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Introduction
The ryanodine receptors (RyR) are a class of calcium release channels in the endoplasmic reticulum or sarcoplasmic
reticulum (SR) membrane. The RyR is regulated by
Ca2+, Mg2+, ATP, calmodulin, FK-506 binding protein, and
voltage-dependent channels such as dihydropyridine recep-
tors[1_4]. The skeletal RyR (RyR1) also contains up to 100 cysteine residues per
monomer[5] and it has become abundantly
clear that the redox status of some of these cysteines can regulate activity of the
RyR[6_9]. Activity of the RyR can be modulated by sulfhydryl modifying agents and the nature of the modification can determine both quantitative and qualitative
changes in the properties of the RyR. These agents include
N-ethyl maleimide[10,11],
diamide[10,11],
glutathione[12], NOC-12[13],
and nitric oxide[13_15]. Apart from glutathione and nitric oxide, however, other agents are exogenous; their relevance to the
physiological and/or pathological state of muscle is not conclusive.
Hydrogen peroxide (H2O2) is a reactive oxygen species produced in living
cells[16,17]. It has been shown that
H2O2 promotes ryanodine binding to SR membranes and
Ca2+ release from SR
vesicles[18]. H2O2
activates RyR single channels reconstituted in the lipid bilayer and increases channel open probability, suggesting that the effect of
H2O2 on SR Ca2+ release
is directly due to an oxidation of the
RyR[19,20]. Aghdasi et al suggested that sulfhydryl modifying agents might induce
inter-subunit cross-linking between RyR monomers to form dimers of RyR
subunits[10,11]. However, the hypothesis is based on
data from SR membranes where hundreds of other proteins exist apart from the RyR, and the cross-linked products that are
identified as dimers of RyR1 subunits are ambiguous. Moreover, the hypothesis was mainly based on experiments using
diamide, an exogenous reagent, as an oxidant. Whether endogenous oxidants produced
in vivo, such as H2O2, also produce
the same or a similar effect remains elusive.
In this study, we treated skeletal SR membranes with
H2O2, isolated the RyR1 complex and investigated the molecular
nature of sulfhydryl modification by
H2O2 on RyR1. Our studies
revealed that: (1)
H2O2 induced inter-subunit cross-linking
within the tetrameric RyR1 molecule; (2) in parallel to inter-subunit cross-linking, the RyR1 molecule changed morphology;
and (3) the chemical and morphological changes were reversible, after reduction by a reducing agent, the RyR1 molecule
regains its original state. These findings suggest that the molecular mechanism of RyR1 channel activity in SR regulated by
H2O2 is through inter-subunit cross-linking within the RyR1 tetrameric molecule, which in turn induces structural changes of
the RyR1.
Materials and methods
Materials [3H]Ryanodine was obtained from Amersham Biosciences (Piscataway, NJ, USA). Anti-dihydropyridine
receptor (DHPR)a1 subunit and anti-triadin monoclonal antibodies, CHAPS, Tween-20 and
protease inhibitors were obtained from Sigma (St Louis, USA), and polyvinylidine difluoride (PVDF) membrane was obtained from Millipore (Billerica, MA,
USA). Anti-RyR antibody, Ab2160, was raised against a consensus sequence of the C-terminus residues of RyR (peptide
sequence FFPAGDCFRKQYEDQL), which is conserved in RyR1 and RyR2. The antibody specifically recognizes the 565 kDa
RyR and the 410 kDa endogenous enzymatic fragment, both containing the peptide sequence FFPAGDCFRKQYEDQL. The
antibody does not cross-react with other proteins that do not contain the peptide sequence, including RyR3. All other
chemicals were of analytical grade.
Preparation of SR vesicles 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 nmol/L aprotinin, 1 µmol/L leupeptin, 1
µmol/L pepstatin, 1 mmol/L benzamidine and 0.2 mmol/L phenylmethylsulfonyl fluoride) as described
previously[21].
