Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Hydrogen peroxide (H2O2) at millimolar concentrations induces Ca2+ release from actively loaded sarcoplasmic reticulum vesicles and induces biphasic [3H]ryanodine binding behavior. High affinity [3H]ryanodine binding is enhanced at concentrations from 100 μM to 10 mM (3-4-fold). At H2O2 concentrations greater than 10 mM, equilibrium binding is inhibited. H2O2 decreased the kd for [3H]ryanodine binding by increasing its association rate, while having no effect on the rate of dissociation of [3H]ryanodine from its receptor. H2O2 (1 mM) also reduced the EC50 for Ca2+ activation from 632 nM to 335 nM. These effects were completely abolished in the presence of catalase, ruthenium red, and/or Mg2+ (mM). H2O2-stimulated [3H]ryanodine binding is not further enhanced by either doxorubicin or caffeine. The direct interaction between H2O2 and the Ca2+ release mechanism was further demonstrated in single-channel reconstitution experiments. Peroxide, at submillimolar concentrations, activated the Ca2+ release channel following fusion of a sarcoplasmic reticulum vesicle to a bilayer lipid membrane. At millimolar concentrations of peroxide, Ca2+ channel activity was inhibited. Peroxide stimulation of Ca2+ channel activity was reversed by the thiol reducing agent dithiothreitol. Paralleling peroxide induced activation of ryanodine binding, Ca2+ transport, and single Ca2+ channel activity, it was observed that the ryanodine receptor formed large disulfidelinked protein complexes that dissociated upon addition of dithiothreitol.


Hydrogen peroxide (H 2 O 2 ) at millimolar concentrations induces Ca 2؉ release from actively loaded sarcoplasmic reticulum vesicles and induces biphasic [ 3 H]ryanodine binding behavior. High affinity [ 3 H]ryanodine
binding is enhanced at concentrations from 100 M to 10 mM (3-4-fold). At H 2 O 2 concentrations greater than 10 mM, equilibrium binding is inhibited. H 2 O 2 decreased the k d for [ 3 H]ryanodine binding by increasing its association rate, while having no effect on the rate of dissociation of [ 3 H]ryanodine from its receptor. H 2 O 2 (1 mM) also reduced the EC 50 for Ca 2؉ activation from 632 nM to 335 nM. These effects were completely abolished in the presence of catalase, ruthenium red, and/or Mg 2؉ (mM).

H 2 O 2 -stimulated [ 3 H]ryanodine binding is not further enhanced by either doxorubicin or caffeine. The direct interaction between H 2 O 2 and the Ca 2؉ release mechanism was further demonstrated in single-channel reconstitution experiments.
Peroxide, at submillimolar concentrations, activated the Ca 2؉ release channel following fusion of a sarcoplasmic reticulum vesicle to a bilayer lipid membrane. At millimolar concentrations of peroxide, Ca 2؉ channel activity was inhibited. Peroxide stimulation of Ca 2؉ channel activity was reversed by the thiol reducing agent dithiothreitol. Paralleling peroxide induced activation of ryanodine binding, Ca 2؉ transport, and single Ca 2؉ channel activity, it was observed that the ryanodine receptor formed large disulfidelinked protein complexes that dissociated upon addition of dithiothreitol.
Reactive oxygen species (ROS) 1 have been shown to mediate various pathological conditions in a variety of tissues (1). These molecular oxygen-derived intermediates may be generated either by electron reduction or energy activation (i.e. light). Among these ROS include hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), hypochlorous acid (HOCl), superoxide radical (O 2 . ), and the hydroxyl radical ( ⅐ OH).
