
channel activity (at concentrations matched for NO release), and
neither compound showed O2 dependence (pO2
~10 mm Hg versus pO2 ~150 mm Hg). However, the underlying mechanism of activation was quite different in each
case. GSNO activated RyR1 via poly-S-nitrosylation and/or oxidation of RyR1 thiols. Cys-3635 and CaM were not essential for
activation. These data are highly reminiscent of the effects of GSNO on
cardiac muscle isoform of RyR (RyR2), except that O2 and
CaM dependence were not explored at that time (20). NO and NOC-12 have little effects on
RyR2.2 In stark contrast,
NOC-12 activates RyR1 via S-nitrosylation of Cys-3635, and
the increases in activity ([3H]ryanodine binding in
intact SR) is CaM dependent, as seen with NO. Specifically, the only
modification of RyR1 by NOC-12 was nitrosylation of a single thiol, and
full-length, heterogeneously expressed RyR1 with a C3635A mutation was
not activated. Unlike NO, however, S-nitrosylation by NOC-12 is seen at
high pO2. Thus we conclude that Cys-3635 of RyR1 is not
oxidized at ambient O2 tension.
Why NO and NOC-12 mediated S-nitrosylation differ in their
pO2-dependence remains unclear. NOC-12 evidently depends on
released NO, because its RyR1-activating effect was inhibited by C-PTIO (a NO scavenger) and was not reproduced by the spent compound. It is
unlikely that differences in half-life of NOC-12 versus NO
(>6 h versus 10 min, Table I) provide an explanation,
because the effect of NOC-12 in single channel recordings was seen
within 1 min. Instead, we favor the idea that access of NO to the
cysteine thiol is responsible for the differences. Hydrophobic domains that concentrate nitrosylating equivalents and the quaternary structure
of the target site are both important determinants for S-nitrosylation (33, 34). NOC-12 may interact with the RyR1 (ionic interactions of these compounds are seen with other
proteins),3 perhaps in a way
that is conducive to nitrosylation irrespective of RyR conformation. An
interaction with the protein would also have the effect of increasing
the effective molarity of the NONOate, thereby potentiating
nitrosylation chemistry involving O2. In contrast, access
of solution NO (i.e. through the protein) to Cys-3635 might
be available in the low pO2 conformation but blocked at
high pO2. The hydrophobic pocket where Cys-3635 resides may even serve to concentrate NO/O2 to produce nitrosylating
equivalents. In this scenario, the allosteric function subserved by low
pO2 is 2-fold: 1) to produce a nitrosylation-responsive
conformation of the RyR1; and 2) to catalyze nitrosylation chemistry
(micellar catalysis).
An intriguing finding was that NOC-12 and GSNO operate by different
mechanisms. NO release from GSNO is evidently necessary for activation
of the RyR1, because the NO scavenger C-PTIO blocked the effects of
GSNO on the RyR1. Nonetheless, it is premature to exclude
transnitrosylation reactions of GSNO, acting alone or in concert with
released NO, in the activating mechanism. Other explanations for GSNO
effects include the possibility that GSNO-mediated oxidation favors the
S-nitrosylation of a specific class of thiols (or vice
versa). It has recently been shown that proteins may have specific
binding sites for GSNO, which would direct the chemistry to thiols in
its vicinity (35).
In summary, NO, NOC-12 and GSNO all activate RyR1 to comparable degrees
in [3H]ryanodine binding and single channel measurements.
However, only the effect of NO is
pO2-dependent. Activation by both NO and NOC-12
involves the CaM-dependent S-nitrosylation of
Cys-3635, whereas GSNO mediated activation (involving
S-nitrosylation/oxidation of up to seven RyR1 thiols) can
occur independent of Cys-3635 and CaM. Thus O2, NO, and
GSNO react with different classes of thiols, and the role of
pO2 in RyR1 S-nitrosylation is likely mediated
through allostery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
iPo,i/N, where N is the total number of
channels, and Po,i is channel open probability
of the ith channel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Peak concentrations and half lifetimes of NO released by NOC-12 and
GSNO
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Fig. 1.
Effects of NO, NOC-12, and GSNO on
[3H]ryanodine binding to skeletal muscle SR vesicles in
pO2 of ~10 mm Hg (A) and ~150 mm Hg
(B). Specific [3H]ryanodine binding
to skeletal SR vesicles was determined in 0.125 M KCl, 20 mM imidazole, pH7.0, 8 µM free
Ca2+, the indicated concentrations of NO
(columns) and NO donors (lines with
symbols), and 5 nM [3H]ryanodine
at 24 °C for 5 h in pO2 ~10 mm Hg (A)
and pO2 ~150 mm Hg (B), respectively. Data are
the mean ± S.D. of four to six experiments. *, p < 0.05; **, p < 0.01 compared with respective control
without NO or NO donor.
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Fig. 2.
