Redox states of type 1 ryanodine receptor alter
Ca2+ release channel response to modulators
Toshiharu
Oba1,
Takashi
Murayama2, and
Yasuo
Ogawa2
1 Department of Physiology, Nagoya City University Medical
School, Nagoya 467-8601; and 2 Department of Pharmacology,
Juntendo University School of Medicine, Tokyo 113-8421, Japan
 |
ABSTRACT |
The type 1 ryanodine
receptor (RyR1) from rabbit skeletal muscle displayed two distinct
degrees of response to cytoplasmic Ca2+ [high- and
low-open probability (Po) channels]. Here, we
examined the effects of adenine nucleotides and caffeine on these
channels and their modulations by sulfhydryl reagents.
High-Po channels showed biphasic
Ca2+ dependence and were activated by adenine nucleotides
and caffeine. Unexpectedly, low-Po channels did
not respond to either modulator. The addition of a reducing reagent,
dithiothreitol, to the cis side converted the
high-Po channel to a state similar to that of
the low-Po channel. Treatment with
p-chloromercuriphenylsulfonic acid (pCMPS) transformed
low-Po channels to a
high-Po channel-like state with stimulation by
,
-methylene-ATP and caffeine. In experiments under redox control
using glutathione buffers, shift of the cis potential toward
the oxidative state activated the low-Po
channel, similar to that of the high-Po or the
pCMPS-treated channel, whereas reductive changes inactivated the
high-Po channel. Changes in trans
redox potential, in contrast, did not affect channel activity of
either channel. In all experiments, channels with higher
Po were stimulated to a great extent by
modulators, but ones with lower Po were
unresponsive. These results suggest that redox states of critical
sulfhydryls located on the cytoplasmic side of the RyR1 may alter both
gating properties of the channel and responsiveness to channel modulators.
calcium-induced calcium release; adenine nucleotide; caffeine; sulfhydryl reagents; redox potential
 |
INTRODUCTION |
RELEASE OF
CA2+ FROM intracellular
Ca2+ stores plays a crucial role in regulation of
intracellular Ca2+, which serves as a second messenger for
many intracellular signaling processes. The ryanodine receptor (RyR) is
one of the Ca2+ release channels located on the endoplasmic
or sarcoplasmic (SR) reticulum (11, 26, 36). Three
isoforms of RyRs (RyR1-3) have been identified in mammalian
tissues (26, 36). All of the RyRs demonstrate
Ca2+-induced Ca2+ release (CICR) activity,
which is modulated by many endogenous and exogenous ligands (4,
14, 17, 20, 26, 27, 33, 38, 41). The RyR channel is activated by
Ca2+ in micromolar amounts and by adenine nucleotides and
caffeine but is inhibited by Ca2+ in the millimolar order
and by Mg2+.
A number of studies have demonstrated that oxidation,
S-nitrosylation, or alkylation of the critical sulfhydryls
in RyR molecules activates Ca2+ release channel activity,
whereas their reduction inhibits it (1, 3, 8-10, 12, 17, 24,
25, 31, 35, 36, 40). Heavy metals such as Hg2+ and
Ag+ have also been reported to activate RyR probably by
directly interacting with sulfhydryls, although there is a difference
in the chemical reaction between heavy metal binding and oxidation or
alkylation of sulfhydryls (2, 31). It has been proposed that the RyR molecule is a redox sensor with a well-defined redox potential (10, 39). In skeletal muscles, the RyR1 channel possesses heterogeneous populations differing in response to cis Ca2+ concentrations, when incorporated into lipid
bilayers (5, 18, 21). Channels termed
"high-Po" show biphasic Ca2+
dependence with relatively high open probability
(Po), and channels termed
"low-Po" display much lower activity, even
at an optimal Ca2+ concentrations, although
Ca2+ is also required for its activation. We have recently
reported that high-Po channels were inhibited by
a sulfhydryl-reducing agent, dithiothreitol (DTT), whereas
low-Po channels were activated by a
sulfhydryl-modifying reagent, p-chloromercuriphenylsulfonic acid (pCMPS; see Ref. 21). The stimulatory effect of pCMPS
was reversed by subsequent addition of DTT. In addition to channel gating, several effects of redox modification on the RyR channels have
been reported (7, 18). Marengo et al. (18)
demonstrated that the redox state of the channel molecule is a decisive
factor in determining the Ca2+ dependence. Donoso et al.
(7) recently reported that oxidation of RyR1 released the
inhibitory effect of Mg2+, resulting in activation of CICR.
These findings indicate that the redox state of the sulfhydryl residues
in RyR1 molecules may have an important impact on the modulation of
channel activity. However, it remains to be elucidated whether redox
states alter the stimulatory effects of CICR modulators, such as
adenine nucleotides and caffeine. Glutathione (GSH) and glutathione
disulfide (GSSG) constitute the major redox buffer system of many
cells, including skeletal muscle (32). The intracellular
redox potential in resting skeletal muscle fibers is maintained in a
highly reduced state of
220 to
230 mV (13), although
the intraluminal side of the SR has been reported to be in an oxidized
state (approximately
180 mV; see Ref. 10). Intracellular
ATP content and GSH concentration ([GSH])-to-GSSG concentration
([GSSG]) ratio are known to be altered depending on exercise strength
(16, 19, 32). Therefore, it is of interest to study
whether redox states of the RyR molecule affect responses to CICR
modulators such as adenine nucleotides and caffeine, as well as
Ca2+.