In various muscle types, ROS have been implicated in alteration of normal Ca 2ϩ homeostasis via disruption of normal sarcoplasmic reticulum (SR) function. This may be accomplished by inhibiting the Ca 2ϩ ATPase pump and/or by activating the Ca 2ϩ release channel. A number of studies have detailed the effects of one or more ROS on whole muscle tissue or on isolated sarcoplasmic reticulum (SR) derived from smooth, cardiac, and/or skeletal muscles (2)(3)(4)(5)(6)(7)(8)(9)(10). Treatment of these tissues with ROS attempt to induce oxidative stress similar to that experienced during ischemia and/or reperfusion. In smooth muscle, O 2 . has been shown to inhibit both Ca 2ϩ -ATPase activity and Ca 2ϩ uptake into the SR while stimulating inositol 1,4,5-trisphosphate-induced Ca 2ϩ release (3,4). In cardiac muscle, HOCl reduced contractile function (7) and inhibited SR Ca 2ϩ uptake (11). On a more microscopic level, H 2 O 2 activated cardiac SR Ca 2ϩ channel gating activity at mM concentrations (9). Superoxide also decreased Ca 2ϩ uptake into cardiac SR vesicles by increasing the Ca 2ϩ permeability of the SR via opening the SR Ca 2ϩ release channel (2). These studies indicate that, while it is not the only intracellular organelle effected, the SR is a likely target for ROS in these tissues. Reduced oxygen species may also interfere with skeletal muscle function. Reperfusion of ischemic muscle, vitamin E deficiency, and muscular dystrophy are important pathological conditions where ROS have been implicated in cytotoxic damage (12). Reactive oxygen intermediates generated during repetitive exercise have also been shown to promote muscle fatigue (13). Despite this evidence, only a few studies have examined the direct effect of ROS on skeletal muscle and SR function. The oxidant peroxydisulfate and the ⅐ OH radical both inhibited Ca 2ϩ uptake into isolated SR vesicles, while peroxydisulfate also increased the Ca 2ϩ permeability of the SR membrane (8). In other work describing the effect of sulfhydryl oxidizing reagents on SR Ca 2ϩ release, HOCl, and the highly reactive 1 O 2 , were shown to stimulate Ca 2ϩ release from actively loaded SR vesicles (10,14). Calcium release induced by ROS was shown to be blocked by SH reducing agents and ruthenium red, indicating the SR Ca 2ϩ release channel was a primary target for their interaction.
Although the mechanism underlying excitation-contraction coupling in skeletal muscle is poorly understood, oxidation of thiols has been shown to regulate the SR Ca 2ϩ release channel, which mediates Ca 2ϩ release preceding muscle contraction (14). Oxidation-induced opening of the SR Ca 2ϩ channel has also been confirmed at the single-channel level following the fusion of SR vesicles to planar lipid bilayer membranes (BLM) (15). In this report, we show that the cellular oxidant, H 2 O 2 , modifies the SR Ca 2ϩ release channel. Moreover, activation of the Ca 2ϩ release channel by H 2 O 2 appears to result from an oxidation of key thiol groups to a disulfide. These results may be significant in understanding muscle cell dysfunction under oxidative stress and may provide insight into the molecular regulation of the Ca 2ϩ release channel.

MATERIALS AND METHODS
Preparation of SR Vesicles-For all studies, sarcoplasmic reticulum vesicles were prepared from rabbit hind leg and back white skeletal muscle according to the method of MacLennan (16). The protein concentration was determined by absorption spectroscopy (17).
Measurement of Ca 2ϩ Fluxes-Ca 2ϩ fluxes across SR vesicles were monitored using a dual wavelength spectrophotometer (14,18), by measuring the differential absorption changes of arsenazo III (ASIII) at 675-685 nm. Ca 2ϩ uptake into SR vesicles (0.2 mg/ml) was carried out in a buffer containing 100 mM KCl, 20 mM Hepes, 1 mM MgCl 2 , 20 M free Ca 2ϩ , and 200 M ASIII. Uptake was initiated by the addition of 0.5 mM Mg 2ϩ -ATP. Upon achieving steady state Ca 2ϩ uptake, release was initiated by the addition of H 2 O 2 , and the free extravesicular Ca 2ϩ concentration was recorded as a function of time. The Ca 2ϩ -ASIII signal was calibrated with the addition of 4 -8 M Ca 2ϩ . Ca 2ϩ uptake and efflux rates were determined from the maximal slope of the extravesicular Ca 2ϩ concentration versus time.