Treatment of SR vesicles with
H2O2 and purification of RyR1
complex SR vesicles were diluted with 0.1 mol/L KCl, 10%
sucrose and 20 mmol/L Na-Pipes, pH 7.1 to a protein concentration of 5 mg/mL, then treated with 1_10 mmol/L
H2O2 for 30 min at room temperature. The
H2O2-treated SR vesicles were pelleted in a Beckman TL-100 ultracentrifuge at 110
000×g for 15 min in a TL-100.3 rotor and re-suspended in 1.0 mol/L NaCl, 100 µmol/L egtazic acid, 150 µmol/L
CaCl2, 0.2 mmol/L PMSF, 1 µmol/L leupeptin, and 20 mmol/L Na-Pipes, pH 7.1 (solubilization buffer). The SR vesicles were incubated with 2 nmol/L
[3H]ryanodine for 1 h to label the RyR1, then the SR vesicles were solubilized with detergent CHAPS. The RyR1 complex was
purified by 5%_20% linear sucrose gradient centrifugation according to published
procedure[22], except that dithiothreitol
(DTT) was omitted from the gradient. The sedimentation position of RyR is defined by its sedimentation coefficient (30 S),
which is determined by using a series of standard proteins with known sedimentation
coefficient[22].
Immunoblotting analysis Samples were solubilized in
non-reducing sample buffer, containing 62.5 mmol/L Tris-HCl, pH
6.8, 20% glycerol (w/v), 1.2% sodium dodecylsulfate (SDS), and 0.05% bromophenol blue, and loaded onto 2.5%_12.0% linear
polyacrylamide sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and resolved by electrophoresis.
After electrophoresis, the proteins were
transferred overnight to a PVDF membrane. The transferred membrane was blocked
in phosphate-buffered saline (PBS), containing 0.1% Tween-20 and 5% non-fat milk, with agitation for 1 h at
room temperature, then incubated with primary antibodies for 2_3 h in blocking solution. The PVDF membrane was washed (3 times×5 min) in
PBS, and incubated with peroxidase-coupled secondary antibody in blocking solution for 1 h, washed as described above,
then developed with 0.6 mg/mL 3,3กฏ-diaminobenzidine, 0.03%
(v/v) H2O2 in PBS.
Electron microscopy Sample (5 µL) was applied to a glow-discharged, carbon-coated 400-mesh electron microscopy
copper grid and allowed to absorb for 1 min. Excess sample was removed by touching the grid from the side with a piece of
Whatman filtration paper. One drop (5 µL) of 1% uranyl acetate was applied to the grid. After 1 min, the grid was washed with
5 drops of 1% uranyl acetate. Excess solution was removed by touching the side of the grid with a piece of Whatman filtration
paper, and the grid was air-dried.
Grids were examined in a Philips CM-12 electron microscope, operating at 100 kV. Pictures were recorded on Kodak S-19
films at a magnification of ×36 000 with exposure time of 1 s. Films were developed in an Ilford D19 developer for 12 min.
Results and discussion
Formation of high-molecular-weight complex SR membranes were treated with
H2O2, which promotes ryanodine
binding to SR membranes and Ca2+ release from SR
vesicles[18]. Similar to diamide, one major target of
H2O2 was the RyR1, as the intensity of the 565 kDa RyR1 peptide and its 410 kDa endogenous enzymatic fragment (fRyR) were dramatically reduced in
H2O2-treated SR membranes (Figure 1A). In contrast to diamide, in which only 2 high-molecular-weight bands (HMWBs)
were observed[10,11], 4 major HMWBs appeared (Figure 1A, bands
a-d). The HMWBs could be classified into 2 types. The
first type (bands a_c) changed their intensities and progressively migrated towards the higher molecular weight range with
increasing H2O2 concentrations. The second type (band d) appeared to have constant intensity throughout the various
H2O2 concentrations used. To reveal whether all HMWBs were derived from RyR1, Western blot analysis was carried out. The
data showed that the three first type HMWBs (bands a_c) were recognized by the anti-RyR antibody Ab2160, whereas the
second type HMWB (band d) was not (Figure 1B, -DTT), indicating HMWBs a_c were derived from RyR1, but HMWB d was
not. After incubation with DTT, a disulphide bond reducing agent, all HMWBs disappeared. In parallel, the intensity of the
565 kDa RyR1 peptide band and the 410 kDa endogenous enzymatic fragment recovered (Figure 1B, +DTT). These data
suggested that, after H2O2 treatment, the RyR1 formed one (or several) high-molecular-weight complex (HMWC) through
cross-linking using disulphide bonds. Notably, the first type HMWBs already existed as faint bands even in the control SR
membrane (Figure 1B, control). This suggested that the RyR1 was very sensitive to reactive oxygen species and could
pre-exist in a partially cross-linked state in the native SR membrane
in vivo, or was oxidized during preparation of SR vesicles.