In the present study, we examined the effects of redox reagents on RyR1
channel activity and its modulations by adenine nucleotide and
caffeine, using a lipid bilayer technique. Attention was also directed
to the effect of cis and/or trans redox
potentials on the RyR1 channel behavior and its modulation by channel
modulators by using a [GSH]/[GSSG] redox buffer. Our present
results suggest that the cytoplasmic redox potential primarily
determines RyR1 channel activity and its responsiveness to channel modulators.
 |
MATERIALS AND METHODS |
Isolation of SR vesicles and purification of RyR1.
Heavy SR vesicles were prepared from rabbit back skeletal muscle in the
presence of a cocktail of protease inhibitors (in µg/ml: 2 aprotinin,
2 leupeptin, 1 antipain, 2 pepstatin A, and 2 chymostatin), as shown
elsewhere (23). RyR1 was purified using sucrose gradients
and Mono-Q anion exchange column chromatography (22).
Purified RyR 1 was free from FK506 binding protein (FKBP12) but
retained the ability to bind FKBP12 (21). The
preparations were quickly frozen in liquid N2 and stored at
80°C until use.
Planar lipid bilayer experiments.
Single-channel recordings were carried out by incorporating purified
RyR1 channels into planar lipid bilayers, as reported previously
(21, 24, 25). Lipid bilayers consisting of a mixture of
L-
-phosphatidylethanolamine,
L-
-phosphatidyl-L-serine, and
L-
-phosphatidylcholine (5:3:2 wt/wt/wt) in n-decane (40 mg/ml) were formed across a hole of ~250 µm in diameter in a
polystyrene partition separating cis and trans
chambers. The cis (1 ml)/trans (1.5 ml)
solutions consisted of 500/50 mM KCl, 20 mM HEPES/Tris (pH 7.4), and
0.1 mM CaCl2. Channel proteins were added to the cis
chamber. After confirming channel incorporation by the occurrence of flickering currents, further incorporation of channels was prevented
by adding an aliquot of 3 M KCl (pH was adjusted to 7.4 by 20 mM
HEPES/Tris) to the trans compartment. Recording of channel
currents was carried out in a symmetrical solution containing 500 mM
KCl, 20 mM HEPES/Tris (pH 7.4), and various concentrations of
Ca2+. The trans side was held at ground
potential, and the cis side was clamped at
40 mV using
1.5% agar bridges in 3 M KCl and Ag-AgCl electrodes. The cytoplasmic
surface of the RyR faced the cis side, as determined by
application of ATP or EGTA (24, 25). Channel activity
ascribed to RyR was confirmed by the responses to ryanodine and
ruthenium red at the end of every experiment. Experiments under redox
control of the cis and/or trans compartments were performed using a [GSH]/[GSSG] buffer solution. Redox potential in
the solution was calculated from the Nernst equation (13) by using the standard redox potential (=
0.24 V). Redox potentials were generated by the following different ratios of [GSH]/[GSSG] (mM/mM): 2:0.469 for
180 mV, 2:0.0196 for
220 mV, and 2:0.0082 for
231 mV. When redox potentials were changed consecutively, shift of
the redox potential from
220 mM to
231 mV was made by addition of
1.096 mM GSH and then to
180 mV by further addition of 1.103 mM GSSG.
Experiments were carried out at room temperature (18-22°C).
Single channel currents were amplified by a patch-clamp amplifier
(Axopatch 1D; Axon Instrument), filtered at 1 kHz using an eight-pole
Bessel filter (model 900; Frequency Devices), and then digitized at 5 kHz for analysis. Data were saved on the hard disk of an IBM personal
computer. The mean Po and lifetime of open and
closed events of the Ca2+ release channel from records of
>2 min were calculated by 50% threshold analysis using pClamp
(version 6.0.4; Axon Instrument) software.
The results are presented as mean ± SD. Statistical analysis was
carried out with ANOVA followed by Fisher's least-significant difference method or Student's t-test. Values of
P < 0.05 were regarded as statistically significant.
Chemicals.
Caffeine (500 mM; Sigma, St. Louis, MO), pCMPS (10 mM; Sigma), GSH (250 mM; Sigma), and GSSG (50 mM; Sigma) were prepared in ultrapure water
(Barnstead, Boston, MA) just before application. Stock solutions of ATP
(disodium salt, 100 mM; Sigma),
,
-methylene-ATP (AMPPCP, disodium
salt, 100 mM; Sigma), and ruthenium red (1 mM; Sigma) were prepared in
water and stored at
20°C. Ryanodine (1 mM; Wako Pure Chemical,
Osaka, Japan) was dissolved in ethanol and stored at
20°C. Other
reagents were of analytical grade.
 |
RESULTS |
Low-Po and high-Po channels of RyR1.
When active RyR1 molecules, which showed a maximal binding of 1 mol
[3H]ryanodine/1 mol tetramer for
[3H]ryanodine binding, were incorporated into lipid
bilayers, the channels belonged to several distinct populations with
different Po at pCa 4. The results of 123 channels examined are summarized in Table
1. For easy analysis, we conveniently
classified channels into two groups. Channels with
Po
0.05 at pCa 4.0 were referred to as
low-Po channels, and ones with
Po > 0.05 were referred to as
high-Po channels. The average
Po values for low-Po and
high-Po channels were 0.019 ± 0.005 (n = 78) and 0.263 ± 0.025 (n = 45), respectively. A clear Ca2+ dependence was not observed
in low-Po channels with
Po<0.01, because the number of open events was
below the analytical limit of our method. However, these channels also
exhibited null activity at Ca2+ concentrations <10 nM.