Ca 2ϩ -stimulated ATPase Activity-Ca 2ϩ -independent, Ca 2ϩ -dependent, and A23187-stimulated Ca 2ϩ ATPase activities were determined spectrophotometrically for untreated and peroxide-treated SR vesicle preparations. The standard assay buffer contained 100 mM KCl, 20 mM Hepes, 1 mM MgCl 2 , 1 mM EGTA, 3 mM NADH, 1 mM phosphoenol pyruvate, 5 units of lactate dehydrogenase, 5 units of pyruvate kinase, and 0.5 mM Mg-ATP at pH 7.0 in a 1-ml volume. This assay was carried out in a stepwise fashion. Ca 2ϩ -independent (Mg 2ϩ -dependent) ATPase activity was initiated by the addition of SR (0.1 mg) to the cuvette, and the absorbance changes at 340 nm were recorded for 2 min (22°C). To measure Ca 2ϩ -stimulated activity, 8 M Ca 2ϩ was added, and again the absorbance changes were monitored for 3 min. The Ca 2ϩ ionophore, A23187, was added and the reaction run to completion. SR (1.0 mg/ml) was incubated at several different H 2 O 2 concentrations for 1 min prior to conducting the various assays. Calcium uptake and Ca 2ϩ ATPase assays were carried out in identical buffers. The data are the average of three independent experiments and are expressed as a percentage of the control.
[ 3 H]Ryanodine Binding-Ryanodine binding measurements were conducted according to the methods of Pessah et al. (19). Briefly, SR membranes (200 g/ml) were incubated at 37°C for 3 h (unless otherwise noted) in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [ 3 H]ryanodine, and 20 mM Hepes at pH 7.1. Depending on the conditions of the assay, Ca 2ϩ (100 M EGTA ϩ various concentrations of Ca 2ϩ ), and H 2 O 2 were present during the incubation procedure. The binding reaction was quenched by rapid filtration through Whatman GF/B glass fiber filters, which were then rinsed with 5 ml of ice-cold buffer containing 50 M Ca 2ϩ . The filters were placed in polytubes (Fisher), filled with 3 ml of scintillation mixture (Beckman, ReadySafe), shaken overnight, and counted the following day. The experiments were repeated at least twice on two different SR preparations with essentially identical results. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ryanodine. For details of individual experiments refer to figure captions.
Measurement of Association/Dissociation Kinetics-The association rate of [ 3 H]ryanodine was measured by quenching the binding reaction by filtration at times ranging from 1 to 480 min after the addition of SR vesicles to the [ 3 H]ryanodine reaction medium. Dissociation of [ 3 H]ryanodine from the equilibrium complex followed equilibration of 1 nM [ 3 H]ryanodine with SR membranes for 2 h at 37°C. The SR was then diluted into a 100-fold excess of the assay medium, or the assay medium containing 1 or 10 mM H 2 O 2 . Determinations of residual specific binding were made at times ranging from 5 to 160 min.
Equilibrium Binding Analysis-Equilibrium binding data from saturation analysis were fit to a one-site model. The dissociation constant, K d , and the maximal binding capacity, B max , were determined using the Enzfitter computer program.