Molecular nature of the HMWC involving RyR1
It has been shown that the RyR1 is regulated by interaction with a
variety of proteins[1_4]. Two of them, DHPR and triadin, have been suggested to directly interact with the
RyR1[23_26]. It is possible that the HMWCs are adducts of RyR1 with these proteins. To reveal the molecular nature of the HMWCs involving
RyR1, the SR membranes treated with
H2O2 (10 mmol/L) were solubilized with detergent CHAPS, labeled with
[3H]ryanodine, and the sample was subjected to sucrose gradient sedimentation analysis. A comparison of
[3H]ryanodine sedimentation profiles in sucrose gradient between the
H2O2-treated sample and the control sample revealed that the sedimentation profiles
were virtually the same. In both cases, the RyR1 complexes sedimented normally as 30S particles (Figure 2, indicated by an
arrow). This sedimentation profile is virtually the same in the
H2O2 concentration used (data not shown). SDS-PAGE under
non-reducing conditions revealed that the 30S fractions were comprised of three HMWBs equivalent to the first type of
HMWBs (bands a-c) observed originally in
H2O2-treated SR membranes (Figure 3A,
DTT). When the fractions were treated with DTT, however, all three HMWBs (bands a_c) disappeared. In parallel with this disappearance there was a recovery of
the 565 kDa RyR1 peptide and the 410 kDa endogenous enzymatic fragment (Figure 3A, +DTT), suggesting that these three
HMWBs were the cross-linked products of RyR1 subunits.
To further confirm that the HMWBs were indeed cross-linked products of RyR1 subunits, Western blot analysis of the
sucrose gradient fractions was carried out under non-reducing conditions. The result showed that all three HMWBs were
specifically recognized by the anti-RyR antibody Ab2160 (Figure 3B,
DTT). Western blot analysis using antibodies against
DHPRa1 and triadin did not recognize any of these HMWBs. After the fractions were treated with DTT, the HMWBs
disappeared; in parallel with this disappearance, the 565 kDa RyR1 peptide and the 410 kDa endogenous enzymatic fragment
were detected by the same anti-RyR antibody, Ab2160 (Figure 3B, +DTT), whereas antibodies against
DHPRa1 and triadin failed to detect any signal in the same fractions containing HMWBs. However, both
DHPRa1 and triadin were indeed detected in the top fractions (fractions 3 for
DHPRa1 and fraction 1 for triadin) of the sucrose gradient and both the
H2O2-treated and control SR membranes (Figure 3B, ±DTT). These data suggest that, after treatment with
H2O2, the RyR1 forms adducts in the SR membrane through cross-linked disulphide bonds, and the cross-linking takes place between subunits of
the RyR1 tetrameric molecule. DHPRa1 and triadin, which have been suggested to directly interact with the RyR1, do not form
adducts with RyR1 through disulphide bonds.
Morphological changes of the RyR1
molecule It has been shown that
H2O2 promotes ryanodine binding to SR membranes,
enhances Ca2+ release from SR vesicles, and increases channel open
probability[18_20]. Hamilton and
coworkers suggested that cross-linking between RyR subunits might be the molecular
mechanism[10,11]. We revealed that
H2O2 indeed induced cross-linking between RyR1 subunits. To see whether the chemical change induced structural change,
we purified the RyR1 complex from
H2O2-treated SR membranes and examined the morphology of RyR1 complex by electron
microscopy. In contrast to the RyR1 particles purified from control SR membranes, which displayed a homogenous
pinwheel-like appearance (Figure 4A), the RyR1 particles purified from
H2O2-treated SR membranes displayed various appearances
(Figure 4B), from pinwheel-like (Figure 4B, a) to square-like (Figure 4B, b) and round-shaped (Figure 4B, c) to windmill-like
(Figure 4B, d). These data demonstrated that, after inter-subunit cross-linking, the structure of the RyR1 molecule changed.
Consistent with the multiple cross-linked products observed by SDS-PAGE (Figures 1,3, bands a_c), different appearances
were present. Despite their different appearances, the overall size of the particles was consistent with the size of the RyR1
molecule, 25_29 nm[27_30]. Intriguingly, the morphological changes were reversible: when disulphide bond reducing agent
DTT was added to the RyR1 particles purified from
H2O2-treated SR membranes, the particles regained their characteristic
pinwheel-like appearance (Figure 4C), indicating that the effect of
H2O2 on the morphology of RyR is reversible. This finding
is consistent with the effect of
H2O2 on the function of RyR, which is also
reversible[18,19].