Low-Po channels with 0.01 < Po
0.05 showed the biphasic
Ca2+ dependence with a peak value around pCa 4.0 (Fig.
1A) and null activity at pCa
>8 (data not shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
Ca2+-dependent low-open probability
(Po) channel activity and their reversible
modulation by sequential additions of
p-chloromercuriphenylsulfonic acid (pCMPS) and
dithiothreitol (DTT). A: control channel activities on
exposure to pCa 7, 5, 4, and 3. B: effects of 50 µM pCMPS
on Ca2+-dependent channel activity. C: reversal
by 100 µM DTT. Closed level of the channel is shown as a short line
on right of each current trace. Calibration, 20 pA and 100 ms.
|
|
Addition of 50 µM pCMPS to the low-Po channel
markedly enhanced Po values in the presence of
various Ca2+ concentrations, and the Ca2+
dependence was biphasic (Fig. 1B). The
Po drastically decreased again on subsequent
application of 100 µM DTT after pCMPS (Fig. 1C). Figure
2 shows the effect of the
cis-side DTT on Ca2+-dependent activity of
high-Po channels. High-Po
channels showed a biphasic Ca2+ dependence with a peak
around pCa 4 (also see Fig. 3). When 50 µM DTT was added to the cis side, the
Po value drastically decreased to 0.001 within 1 min. Note that the channel on that occasion was apparently insensitive
to the change in cis Ca2+ (Fig. 2B).
Addition of DTT to the trans side, in contrast, did not
cause such changes for at least 5 min (data not shown). These channels
were inactive at 10 nM Ca2+ or less (data not shown),
suggesting that they still required Ca2+ for activation, as
is true of low-Po channels with
P < 0.01. The Po of the channel
markedly increased again on subsequent addition of 100 µM pCMPS after
DTT (Fig. 2C); thus, the effect of DTT is reversible. All
experiments similar to those in Figs. 1 and 2 are summarized in Fig. 3.
Ca2+ dependencies of the pCMPS-activated
low-Po channels and
high-Po channels were similar, although the
former showed less inhibition at high Ca2+ concentrations.
Peak values were also comparable to each other. A definite
Ca2+ dependence could not be obtained for
low-Po channels and DTT-treated channels because
of their small Po values and relatively large variations, respectively. These results suggest that the RyR channels with similar Po values may have similar gating
mechanisms. To examine this possibility, kinetics of channel activity
were examined.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
Ca2+-dependent high-Po
channel activity and its modulation by DTT and pCMPS. A:
control channel activities on exposure to cytoplasmic pCa 7, 4, and 3. B: reduction by 50 µM DTT. Note that the channel activity
became apparently independent of Ca2+ in the presence of 50 µM DTT. C: subsequent addition of 100 µM pCMPS in pCa 3 caused a marked increase in channel activity.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
pCa-Po relationship of each type
of channel in various states as follows: low-Po
( ), high-Po ( ),
pCMPS-treated low-Po ( ), and
DTT-treated high-Po ( ) channels.
Numbers represent the no. of determinations. Data are means ± SD.
pCa-Po relationships for
high-Po channels and pCMPS-activated
low-Po channels appeared to be similar, although
the latter channels were inhibited less at high Ca2+
concentrations. Low-Po channels and DTT-treated
high-Po channels, in contrast, showed unclear
Ca2+ dependence.
|
|
Results obtained from high-Po channels and
pCMPS-activated low-Po channels are summarized
in Table 2. The open time distribution of
the high-Po channel was best fit by the sum of
two exponentials as follows:
O1 = 0.44 ms (80.2%
in relative area) and
O2 = 2.27 ms (19.8%). The
low-Po channel after exposure to 50 µM pCMPS
displayed a new, third time constant of
O3 = 7.81 ms (9.2%) in addition to two time constants of
O1 = 0.34 ms (53.0%) and
O2 = 1.73 ms (37.8%). The
closed time distribution was best fit by three exponentials with
similar values between channels. In addition to the occurrence of the
long open time constant, another characteristic was a significant
decrease in the relative area of
O1 and an increase in
that of
O2. Application of pCMPS to the
low-Po channel caused prolongation of the open
time, which may account for the increase in Po.
Redox reagents and gating behavior of low- and high-Po
channels.
When applied to the cis compartment, 10 µM pCMPS slightly
activated the low-Po channel in the presence of
10 µM cis Ca2+ (Fig.
4A). An increase in pCMPS to
50 µM induced an ~20-fold increase in the Po
to 0.243. Results obtained using pCMPS over a range of 1-500 µM
are summarized in Fig. 4C. Two out of six channels examined
showed increased Po on exposure to pCMPS as low
as 10 µM, with an average of 0.027 ± 0.019 (n = 6). An increase in pCMPS to 25 µM significantly increased the
Po to 0.092 ± 0.030 (n = 4; P < 0.05), indicating that the threshold
concentration of pCMPS required for activation of the
low-Po channel is near 10 µM, although there
was a large channel-to-channel variation. Figure 4C shows
that the EC50 of pCMPS was ~26 µM. The
high-Po channel was also sensitive to pCMPS
(Po = 0.111 ± 0.075 in controls in
pCa 6, 0.132 ± 0.096 in 5 µM pCMPS, and 0.273 ± 0.106 in
50 µM, n = 5), suggesting that it was more sensitive
than the low-Po channel.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of redox reagents on
low-Po and high-Po
channel activities. A: low-Po channel
activities activated by 10 and 50 µM pCMPS. B:
high-Po channel activities reduced by 10, 100, and 500 µM DTT. C: dose-dependent changes in
Po. , pCMPS; ,
DTT. Data are means ± SD (no. of determinations are shown for
each symbol). Brackets denote concentration.
|
|
Dose-dependent inhibition of high-Po channel
activity by treatment with DTT is demonstrated in Fig. 4B.