Single Ca 2ϩ Channel Analysis-Ca 2ϩ release channel reconstitution into a bilayer membrane was carried out as described elsewhere (21). Briefly, isolated SR vesicles, suspended in 0.3 M sucrose, were added to the cis side of a dual chamber bilayer set-up, separated by a planar bilayer lipid membrane (BLM) composed of a 5:3 mixture of phosphatidylethanolamine and phosphatidylserine at 50 mg/ml suspended in decane. The cis chamber contained 500 mM CsCl, 200 M CaCl 2 , 10 mM Hepes, pH 7.2, while the trans side contained 100 mM CsCl, 10 mM Hepes, pH 7.2. Following the fusion of a single vesicle, 200 M EGTA was added to the cis chamber to stop further fusions and the chamber was perfused with an identical buffer containing no added Ca 2ϩ or EGTA. Subsequent channel activity was measured at a holding potential of ϩ25 mV with respect to the trans (ground) side. In bilayer experiments carried out with H 2 O 2 , following addition of the oxidant, the cis chamber was stirred for at least 2 min prior to recording data. The single-channel data were then processed, stored on VCR tape, and analyzed by computer for channel activity (15). All single-channel analyses were performed using pClamp 5.0 software (Axon Instruments, Burlingame, CA).
Sample Preparation-SR vesicles (80 g) were suspended in 0.01 ml of buffer containing 20 mM Hepes, 100 mM KCl, pH 7.0. To duplicate samples, 0, 1, 10, or 100 mM H 2 O 2 was added and incubated for 10 min at room temperature. Subsequently, catalase at 330 units/ml (final) was added, and the samples were again incubated for 10 min. These samples were then boiled for 1 min with Laemmli sample buffer, with and without the reducing agent DTT (2 mM), and electrophoresed. The gels were stained overnight in 45% methanol, 10% acetic acid, and 0.10% Coomassie Blue and destained in an identical solution without Coomassie Blue.
Materials-All reagents were analytical grade. Hepes was obtained from Research Organics (Cincinnati, OH). [ 3 H]Ryanodine was purchased from DuPont NEN, and ryanodine-dehydroryanodine was purchased from Agrisystems Int. (Wind Gap, PA). All other chemicals were obtained from Sigma.

RESULTS
Ca 2ϩ Transport Measurements-Steady-state Ca 2ϩ -dependent ATPase activity of intact SR vesicles was observed to be stimulated biphasically following exposure to H 2 O 2 for 1 min. The stimulation of ATPase activity induced by H 2 O 2 (1-20 mM) parallels a reduction in both the rate and amount of Ca 2ϩ uptake when the SR membrane was pretreated with H 2 O 2 ( Fig.  1). However, in the presence of the Ca 2ϩ ionophore A23187, ATPase activity was not modified by H 2 O 2 concentrations up to 80 mM. It appeared that, in the absence of the Ca 2ϩ ionophore, modification of ATPase activity and Ca 2ϩ uptake were due to alterations in the Ca 2ϩ permeability of the SR induced by H 2 O 2 .
In Fig. 2, it was demonstrated that H 2 O 2 directly stimulated Ca 2ϩ release from actively loaded SR vesicles. Calcium was released at concentrations of H 2 O 2 as low as 1 mM, while the release rates appeared to saturate between 50 and 80 mM H 2 O 2 . The maximal release rate derived from the kinetic eval- In the absence of H 2 O 2 , Ca 2ϩ -dependent ATPase activity ϭ 0.45 mol/ mg/min; A23187-stimulated ATPase activity ϭ 1.20 mol/mg/min; total amount of Ca 2ϩ uptake ϭ 111 nmol/mg; and the maximal rate of Ca 2ϩ uptake ϭ 12.98 nmol/mg/s. uation using the program ENZFITTER (Elsvier-Biosoft) was 7.28 Ϯ 0.51 nmol/mg/min, while the EC 50 was 12.9 Ϯ 2.8 mM.
When Ca 2ϩ release was measured in the presence of the Ca 2ϩ channel inhibitor, ruthenium red (10 M), no release of Ca 2ϩ was observed with up to 80 mM H 2 O 2 . The ability to block Ca 2ϩ release with a specific channel inhibitor suggests that H 2 O 2 was affecting the Ca 2ϩ release mechanism of SR, and was not causing an increase in the SR Ca 2ϩ permeability by nonspecific effects such as lipid peroxidation.