Sulfhydryl modifying agents have been shown to affect ryanodine binding to, and
Ca2+ release from, SR vesicles. This led
to suggestions that redox potential might be involv-ed in the regulation of
Ca2+ release from SR and excitation-contraction
coupling in muscle[6_9]. The precise
mechanism, however, might be different from agent to
agent[14]. There are 3 possible explanations for the modulation of
Ca2+ release from SR by
H2O2: (1) oxidation of sulfhydryl might induce the RyR1 molecule
to form adducts with other proteins, such as DHPRa1 and/or triadin, which have been shown to directly interact with
RyR1[23_26]; (2) oxidation of sulfhydryl might induce the RyR1 molecule to form adducts with other RyR1 molecules, as the RyR1
oligomers are organized as ordered arrays in the SR membrane
in situ[27,28] and physically coupled with each
other[31,32]; and (3) oxidation of sulfhydryl might induce RyR1 to form a complex within the tetrameric molecule.
The data presented in this work indicate that possibility (1) is unlikely to be correct because, if it is correct, then
RyR1_DHPRa1 and/or RyR1-triadin complexes will form in the SR membrane after treatment with
H2O2. Neither SDS-PAGE nor Western blot analysis detected these complexes (Figure 3).
Possibility (2) is also ruled out for the following reasons: (1) if possibility (2) is correct, RyR1 will form giant RyR1
molecular complexes with single RyR1 molecules of multiple sizes, so the sedimentation coefficient will be a multiple of 30 S
(such as 60 S, 90 S, or 120 S) that will sediment at marked different positions in
5%-20% linear sucrose gradient. The sedimentation profile of the RyR1 complex purified from
H2O2-treated SR membranes is virtually the same as that of the RyR1
molecule purified from control SR membranes (Figures 2, 3), indicating the RyR1 complex formed after treatment with
H2O2 is still 30S; (2) consistent with sedimentation analysis, electron microscopy of the RyR1 particles purified from
H2O2-treated SR membranes revealed that the size of RyR1 particles is in the range of
25-29 nm (Figure 4), which is the typical size of a single
RyR1 molecule[27_30].
It seems that the correct mechanism is possibility (3), and indeed all the data presented in this work conform to this
possibility. Hamilton and coworkers proposed a similar mechanism of RyR modification by
diamide[10,11]. They found that when SR membrane was treated with diamide, 2 HMWBs were detected in SDS-PAGE, which they assigned as the dimer of the
565 kDa RyR1 peptide and the dimer of the 410 kDa fragment of RyR1 peptide, according to their
mobility[10,11]. From these data, they suggested that cross-linking of sulfhydryl between RyR1 subunits might play a role in the modulation of
Ca2+ release from SR[8]. We demonstrated that
H2O2, which is produced in living cells, produced similar effects. Our data are based on
isolated RyR1 complex, which unambiguously identified the HMWBs as cross-linked RyR1 subunits (Figure 3). Furthermore,
our data demonstrated that the cross-linking took place within the tetrameric RyR1 molecule, but not between the RyR1
oligomers (Figures 2, 4).
In conclusion, the data in this work revealed that, after oxidation by
H2O2, disulphide bonds between subunits within the
tetrameric RyR1 molecule formed. The inter-subunit cross-linking triggered structural changes that might alter ryanodine
binding and channel activity.
References
1 Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol
Rev 1997; 77: 699_729.
2 Fill M, Copello JA. Ryanodine receptor calcium release channels. Physiol Rev 2002; 82: 893_922.
3 Meissner G. Molecular regulation of cardiac ryanodine receptor ion channel. Cell Calcium 2004; 35: 621_8.
4 Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol 2004; 37: 417_29.
5 Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa K, Minamino N,
et al. Primary structure and expression from
complementary DNA of skeletal muscle ryanodine receptor. Nature 1989; 339: 439_45.
6 Pessah IN, Feng W. Functional role of hyperreactive sulfhydryl moieties within the ryanodine receptor complex. Antioxid Redox Signal
2000; 2: 17_25.
7 Anzai K, Ogawa K, Ozawa T, Yamamoto H. Oxidative modification of ion channel activity of ryanodine receptor. Antioxid Redox Signal
2000; 2: 35_40.
8 Hamilton SL, Reid MB. RyR1 modulation by oxidation and calmodulin. Antioxid Redox Signal 2000; 2: 41_5.
9 Morad M, Suzuki YJ. Redox regulation of cardiac muscle calcium signalling. Antioxid Redox Signal 2000; 2: 65_71.
10 Aghdasi B, Zhang JZ, Wu Y, Reid MB, Hamilton SL. Multiple classes of sulfhydryl modulate the skeletal muscle
Ca2+ release channel. J Biol Chem 1997; 272: 3739_48.