Addition of 10 µM DTT to the cis chamber decreased the
Po by a factor of three. DTT inhibited channel
activity in a dose-dependent manner (Fig. 4C). In the
high-Po channel, 200 µM DTT inhibited channel
activity maximally. The results in Fig. 4C indicated that
the IC50 for DTT was ~6 µM.
Effects of adenine nucleotides and caffeine on low- and
high-Po channels with or without redox reagent treatment.
When added to the cis side of the
low-Po channel, AMPPCP, a nonhydrolyzable analog
of ATP, did not alter channel activity in 10 µM Ca2+
(Fig. 5A). In the presence of
3 mM AMPPCP, subsequent addition of 10 mM caffeine also failed to
activate this channel. In contrast, the high-Po
channel in pCa 6 (Po = 0.010) responded to
3 mM AMPPCP with an ~10-fold increase in Po
(Fig. 5B). Subsequent exposure to 10 mM caffeine further
increased the Po to 0.461, indicating a
potentiating action of adenine nucleotide and caffeine. In separate experiments, substituting 1 mM ATP for AMPPCP similarly stimulated high-Po channels (Po = 0.072 ± 0.020 at pCa 6 to 0.332 ± 0.045, n = 5) but had no effects on low
Po (Po = 0.007 ± 0.002 at pCa 5 to 0.007 ± 0.002, n = 3).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 5.
Differential responses of low-Po
(A) and high-Po (B)
channels to , -methylene-ATP (AMPPCP) and caffeine. AMPPCP and
caffeine were added in sequence. Note that
low-Po channels were not activated by 3 mM
AMPPCP or 10 mM caffeine (A), whereas
high-Po channels were markedly activated
(B).
|
|
The addition of 10 µM pCMPS to a low-Po
channel led to a moderate increase in Po (Fig.
6A, left). When
exposed to 1 mM AMPPCP, the channel activity was enhanced. In contrast,
when the Po of a high-Po
channel was reduced by application of 30 µM DTT, addition of AMPPCP
failed to increase the Po (Fig. 6A,
right). The effects of caffeine on the redox reagent-treated
channels were similar to those of AMPPCP. After a moderate activation
with pCMPS, the low-Po channel was activated on
application of 3 mM caffeine (Fig. 6B, left). In
contrast, the DTT-treated high-Po channel no
longer showed increased activity in response to 3 mM caffeine (Fig.
6B, right). Results were similar with 10 mM
caffeine (Po = 0.036 ± 0.028 from
Po = 0.033 ± 0.017 in DTT,
n = 5, P > 0.05).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of AMPPCP (A) and caffeine
(B) on the pCMPS-activated low-Po and
the DTT-treated high-Po channels.
Left: low-Po channels.
Right: high-Po channels. Note marked
stimulation by AMPPCP and caffeine of the pCMPS-treated
low-Po channel, whereas no response to
modulators occurred after reduction of the
high-Po channel.
|
|
Similar experiments were repeated using different doses of AMPPCP (Fig.
7A) and caffeine (Fig.
7B). AMPPCP and caffeine enhanced the
high-Po channel activity in a dose-dependent
manner. Application of 0.3 mM AMPPCP to the
high-Po channel significantly increased the
Po to 0.313 ± 0.130 (P < 0.05, n = 9) from 0.053 ± 0.011 in controls. A
further increase in AMPPCP produced only a slight activation of the
channel, and no significant difference was observed between 1 and 3 mM
because of large variations (P > 0.05). The minimum
effective concentration of caffeine required for significant activation
of the high-Po channel was 1 mM
(P < 0.05). The increase in caffeine induced further
increases in Po in a dose-dependent manner
(Po = 0.163 ± 0.032 at 3 mM and
Po = 0.220 ± 0.059 at 10 mM,
n = 9).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
Dose-dependent effects of AMPPCP (A) and
caffeine (B) on Po of various states
of RyR1 channels. , High-Po
channel activity; , low-Po
channel activity; , low-Po
channels activated in the presence of 10 µM pCMPS; ,
high-Po channels inhibited by 30-100 µM
DTT. P < 0.05 and  P < 0.01, different from corresponding control values before application of
AMPPCP or caffeine. No significant difference was observed between the
effects of 1 and 3 mM AMPPCP on high-Po channels
(A).
|
|
Exposure of the pCMPS-activated low Po-channels
to 0.3 mM AMPPCP produced significant increases in
Po and mean open time
(Po = 0.144 ± 0.018 and 4.08 ± 1.03 ms from Po = 0.056 ± 0.023 and 2.95 ± 0.79 ms before AMPPCP treatment, P < 0.05). AMPPCP significantly decreased the mean closed time (70.55 ± 17.33 to 43.28 ± 15.08 ms, P < 0.05) and
markedly increased numbers of open events (13.4 ± 3.2 to
27.9 ± 6.7/s, P < 0.05). An increase in AMPPCP
to 1 mM led to further increases in Po
(Po = 0.171 ± 0.060) and numbers of
open events (48.0 ± 16.1/s) and a decrease in the mean closed time (25.58 ± 10.37 ms). Exposure of a 1 mM ATP-treated
low-Po channel to 50 µM pCMPS also markedly
increased the Po (0.005 ± 0.003 to
0.185 ± 0.064, n = 3).