[ 3 H]Ryanodine Binding Studies-A more direct assay to monitor the interaction between the Ca 2ϩ release protein and H 2 O 2 was obtained through the use of the highly specific Ca 2ϩ channel probe, ryanodine. H 2 O 2 caused a biphasic interaction between [ 3 H]ryanodine and its receptor (Fig. 3 When catalase, an enzyme that detoxifies H 2 O 2 by converting it to H 2 O and O 2 , was present at 5 units/ml in the incubation medium, no stimulation or reduction in binding was observed (Fig. 3). In addition, when assayed in the presence of 5 M ruthenium red, H 2 O 2 failed to enhance [ 3 H]ryanodine binding (Table II). These data further suggest that H 2 O 2 interacts directly with the SR Ca 2ϩ release channel.
In order to more fully describe the interaction between H 2 O 2 and the ryanodine receptor, time-dependent association and dissociation experiments were conducted. Increasing H 2 O 2 concentrations stimulated the rate of association of [ 3 H]ryanodine to its receptor ( Fig. 4a and Table I). A linear transformation of the association data highlighted this more clearly (Fig. 4b).
Interestingly, at 100 mM H 2 O 2, when equilibrium measurements (3-h incubation) indicated only a small amount of ryanodine binding, time-dependent measurements show a rapid acceleration in the rate of ligand binding followed by a rapid dissociation.
Experiments (at 1 and 10 mM H 2 O 2 ) were also carried out to determine the influence of H 2 O 2 on the dissociation of bound [ 3 H]ryanodine from its receptor (Fig. 5 and Table I). SR vesicles were labeled with [ 3 H]ryanodine in the absence of H 2 O 2 and allowed to equilibrate for 2 h at 37°C. Aliquots were subsequently diluted 100-fold into a buffer that contained 0, 1, or 10 mM H 2 O 2 without ryanodine. Peroxide in the dissociation buffer had no effect on the dissociation of [ 3 H]ryanodine from its receptor (Fig. 5 and Table I). These data coupled with the H 2 O 2 -dependent increase in the rate of association suggested that the increase in [ 3 H]ryanodine binding caused by H 2 O 2 (Fig. 3) was due solely to an enhancement of the ryanodine association kinetics.
An increase in association rate with little or no change in the dissociation rate should result in a decrease in the equilibrium dissociation constant, K d . In equilibrium binding studies (Fig.  6), we observed that 1 mM H 2 O 2 reduced the K d , from 10.19 to 7.17 nM, a result in agreement with the K d calculated from the ratio of K Ϫ1 /K ϩ1 (Table I). With either method of calculating the K d , there is a small increase in the affinity of the receptor for binding ryanodine in the presence of peroxide (1 mM). As shown in Fig. 6, there is also an increase in the number of high affinity ryanodine binding sites (B max :control ϭ 4.2; 1 mM peroxide, 5.4 pmol/mg).
Several activators, including caffeine, have been shown to alter the Ca 2ϩ -dependent binding of ryanodine to its receptor. In Fig. 7, it is demonstrated that H 2 O 2 sensitized the ryanodine receptor to activation by Ca 2ϩ . In the absence of H 2 O 2 , the EC 50 for Ca 2ϩ binding was determined to be 632 nM, while in the presence of 1 mM H 2 O 2 , the EC 50 was 335 nM Ca 2ϩ . It was also observed that known activators and inhibitors of Ca 2ϩ release similarly affect peroxide's ability to stimulate high affinity [ 3 H]ryanodine binding to SR (Table II). Activation of ryanodine binding by H 2 O 2 was considerably decreased by two Ca 2ϩ channel inhibitors, Mg 2ϩ and ruthenium red. H 2 O 2 had no effect on the ability of doxorubicin to stimulate binding, since binding had already been maximally activated by doxorubicin. However, H 2 O 2 stimulated binding well beyond that induced by 5 mM caffeine.