Effects of CaM, NOC-12, and GSNO on
[3H]ryanodine binding of skeletal muscle SR
vesicles. Skeletal muscle SR vesicles were pretreated in the
presence of 100 µM Ca2+ without or with 1 µM CaM or 1 µM CaM binding peptide
(CaMBP) at 24 °C for 30 min. Specific
[3H]ryanodine binding was assayed at 8 µM
Ca2+ (A) or 0.3 µM
Ca2+ (B) as described in Fig. 1 in the absence
and presence of 0.1 mM NOC-12 or 0.2 mM GSNO in
pO2 ~150 mm Hg. Data are the mean ± S.D. of three
to four experiments. *, p < 0.05; **,
p < 0.01 compared with controls without NO donor in
each group.
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Fig. 3.
Effects of NOC-12 and GSNO on RyR1
activities. SR vesicles were fused with lipid bilayers in
pO2 ~150 mm Hg. Single RyR1 channel currents, shown as
downward deflections from closed levels (solid lines) to
open levels (dotted lines), were recorded in symmetric 0.25 M CsCH3SO3, 10 mM
cesium HEPES buffer, pH7.3, at a holding potential of 35 mV.
Top traces, control with 2 µM free
Ca2+ and 1 µM CaM; bottom traces,
after the addition of 0.1 mM NOC-12 (A) or 0.2 mM GSNO (B) to the cytosolic side. C,
normalized Po before and after the addition of
0.1 mM NOC-12 or 0.2 mM GSNO. Data are the
mean ± S.E. of the number of recordings indicated in parentheses.
*, p < 0.05 compared with control (without NO
donors).
Free thiol (SH) and S-nitrosothiol (SNO) contents of RyR1 and
[3H]ryanodine binding levels in the absence and presence of
NOC-12 and GSNO
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Fig. 4.
Effects of NOC-12 and GSNO on WT RyR1 and
C3635A RyR1 activities. Specific [3H]ryanodine
binding to membrane fractions prepared from HEK293 cells expressing WT
or C3635A RyR1s was determined in 8 µM free
Ca2+ medium as described in the Fig. 1 legend in the
presence of indicated concentrations of NOC-12 (A) or GSNO
(B) in pO2 ~150 mm Hg.
[3H]Ryanodine binding data are the mean ± S.D. of
three to five experiments. *, p < 0.05; **,
p < 0.01, compared with each control (without NO
donors).
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Fig. 5.
Effects of NOC-12 on single channel
activities of WT RyR1 and C3635A RyR1. A and
B, membrane fractions containing WT and C3635A RyR1s were
fused with lipid bilayers in pO2 ~150 mm Hg. Left
side, single channel currents of two WT RyR1 channels, shown as
downward deflections from closed levels (solid lines) to
open levels (dotted lines; the two dotted lines in each
trace indicate the amplitude of two channels), were recorded in
symmetric 250 mM KCl, 20 mM potassium Hepes
buffer, pH 7.4, at holding potential of 35 mV. Top
traces, control with 2 µM free
Ca2+ and 1 µM CaM; bottom traces,
after the addition of 0.2 mM NOC-12 (A) or 0.2 mM GSNO (B) to the cytosolic side. Right
side, single channel currents of two C3635A RyR1 channels recorded
in same condition as WT RyR1. Top traces, control
with 2 µM free Ca2+ and 1 µM
CaM; bottom traces, after the addition of 0.2 mM NOC-12 (A) or 0.2 mM GSNO
(B) to the cytosolic side. C, normalized
Po before and after the addition of 0.2 mM NOC-12 or 0.2 mM GSNO. Data are the
mean ± S.E. of the number of experiments indicated in
parentheses. *, p < 0.05, compared with control
(without NO donors).
View larger version (20K):
[in a new window]
Fig. 6.
Immunoblots for nitrosocysteine and
RyR1. Skeletal SR vesicles (A, 10 µg protein/lane)
and cell membrane fractions (B and C, 20 µg
protein per lane) containing WT RyR1 (B) or C3635A RyR1
(C) were incubated for 1 h at 24 °C in
pO2 ~150 mm Hg with 8 µM free
Ca2+ in the absence or presence of indicated concentrations
of NOC-12 or GSNO. The proteins were separated by 3-12% gradient
SDS-PAGE and transferred to polyvinylidene difluoride membranes
overnight at 4 °C. A polyclonal anti-S-nitrosocysteine
antibody (left panel, 1:500 dilution) was used to detect an
S-nitrosylation signal in the protein band region of RyR1
probed with D110 monoclonal anti-RyR1 (right panel, 1:10
dilution). The data are representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL52529 and HL59130 (to J. J. S.) and AR18687 and HL27430 (to G. M.).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.
¶ To whom correspondence should be addressed. Tel.: 919-966-5021; Fax: 919-966-2852; E-mail: meissner@med.unc.edu.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211940200
2 J. Sun and G. Meissner, unpublished studies.
3 J. S. Stamler, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal muscle isoform of RyR; pO2, O2 tension; CaM, calmodulin; NO, nitric oxide; SNO, S-nitrosothiol; GSNO, S-nitrosoglutathione; SR, sarcoplasmic reticulum; NOC-12, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine; WT, wild type; HEK293, human embryonic kidney 293.
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