When exposed to 0.3 mM caffeine, pCMPS-activated low
Po-channels produced a significant increase in
Po (Po = 0.138 ± 0.007 from Po = 0.093 ± 0.025 in
pCMPS-treated channel, n = 8, P < 0.05; Fig. 7B) and a marked decrease in the mean closed time
(10.66 ± 3.23 from 26.51 ± 10.53 ms, P < 0.05). In addition, an increase in the numbers of open events was
observed after exposure to 1 and 3 mM caffeine (49.5 ± 19.7/s at
1 mM and 58.4 ± 24.1/s at 3 mM from 35.6 ± 18.3/s in
pCMPS, P < 0.05). On the other hand, low-Po and DTT-treated
high-Po channels did not respond to AMPPCP and
caffeine at all concentrations used here. These results suggest that
responsiveness to adenine nucleotides and caffeine may depend on the
redox state of sulfhydryls in the RyR, which in turn may determine the
channel activity.
Effects of redox potentials on low- and high-Po
channels and response to adenine nucleotide.
Until now, we have described the results of experiments where redox
reagents were added on the cis side alone. A recent study, however, indicated that the redox potential difference between cis and trans sides is critical for channel
activity. In that study (10), application of the reagents
to the cis side alone did not affect activity. Thus we
performed separate experiments under redox control using a
[GSH]/[GSSG] redox buffer (see MATERIALS AND METHODS).
The Po of low-Po channels
was markedly increased by defining cis redox potential at
180 mV (0.004 ± 0.003 in control to 0.079 ± 0.015, n = 3; Fig.
8A). Subsequent fixation of
the trans potential at
180 mV (a symmetrical
cis/trans potential of
180 mV), however, did not affect
channel activity (0.070 ± 0.002; Fig. 8A). The channel
activity was increased further to Po = 0.161 ± 0.061 (n = 3) on subsequent addition of
0.3 mM AMPPCP (Fig. 8A). In contrast, when trans
potential was set to
180 mV without defining cis
potential, the effect on Po was negligible (0.004 ± 0.003 before redox fixation to 0.015 ± 0.008, n = 7). Under this condition, Po
was increased by fixation of the cis potential at
180 mV
(0.069 ± 0.010, n = 3). Fixation of the cis potential at
220 mV instead of
180 mV did not affect the
channel activity, irrespective of the trans potential
(undefined or
180 mV; Po = 0.010 ± 0.006 to 0.013 ± 0.006, n = 4). These results strongly indicate that activation of low-Po
channels is primarily caused by the cis potential.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of transmembrane redox potential differences.
A: a low-Po channel at pCa 4. B: high-Po channel at pCa 6. Experiments were carried out in sequence as shown in each column. Redox
potentials were defined by [GSH]/[GSSG] buffer solutions. Oxidation
or reduction on the cis side alone was effective for
modulation of the channel activity. AMPPCP could activate the state
with an increased Po but not the state with a
lowered Po.
|
|
Po of the high-Po channel
in pCa 6-6.3, in turn, was apparently unchanged when the redox
potential on the trans side was set at
180 mV (0.118 ± 0.045 in undefined control to 0.080 ± 0.045, n = 7; Fig. 8B). Subsequent reduction of the cis
potential to
220 mV had a minor effect on
Po (0.122 ± 0.051). Further reduction in
the cis potential to
231 mV drastically decreased the
Po (0.012 ± 0.007; Fig. 8B).
Under this condition, AMPPCP up to 3 mM failed to enhance
Po of the channel further (0.012 ± 0.007 at 0.3 mM AMPPCP and 0.016 ± 0.012 at 3 mM AMPPCP), as is true
with the low-Po channel and the DTT-treated
high-Po channel. These observations indicate
that the channel activity of the high-Po channel
also may be mainly determined by cis redox potential.
 |
DISCUSSION |
The present study provides evidence that 1) transition
between low-Po and
high-Po states of skeletal muscle RyR1 channels is primarily regulated by the cytoplasmic redox potential alone, which
may be caused by oxidation/reduction of critical sulfhydryls of the
channel rather than by trans potential or transmembrane redox potential gradients, 2) pCMPS and DTT may act via
these sulfhydryls on the cis side of the channel, and
3) a marked reduction in these sulfhydryls on the RyR
molecule makes the channel unresponsive to CICR modulators such as
adenine nucleotide and caffeine, whereas an oxidation leads to
increased responsiveness to modulators.
When redox potentials were defined using a [GSH]/[GSSG] buffer, the
low-Po channel was markedly activated by setting
cis redox potential to
180 mV but was affected negligibly
at
220 mV (Fig. 8). High-Po channel activity
was not significantly altered at a cis redox potential of
220 mV but was inhibited markedly by the decrease from
220 to
231
mV. On the other hand, defining the trans redox potential to
180 mV under the control of cis redox potential failed to
affect low-Po or high-Po
channel activity. These results indicate that channel activity may be
primarily regulated by cis redox potential and that the
trans potential or the redox potential gradient across the
membranes may have minor effects. This is consistent with previous
observations that GSH inhibited and GSSG stimulated channel activity
when applied to the cytoplasmic, but not the luminal, side of the RyR
(40). Our results also indicate that channel activity
depended on thiol modification on the channel induced by application of
pCMPS/DTT in the cis side of the RyR (Figs. 1 and 2 and see
Ref. 24). Similar activation of the RyR channels has been
reported by oxidation of the cis, but not the
trans, side by exposure to another mercurial water-soluble
oxidant, thimerosal, indicating again that the sulfhydryl (SH) residues
involved in the observed Po changes were only
accessible from the cis side (3, 18).