Peroxide's interaction with the Ca 2ϩ release mechanism from SR could result from an oxidation of critical thiols on the receptor, which might be expected to be reversible upon addition of reducing agents. To determine if the stimulatory effect   (Fig.  8). This suggested that H 2 O 2 -stimulated binding was due to an oxidation of sulfhydryl groups, associated with the ryanodine receptor, to disulfides. When catalase alone was added following the initial 30-min incubation period, the stimulatory effect of H 2 O 2 persisted. The oxidation-induced stimulatory effect of H 2 O 2 on [ 3 H]ryanodine binding occurred during the initial incubation period and continued throughout the course of the binding experiment. This effect was persistent unless DTT reduced the oxidized SH groups to their native state.
SDS-PAGE-Polyacrylamide gel electrophoresis revealed a distinct loss of the 450-kDa protein at 1, 10, and 100 mM H 2 O 2 (10-min exposure followed by catalase treatment) (Fig. 9). This band was confirmed to be the ryanodine receptor/Ca 2ϩ channel by Western blotting techniques using monoclonal antibody probe specific to the ryanodine receptor (not shown). When the SR was subjected to reducing conditions (2 mM DTT) following the H 2 O 2 exposure, the mobility of the 450-kDa protein returned to that of control (untreated with H 2 O 2 ).
Single-channel Measurements-Stimulation of single-channel activity of a reconstituted SR Ca 2ϩ release channel following exposure to 0.1 mM H 2 O 2 was observed in Fig. 10. At higher peroxide concentrations (1-10 mM), Ca 2ϩ channel activity was inhibited (Fig. 10, traces C and D). The peroxide-inactivated channel could not be reactivated by addition of 100 M Ca 2ϩ (trace E).
In the range of H 2 O 2 concentrations (0.1-0.5 mM) for Ca 2ϩ channel activation, addition of the reducing agent DTT inhibited channel activity by decreasing the open probability (P o ). Neither the H 2 O 2 or DTT had any effect on the single-channel conductance. As shown in Fig. 11

DISCUSSION
Recently, ROS have drawn attention for their potential to disrupt normal muscle function by targeting specific proteins for modification. H 2 O 2 is reportedly produced physiologically in muscle cells undergoing oxidative stress (ischemia, reperfusion, exercise, etc.). Modification of the Ca 2ϩ release mechanism of sarcoplasmic reticulum by H 2 O 2 may promote muscle dysfunction during such pathophysiological conditions. We have examined the interaction between hydrogen peroxide and the Ca 2ϩ transport mechanisms from skeletal muscle SR.
When ATPase activity and Ca 2ϩ uptake were assayed as a function of H 2 O 2 concentration with intact SR vesicles, a biphasic concentration dependence was observed. Peroxide concentrations up to 20 mM increased the permeability of intact SR vesicles, which resulted in an increased Ca 2ϩ -dependent ATPase activity and a decreased net accumulation of Ca 2ϩ . This was not due to an interaction with the Ca 2ϩ pump, since peroxide did not affect the ability of the Ca 2ϩ -ATPase to hydrolyze ATP when analyzed in the presence of the Ca 2ϩ ionophore, A23187. Exposure of the SR to H 2 O 2 concentrations greater than 20 mM apparently decreased the Ca 2ϩ permeability of the SR, which stimulated Ca 2ϩ uptake into SR vesicles and inhibited the steady-state ATPase activity.
