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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta ,gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -phosphatidylethanolamine, L-alpha -phosphatidyl-L-serine, and L-alpha -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), beta ,gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Distribution of Po values at pCa 4 of RyR 1 channels



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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.


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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.



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Fig. 3.   pCa-Po relationship of each type of channel in various states as follows: low-Po (open circle ), high-Po (), pCMPS-treated low-Po (black-down-triangle ), and DTT-treated high-Po (black-triangle) 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: tau O1 = 0.44 ms (80.2% in relative area) and tau O2 = 2.27 ms (19.8%). The low-Po channel after exposure to 50 µM pCMPS displayed a new, third time constant of tau O3 = 7.81 ms (9.2%) in addition to two time constants of tau O1 = 0.34 ms (53.0%) and tau 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 tau O1 and an increase in that of tau O2. Application of pCMPS to the low-Po channel caused prolongation of the open time, which may account for the increase in Po.

                              
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Table 2.   Comparison of time constants of high-Po RyR1 with those of pCMPS-activated low-Po RyR1 with similar 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.


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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; open circle , 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).


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Fig. 5.   Differential responses of low-Po (A) and high-Po (B) channels to beta ,gamma -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).


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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).


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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; triangle , low-Po channels activated in the presence of 10 µM pCMPS; diamond , high-Po channels inhibited by 30-100 µM DTT. star P < 0.05 and star star 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.


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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 congruent  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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