In addition, the dose-dependent inhibitory effect of DTT (Fig. 4) seems
to be consistent with results from redox potential fixed by a
[GSH]/[GSSG] buffer solution (Fig. 8). Taken together, our results
suggest that cis redox state plays a primary role in
controlling channel activity, irrespective of the use of a
[GSH]/[GSSG] buffer solution or a pCMPS/DTT solution. Our results
are in marked contrast to the recent report by Feng et al.
(10) in which channel activity was primarily determined by
the transmembrane redox potential gradient but not by either the
cis or trans redox potential by itself. The
discrepancy between Feng et al. (10) and results presented
here remains to be resolved. We used purified proteins, whereas they
used SR vesicles. This difference of RyR preparations, however, cannot
be the explanation for the discrepancy, because effects of pCMPS in
the cis side alone on RyR channel activation in SR vesicles
were already reported (21, 24).
Previously, the occurrence of at least two groups of RyR channels with
low and high Po values, when the RyR was
incorporated into planar lipid bilayers, has been reported (10,
18, 21). In this study, we found that the RyR1 channels
consisted of several populations with different
Po (0
Po
0.5) at
the optimum Ca2+ concentration of 0.1 mM (Table 1). This
indicates that many stepwise states of channel activity may occur in
the RyR channels. pCMPS enhanced activity of the
low-Po channel in a dose-dependent manner (Fig.
4). The effect of pCMPS was reversed by addition of a reducing reagent,
DTT (Fig. 1C and see Ref. 21). Exposure to DTT,
on the other hand, dose-dependently decreased
high-Po channel activity, and subsequent
application of pCMPS increased activity again (Fig. 2), indicating that
pCMPS/DTT may act via the SH residues. In addition, treatment of the
low-Po channel with pCMPS (Fig. 4A)
or addition of DTT to the high-Po channel (Fig.
4B) caused several intermediate states in
Po at the single channel level. These findings
suggest that there are multiple SH residues involved in activation and
inhibition of RyR1 channels and that many stepwise states of channel
activity (as shown in Table 1) may be attributable to
oxidation/reduction states of these multiple SH residues. This is
consistent with the recent finding that RyR1 channel activity
correlates with the redox state (or numbers of SH groups) on the
channel molecule, i.e., oxidation of ~10 thiols had little effect on
channel activity, the loss of 10~25 thiols activated the channel in a
number-dependent manner, and further loss of thiols irreversibly
inactivates the channel (36). The multiple
oxidation/reduction of these reactive SH residues (probably with
different thresholds) may enable subtle regulation of the RyR1 channel
activity by redox potential. For ease of analysis, in this study, we
conveniently classified channels into the following two groups:
high-Po (Po
0.05 at pCa 4) and low-Po
(Po < 0.05 at pCa 4) channels (Table 1).
This separation was satisfactory to analyze the properties of RyR
channels. The two groups showed obviously distinct behaviors in
response to Ca2+ (Fig. 3) and to modulators of CICR (Fig.
5).
It has been accepted that SH oxidation and reduction modifies RyR
channel activity (i.e., the activity at the optimal Ca2+;
see Refs. 1, 3, 8-10,
21, 25, 31, 35,
40). In addition, Marengo et al. (18)
demonstrated that the Ca2+ dependence also depends on the
redox state of the RyR channel. Donoso et al. (7) recently
found that oxidation of the RyR1 with thimerosal suppressed the
inhibitory effect of Mg2+ on CICR. In this study, we found
that channels in the high-Po state (Fig. 5),
channels activated by exposure to pCMPS (Fig. 6), and channels
activated by setting cis redox potential to
180 mV (Fig.
8) were all stimulated by AMPPCP and/or caffeine, whereas channels with
low Po activity were apparently insensitive to
these drugs (Figs. 5-8). These observations suggest that actions
of adenine nucleotides and caffeine on the RyR1 channel may also depend
on the redox state. Adenine nucleotides increase CICR activity without changing Ca2+ sensitivity. Caffeine sensitizes RyR channels
to Ca2+ for activation and also increases the activity at
the optimum Ca2+ concentrations (for example, caffeine
increased Po of the pCMPS-activated channel at
pCa 5 in Fig. 6B). Thus both drugs have a common positive effect on channel activity, irrespective of the Ca2+
sensitivity. Recently, Xia et al. (39) reported that the
initial rate of ryanodine binding to the RyR vesicles depends on redox potentials in a solution without changing Ca2+ sensitivity;
oxidation increased the initial binding rate, whereas reduction
decreased it (see Fig. 3 in Ref. 39). They suggested that
the magnitude of the channel response may be set by the cellular redox
potential. In addition, they showed that Ca2+,
Mg2+, and caffeine modulate the redox potential of SH
residues involved in activation of the RyR. Therefore, it is reasonable
to assume that the redox potential is a primary factor determining the
maximum channel activity and may alter the effects of CICR modulators. If this assumption is true, then the redox states might greatly modulate the channel-activating action of caffeine or adenine nucleotides. At the present time, however, definite conclusions cannot
be reached, partly because of large variations of
Po. This primary modulating effect of redox
potential may explain why an extreme reduction of the RyR molecule
produced by exposure to DTT or setting redox potential to
231 mV
failed to activate the channel on application of adenine nucleotide or
caffeine (Figs. 5 and 8).