An apparent discrepancy between the biphasic effect of H 2 O 2 on ATPase activity and active Ca 2ϩ uptake (Fig. 1) and the monophasic stimulation of Ca 2ϩ release (Fig. 2) can be explained by differences in the manner in which these assays were carried out. In Fig. 2, the SR vesicles were first actively loaded with Ca 2ϩ , and then exposed to H 2 O 2 . The initial Ca 2ϩ release rate is a measure of the initial rate at which H 2 O 2 reacts with the Ca 2ϩ release mechanism and opens the Ca 2ϩ release channel. In contrast, the data presented in Fig. 1 were  The K obs and K Ϫ1 were determined from nonlinear regression of the data shown in Fig. 4 using the program ENZFITTER. K ϩ1 ϭ (K obs Ϫ K Ϫ1 )/[L] where [L] is the ligand concentration. The K d (Scatchard) was derived from the plot in Fig. 6. taken over a period of several minutes and represent measurements of ATPase activity and net Ca 2ϩ uptake following exposure of SR vesicles to H 2 O 2 . As shown in Fig. 4a, high concentrations of H 2 O 2 (10 -100 mM) first activated the release mechanism and then inhibited the Ca 2ϩ channel as a function time. Thus, the effect of high concentrations of H 2 O 2 was to stimulate rapid initial release of Ca 2ϩ from SR vesicles. However, the Ca 2ϩ ATPase activity and Ca 2ϩ uptake, which was measured on the time scale of several minutes, displayed a biphasic H 2 O 2 concentration dependence (Fig. 1). While activation of Ca 2ϩ release by H 2 O 2 (EC 50 Х 13 mM) and modification of Ca 2ϩ -dependent ATPase activity occurs at [H 2 O 2 ] of approximately 15 mM, submillimolar concentrations of H 2 O 2 were observed to activate high affinity ryanodine binding (EC 50 Х 0.65 mM). A higher concentration of H 2 O 2 is required to modify Ca 2ϩ fluxes across SR vesicles, than to alter ryanodine binding. A likely explanation for this discrepancy is that the ryanodine binding measurements, which were carried out over a time scale of hours, represent equilibrium measurements. SR thiols were oxidized in less than 30 min (Fig. 8). The transport assays, however, do not reflect equilibium measure-ments. A relatively large excess of H 2 O 2 was required to oxidize thiols and modify transport on the time scale of the assays shown in Figs. 1 and 2.
The mechanism underlying H 2 O 2 -induced Ca 2ϩ release appears to involve a direct interaction with the ryanodine receptor from the terminal cisternae. Peroxide had a biphasic effect on high affinity [ 3 H]ryanodine binding to the receptor (Fig. 3).   (Fig. 6) and induced rapid Ca 2ϩ release (Fig. 2). H 2 O 2 slightly decreased the K d for [ 3 H]ryanodine binding (Fig. 6) by accelerating the rate at which ryanodine binds to its receptor (Fig. 4, a and b) (Fig. 4). This time-dependent loss of ryanodine binding led to complete inactivation of the receptor, which suggests that the receptor was damaged via intermolecular cross-linking, extensive protein oxidation, or destruction of its supporting membrane by lipid peroxidation.
Peroxide-induced stimulation of Ca 2ϩ release, [ 3 H]ryanodine binding, and single-channel activity appears to be mediated by sulfhydryl interactions on the Ca 2ϩ release protein. In Figs. 8 and 11, it is demonstrated on the level of ryanodine binding and single-channel activity that the stimulatory effect of H 2 O 2 was reversible upon addition of thiol reducing agents. Following incubation with H 2 O 2 , the oxidant may be removed (by the addition of catalase) without altering its stimulatory effect (Fig. 8). Only when DTT was added did the stimulation of [ 3 H]ryanodine binding return to normal levels. It appears that activation of the Ca 2ϩ release channel via peroxide was mediated by a direct SH oxidation, and that reduction of this disulfide restores the receptor to its native state. This was also supported by the data derived from the electrophoresis experiments (Fig. 9).