11 Aghdasi B, Reid MB, Hamilton SL. Nitric oxide protects the skeletal muscle
Ca2+ release channel from oxidation induced activation. J Biol
Chem 1997; 272: 25462_7.
12 Zable AC, Favero TC, Abramson JJ. Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum: evidence for
redox regulation of the Ca2+ release mechanism. J Biol Chem 1997; 272: 7069_77.
13 Sun J, Xu L, Eu JP, Stamler JS, Meissner G. Nitric oxide, NOC-12, and
S-nitrosoglutathione modulate the skeletal muscle calcium release
channel/ryanodine receptor by different mechanisms: an allosteric function for
O2 in S-nitrosylation of the channel. J Biol Chem 2003;
278: 8184_9.
14 Xu L, Eu JP, Meissner G, Stamler JS. Activation of the cardiac calcium release channel (ryanodine receptor) by
poly-S-nitrosylation. Science 1998; 279: 234_7.
15 Eu JP, Sun JH, Xu L, Stamler JS, Meissner G. The skeletal muscle calcium release channel: coupled
O2 sensor and NO signaling functions. Cell 2000; 102: 499_509.
16 Reid MB, Khaeli FA, Moody MR. Reactive oxygen in skeletal muscle. III. Contractility of unfatigued muscle. J Appl Physiol 1993; 75:
1081_7.
17 Munns SE, Lui JK, Arthur PG. Mitochondrial hydrogen peroxide production alters oxygen consumption in an
oxygen-concentration-dependent manner. Free Radic Biol Med 2005; 38: 1594_603.
18 Favero TG, Zable AC, Abramson JJ. Hydrogen peroxide stimulates the
Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.
J Biol Chem 1995; 270: 25557_63.
19 Boraso A, Williams AJ. Modification of the gating of the cardiac sarcoplasmic reticulum
Ca2+-release channel by
H2O2 and dithiothreitol. Am J Physiol 1994; 267: H1010_6.
20 Oba T, Ishikawa T, Yamaguchi M. Sulfhydryl associated with
H2O2-induced channel activation are on luminal side of ryanodine receptors.
Am J Physiol 1998; 274: C914_21.
21 Saito A, Seiler S, Chu A, Fleischer S. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.
J Cell Biol 1984; 99: 875_85.
22 Lai FA, Erickson HP, Rousseau E, Liu QY, Meissner G. Purification and reconstitution of the calcium release channel from skeletal muscle.
Nature 1988; 331: 315_9.
23 Guo W, Campbell KP. Association of triadin with the ryanodine receptor and calsequestrin in the lumen of the sarcoplasmic reticulum. J
Biol Chem 1995; 270: 9027_30.
24 Groh S, Marty I, Ottolia M, Prestipino G, Chapel A, Villaz M,
et al. Functional interaction of the cytoplasmic domain of triadin with the
skeletal ryanodine receptor. J Biol Chem 1999; 274: 12278_83.
25 Marty I, Robert M, Villaz M, De Jongh K, Lai Y, Catterall WA,
et al. Biochemical evidence for a complex involving dihydro-pyridine
receptor and ryanodine receptor in triad junctions of skeletal muscle. Proc Natl Acad Sci USA 1994; 91: 2270_4.
26 Mouton J, Ronjat M, Jona I, Villaz M, Feltz A, Maulet Y. Skeletal and cardiac ryanodine receptors bind to the
Ca2+-sensor region of dihydropyridine receptor
alpha1C subunit. FEBS Lett 2001; 505:
441_4.
27 Ferguson DG, Schwartz HW, Franzini-Armstrong C. Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying,
rotary-shadowing study. J Cell Biol 1984; 99:
1735_42.
28 Saito A, Inui M, Radermacher M, Frank J, Fleischer S. Ultrastructure of the calcium release channel of sarcoplasmic reticulum. J Cell Biol
1988; 107: 211_9.
29 Samso M, Wagenknecht T, Allen PD. Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel
by cryo-EM. Nat Struct Mol Biol 2005; 12:
539_44.
30 Ludtke S, Serysheva I, Hamilton S, Chiu W. The pore structure of the closed RyR1 channel. Structure 2005; 13:
1203_11.
31 Yin CC, Lai FA. Intrinsic lattice formation by the ryanodine
receptor calcium-release channel, Nat Cell Biol 2000; 2:
669_71.
32 Yin CC, Blayney LM, Lai FA. Physical coupling between ryanodine receptor-calcium release channels. J Mol Biol 2005; 349:
538_46.
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