Total GSH (GSH + GSSG) concentration in skeletal muscle has been
estimated to be ~3 mM and GSSG to be ~50 µM (16,
32). Redox potential in the cell cytosol is estimated to be
about
220 to
230 mV, although the intraluminal side of the SR has
been reported to be in an oxidative state (redox potential
approximately
180 mV; see Ref. 10). This means that
cytoplasm of resting muscle cells probably is in a reduced state. In
the present experiment, fixation of the cis potential to
220 mV using a [GSH]/[GSSG] buffer slightly decreased the
Po of the high-Po
channel, but further reduction to
231 mV led to a marked decrease in
Po (Fig. 8). Such low-Po
channels did not respond to AMPPCP. No response of the channels to
AMPPCP was observed when trans redox potential of the
low-Po channel was fixed at
180 mV. Thus, in
resting muscle cells, the CICR activity of the RyR channel may be set
at a lower level by sulfhydryl reduction, even in the presence of high
concentrations of intracellular ATP (~5 mM; see Ref. 19)
and even if the intraluminal side of the SR were in an oxidized state
(10). In contrast, setting the cis potential to
180 mV markedly increased Po of the
low-Po channel and sensitized channels to AMPPCP
(Fig. 8). Therefore, if the redox potential of cytoplasm would become
more oxidized, the activity of the RyR channel would be enhanced.
Repetitive muscle contractions, as occur in strenuous exercise, produce
reactive oxygen species (6, 15, 28, 30, 34) that oxidize
GSH to increase in intracellular GSSG, resulting in a shift of the intracellular redox balance toward an oxidative state. It has been
reported that skeletal muscle dysfunction may occur after intense
exercise (29, 30). The activation of RyR channels through
the shift in the myoplasmic redox potential might also be involved in
the pathophysiological changes of skeletal muscles.
 |
ACKNOWLEDGEMENTS |
This work was supported by Grants-in-aid for Scientific Research
09470013 and 1267044 (to T. Oba) from the Japan Society for the
Promotion of Science.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: T. Oba,
Dept. of Physiology, Nagoya City Univ. Medical School, Mizuho-ku, Nagoya 467-8601, Japan (E-mail: tooba{at}med.nagoya-cu.ac.jp).
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.
10.1152/ajpcell.01273.2000
Received 17 October 2000; accepted in final form 22 October 2001.
 |
REFERENCES |
1.
Abramson, JJ,
and
Salama G.
Sulfhydryl oxidation and Ca2+ release from sarcoplasmic reticulum.
Mol Cell Biochem
82:
81-84,
1988[ISI][Medline].
2.
Abramson, JJ,
Trimm JL,
Weden L,
and
Salama G.
Heavy metals induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle.
Proc Natl Acad Sci USA
80:
1526-1530,
1983[Abstract].
3.
Abramson, JJ,
Zable AC,
Favero TG,
and
Salama G.
Thimerosal interacts with the Ca2+ release channel ryanodine receptor from skeletal muscle sarcoplasmic reticulum.
J Biol Chem
270:
29644-29647,
1995[Abstract/Free Full Text].
4.
Ahern, GP,
Junankar PR,
and
Dulhunty AF.
Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.
Biophys J
72:
146-162,
1997[Abstract].
5.
Copello, AJ,
Barg S,
Onoue H,
and
Fleischer S.
Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.
Biophys J
73:
141-156,
1997[Abstract].
6.
Davies, K,
Quintanilha A,
Brooks G,
and
Packer L.
Free radicals and tissue damage produced by exercise.
Biochem Biophys Res Commun
107:
1198-1205,
1982[ISI][Medline].
7.
Donoso, P,
Anacena P,
and
Hidalgo C.
Sulfhydryl oxidation overrides Mg2+ inhibition of calcium induced calcium release in skeletal muscle triads.
Biophys J
79:
279-286,
2000[Abstract/Free Full Text].
8.
Eager, KR,
Roden LD,
and
Dulhunty AG.
Actions of sulfhydryl reagents on single ryanodine receptor Ca2+-release channels from sheep myocardium.
Am J Physiol Cell Physiol
272:
C1908-C1918,
1997[Abstract/Free Full Text].
9.
Favero, TG,
Zable AC,
and
Abramson JJ.
Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.
J Biol Chem
270:
25557-25563,
1995[Abstract/Free Full Text].
10.
Feng, W,
Liu G,
Allen PD,
and
Pessah IN.
Transmembrane redox sensor of ryanodine receptor complex.
J Biol Chem
275:
35902-35907,
2000[Abstract/Free Full Text].
11.
Franzini-Armstrong, C,
and
Protasi F.
Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions.
Physiol Rev
77:
699-729,
1997[Abstract/Free Full Text].
12.
Haarmann, CS,
Fink RHA,
and
Dulhunty AF.
Oxidation and reduction of pig skeletal muscle ryanodine receptors.
Biophys J
77:
3010-3022,
1999[Abstract/Free Full Text].
13.
Hwang, C,
Sinskey AJ,
and
Lodish HF.
Oxidized redox state of glutathione in the endoplasmic reticulum.
Science
257:
1496-1502,
1992[ISI][Medline].
14.
Ikemoto, N,
Ranjat M,
Meszaros LG,
and
Koshita M.
Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum.
Biochemistry
28:
6764-6771,
1989[ISI][Medline].
15.
Jackson, MJ,
Edwards RHT,
and
Symons MCR
Electron spin resonance studies of intact mammalian skeletal muscle.
Biochim Biophys Acta
845:
185-190,
1985.
16.
Kondo, H,
Miura M,
Kodama J,
Ahmed SM,
and
Itokawa Y.
Role of ion in oxidative stress in skeletal muscle atrophied by immobilization.
Pfleugers Arch
421:
295-297,
1992[ISI][Medline].