Analysis of SR proteins by SDS-PAGE strongly suggests that activation of the release mechanism by H 2 O 2 induced the formation of disulfide-linked high molecular mass aggregates or multi-protein complexes. As the concentration of H 2 O 2 was increased from 1 to 100 mM, we observed a progressive decrease in the density of protein bands at 450 kDa (ryanodine receptor) and 170 kDa. At 100 mM H 2 O 2 , both bands completely disappeared. These data parallel the concentration-dependent increase in association of [ 3 H]ryanodine to its receptor (Fig. 4, a  and b), peroxide-stimulated Ca 2ϩ release rate (Fig. 2), and activation of Ca 2ϩ channel activity by peroxide (Fig. 10). These data directly demonstrate that during activation of the SR Ca 2ϩ release channel by H 2 O 2 , several proteins form a complex via oxidation of protein thiols localized on the SR Ca 2ϩ release channel.
It has been previously demonstrated that oxidation of SH groups to disulfides activates the Ca 2ϩ release mechanism of SR, while reduction of the disulfides inhibits Ca 2ϩ release (14). As shown in Fig. 8, addition of the reducing agent DTT to peroxide-treated SR reduced ryanodine binding to a level equal to that observed under control conditions. The addition of DTT also restored the high molecular mass complex containing the 450-kDa junctional foot protein to its native state (Fig. 9). Recently, it has been shown that during activation of Ca 2ϩ release by a number of known channel activators (Ca 2ϩ , caffeine, ATP, and ryanodine), hyperreactive thiols associated with the ryanodine receptor and triadin no longer interact with nanomolar concentrations of the fluorescent maleimide, 7-di- ethylamino-3(4Ј-maleimidyl phenyl)-4-methycoumarin (21). Moreover, activation leads to the formation of a high molecular weight complex linked via disulfide bridges (22). These results strongly suggest that during physiological activation of the Ca 2ϩ release process, the Ca 2ϩ release channel is disulfidelinked to other key SR proteins, and that reduction of these disulfide linkages results in channel closure. The formation of a disulfide-linked protein complex induced by the addition of H 2 O 2 may reflect a normal protein-protein interaction that occurs during Ca 2ϩ release channel activation.
Lower concentrations of H 2 O 2 were required to activate and inhibit Ca 2ϩ channel activity in single-channel measurements (Figs. 10 and 11) than were needed to modify [ 3 H]ryanodine binding (Fig. 3) and Ca 2ϩ release from SR vesicles (Fig. 2). Although the reasons for this higher sensitivity of the reconstituted channel are not well understood, this phenomenon has been previously observed with other agonists in this and other laboratories (23). Interestingly, the concentrations of peroxide required to activate single-channel gating in skeletal SR are substantially lower than those required to activate Ca 2ϩ channel gating in single cardiac Ca 2ϩ channels. At micromolar Ca 2ϩ concentrations, 3-5 mM peroxide was required to activate single-channel activity (9). Consistent with our results, cardiac SR Ca 2ϩ channel activation was also inhibited by the addition of the disulfide reducing agent DTT.
Oxidation of protein thiols by ROS may be relevant in various pathophysiological conditions. Harmful oxygen species have been shown to disrupt normal cell Ca 2ϩ concentrations during reperfusion of cardiac (24) and skeletal muscle tissue (25) following an extended period of ischemia (24). In heart muscle, this may also be accompanied by a modification of the Ca 2ϩ transient (26). The increase in resting Ca 2ϩ is most likely mediated by Ca 2ϩ entry via a damaged sarcolemma and/or by the disruption of the internal SR membrane systems.
While it has not been conclusively established that endogenous sulfhydryl reactivity regulates the skeletal muscle Ca 2ϩ release channel in vivo, our current understanding of excita-tion-contraction coupling from experiments performed in vitro strongly suggest that modification of SH groups can control the gating pattern of the SR Ca 2ϩ release channel. The continued use of SH probes may provide us with more detailed information regarding SR Ca 2ϩ channel function.