17.
Liu, G,
Abramson JJ,
Zable AC,
and
Pessah IN.
Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3- (4'-maleimidylphenyl)
4-methylcoumarin.
Mol Pharmacol
45:
189-200,
1994[Abstract].
18.
Marengo, JJ,
Hidalgo C,
and
Bull R.
Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells.
Biophys J
74:
1263-1277,
1998[Abstract/Free Full Text].
19.
McCartney, N,
Spriet LL,
Heigenhauser GJ,
Kowalchuk JM,
Sutton JR,
and
Jones NL.
Muscle power and metabolism in maximal intermittent exercise.
J Appl Physiol
60:
1164-1169,
1986[Abstract/Free Full Text].
20.
Meissner, G.
Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors.
Annu Rev Physiol
56:
485-508,
1994[ISI][Medline].
21.
Murayama, T,
Oba T,
Katayama E,
Oyamada H,
Oguchi K,
Kobayashi M,
Otsuka K,
and
Ogawa Y.
Further characterization of type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.
J Biol Chem
274:
17297-17308,
1999[Abstract/Free Full Text].
22.
Murayama, T,
and
Ogawa Y.
Purification and characterization of two ryanodine-binding protein isoforms from sarcoplasmic reticulum of bullfrog skeletal muscle.
J Biochem (Tokyo)
112:
514-522,
1992[Abstract].
23.
Murayama, T,
and
Ogawa Y.
Characterization of type 3 ryanodine receptor (RyR3) of sarcoplasmic reticulum from rabbit skeletal muscles.
J Biol Chem
272:
24030-24037,
1997[Abstract/Free Full Text].
24.
Oba, T,
Ishikawa T,
and
Yamaguchi M.
Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors.
Am J Physiol Cell Physiol
274:
C914-C921,
1998[Abstract/Free Full Text].
25.
Oba, T,
Koshita M,
and
Yamaguchi M.
H2O2 modulates twitch tension and increases Po of Ca2+ release channel in frog skeletal muscle.
Am J Physiol Cell Physiol
271:
C810-C818,
1996[Abstract/Free Full Text].
26.
Ogawa, Y.
Role of ryanodine receptors.
Crit Rev Biochem Mol Biol
29:
229-274,
1994[Abstract].
27.
Ogawa, Y,
Kurebayashi N,
and
Murayama T.
Ryanodine receptor isoforms in excitation-contraction coupling.
Adv Biophys
36:
27-64,
1999[ISI][Medline].
28.
O'Neill, CA,
Stebbins CL,
Bonigut S,
Halliwell B,
and
Longhurst JC.
Production of hydroxyl radicals in contracting skeletal muscle of cats.
J Appl Physiol
81:
1197-1206,
1996[Abstract/Free Full Text].
29.
Rajgura, SU,
Yeargans GS,
and
Seidler NW.
Exercise causes oxidative damage to rat skeletal muscle microsomes while increasing cellular sulfhydryls.
Life Sci
54:
149-157,
1994[ISI][Medline].
30.
Reid, MB,
Haack KE,
Franchek KM,
Valberg PA,
Kobzik L,
and
West MS.
Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro.
J Appl Physiol
75:
1081-1087,
1993[Abstract].
31.
Salama, G,
and
Abramson J.
Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum.
J Biol Chem
259:
13363-13369,
1984[Abstract/Free Full Text].
32.
Sen, CK.
Oxidants and antioxidants in exercise.
J Appl Physiol
79:
675-686,
1995[Abstract/Free Full Text].
33.
Shou, W,
Aghdasi B,
Armstrong DL,
Guo Q,
Bao S,
Charng MJ,
Mathews LM,
Schneider MD,
Hamilton SL,
and
Matzuk MM.
Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12.
Nature
391:
489-492,
1998[ISI][Medline].
34.
Sjodin, B,
Westing YH,
and
Apple FS.
Biochemical mechanisms for oxygen free radical formation during exercise.
Sports Med
10:
236-254,
1990[ISI][Medline].
35.
Stoyanovsky, D,
Murphy T,
Anno PR,
Kim YM,
and
Salama G.
Nitric oxide activates skeletal and cardiac ryanodine receptors.
Cell Calcium
21:
19-29,
1997[ISI][Medline].
36.
Sun, J,
Xu L,
Eu JP,
Stamler JS,
and
Meissner G.
Classes of thiols that influence the activity of the skeletal muscle calcium release channel.
J Biol Chem
276:
15625-15630,
2001[Abstract/Free Full Text].
37.
Sutko, JL,
and
Airey JA.
Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function?
Physiol Rev
76:
1027-1071,
1996[Abstract/Free Full Text].
38.
Timerman, AP,
Ogunbunmi E,
Freund EA,
Wiederrecht G,
Marks AM,
and
Fleischer S.
The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum.
J Biol Chem
268:
22992-22999,
1993[Abstract/Free Full Text].
39.
Xia, R,
Stangler T,
and
Abramson JJ.
Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators.
J Biol Chem
275:
36556-36561,
2000[Abstract/Free Full Text].
40.
Zable, AC,
Favero TG,
and
Abramson JJ.
Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum.
J Biol Chem
272:
7069-7077,
1997[Abstract/Free Full Text].
41.
Zhang, L,
Kelley J,
Schmeisser G,
Kobayashi YM,
and
Jones LR.
Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor.
J Biol Chem
272:
13389-13397,
1997.
Am J Physiol Cell Physiol 282(4):C684-C692
0363-6143/02 $5.00
Copyright © 2002 the American Physiological Society