Skeletal Muscle Ryanodine Receptor Is a Redox Sensor with a Well Defined Redox Potential That Is Sensitive to Channel Modulators*

Hyperreactive sulfhydryl groups associated with the Ca2+ release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca2+ channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the Ca2+ release protein. In contrast, channel inhibitors increase the redox potential, which favors the reduction of disulfides and the closure of the release protein. Modulation of redox potential of reactive thiols may be a general control mechanism by which sarcoplasmic/endoplasmic reticulum, ryanodine receptors/IP3 receptors, control cytoplasmic Ca2+ concentrations.

The sarcoplasmic reticulum (SR) 1 is a subcellular organelle that controls the contractile state of muscle by regulating the Ca 2ϩ concentration in the cytosol. With hydrolysis of ATP, the SR actively accumulates Ca 2ϩ into its lumen leading to muscle relaxation. Depolarization of the transverse tubule membrane results in the release of Ca 2ϩ from the SR and muscle contraction. The Ca 2ϩ release protein is pharmacologically characterized by its ability to bind the plant alkaloid ryanodine with high affinity and high specificity, and hence this protein is now known as the ryanodine receptor (RyR). [ 3 H]ryanodine has been used to identify the Ca 2ϩ release protein and is important in characterizing this receptor (1,2). It has been repeatedly demonstrated that reagents that open the Ca 2ϩ release channel, increase equilibrium binding of ryanodine. The binding of ryanodine has become a functional probe to characterize the open versus closed state of the Ca 2ϩ release mechanism.
Recently, a great deal of attention has focused on understanding the sensitivity of the Ca 2ϩ release mechanism to cellular redox changes (3). It is well established that oxidation of critical thiol groups activates the Ca 2ϩ release mechanism, whereas addition of thiol reducing agents close down the Ca 2ϩ channel (4 -6). Oxidative modification of Ca 2ϩ channel function has been observed at the level of skinned fibers in Ca 2ϩ flux measurements in single channel measurements and at the level of high affinity ryanodine binding measurements (4 -9). Moreover, it has been observed that reactive oxygen species activate Ca 2ϩ release from SR (10 -13) and may act as redox active signaling molecules to activate Ca 2ϩ transport (14). It is clear from the above studies that redox reactions may play a critical role in controlling the kinetics of the Ca 2ϩ release mechanism. Furthermore, from experiments carried out with a fluorescent maleimide, it has been shown that the reactivity of these hyperreactive thiols is very sensitive to the concentration of Ca 2ϩ channel modulators such as Ca 2ϩ , Mg 2ϩ , and caffeine (15).
The redox potential within the cell is controlled by the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), NAD ϩ , and NADH. Zable et al. (16) have shown that GSH inhibits Ca 2ϩ channel activity and equilibrium ryanodine binding, whereas GSSG stimulates the activity of the receptor. This suggests that changes in the cellular redox potential may influence the degree of activation of the Ca 2ϩ release mechanism and effect the myoplasmic Ca 2ϩ concentration and the contractile state of muscle.
Instead of using the more traditional method of measuring equilibrium ryanodine binding, in this study a model was developed in which the rate of ryanodine binding was related to the redox state of the receptor. For the first time, hyperreactive sulfhydryl groups associated with the Ca 2ϩ release mechanism from skeletal muscle SR were shown to have a well defined redox potential, and this redox potential was controlled by physiologically relevant Ca 2ϩ channel activators and inhibitors. Although it is unlikely that the redox state of these thiols controls excitation-contraction coupling, our results suggest that during oxidative stress, these hyperreactive thiols oxidize and activate the SR Ca 2ϩ release mechanism and alter the Ca 2ϩ sensitivity of the release channel. Under mild oxidative stress, relatively small changes in the cellular redox potential can contribute to significant stimulation of the ryanodine receptor.
EXPERIMENTAL PROCEDURES SR vesicles were isolated from rabbit fast twitch skeletal muscle by the method of MacLennan (17) with small modifications. 50 M dithiothreitol and 0.2 g/ml leupeptin were included in all buffers except for the final SR resuspension buffer. Samples were stored in liquid N 2 .
through Whatman GF/B filters mounted on a 48-well Brandel Cell Harvester. Filters were rinsed twice with binding buffer containing 50 M Ca 2ϩ . Scintillation vials were filled with scintillation fluid, shaken overnight, and counted the following day. The initial binding rate was calculated from a linear regression fit of 4 time-dependent measurements of bound ryanodine. The derived slope is the initial rate of ryanodine binding. Subtraction of nonspecific binding does not affect the rate of binding, and therefore no subtraction was made.
SR was labeled with 7-diethylamino-3-(4Ј-maleimidylphenyl)-4methylcoumarin (CPM) using a method similar to that of Liu et al. (15). SR at 0.1 mg/ml was incubated in standard ryanodine binding buffer (without ryanodine) containing 1.0 mM CaCl 2 and 20 nM CPM at room temperature with rigorous stirring for 3 min. The reaction was quenched by addition of EGTA and 0.1-0.3 mM GSH, and samples were then incubated for 10 min at room temperature. Ca 2ϩ -dependent binding of ryanodine was fit to a Hill equation of the form, where BЈ is the initial rate of ryanodine binding (pmol/mg/min), BЈ max is the maximum rate of binding, K d is the apparent affinity for Ca 2ϩ of the receptor, and n H is the Hill coefficient, a measure of the degree of cooperativity for Ca 2ϩ activation of receptor binding. Data were fit using a nonlinear regression curve fitting routine (Sigma Plot for Windows-version 5.0). EC 50 ϭ (K d ) 1/nH . CPM was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma.

RESULTS
The redox potential of the SR sulfhydryl groups that control ryanodine binding was determined by measuring the rate of [ 3 H]ryanodine binding versus the redox potential of the aqueous environment. As shown in Fig. 1A, the amount of binding increased linearly with time over the first 12 min of exposure to [ 3 H]ryanodine. Also, the initial rate of ryanodine binding increased as the solution redox potential became more positive (more oxidizing). Although only four traces are shown in Fig.  1A, in a typical redox titration the initial rate of ryanodine binding was measured at twelve different redox potentials.
To determine the redox potential of receptor thiols, the rate of ryanodine binding was measured as a function of the solution redox potential. To fit these data, a simple model is proposed in which two rate constants, k ϩ and k ϩ Ј, describe the rate of ryanodine binding to the reduced receptor, R red and the oxidized receptor, R ox , respectively. As the solution redox potential becomes more positive, the fraction of receptors in the oxidized state increases and the initial rate of ryanodine binding BЈ increases according to Equation 2.
The maximum rate of binding, BЈ max ϭ k ϩ Ј(Ry)(R tot ), occurs when all of the receptors are oxidized (R ox ϭ R tot ), and the minimum rate of binding, BЈ min ϭ k ϩ (Ry)(R tot ) occurs when all of the receptors are reduced (R red ϭ R tot ). Combining these terms yields the following equations.
In a typical redox titration, as shown in Fig. 1B, k ϩ Ј/k ϩ ϭ 5.2 Ϯ 1.4 (based on 21 independent experiments at different Ca 2ϩ concentrations). Interestingly, the ratio of k ϩ Ј/k ϩ was found to be independent of the experimental conditions. Although BЈ min varied slightly with Ca 2ϩ , caffeine, and Mg 2ϩ concentration, the ratio of BЈ max /BЈ min (k ϩ Ј/k ϩ ) was always approximately equal to 5.2.
If two protein sulfhydryl groups are oxidized to a disulfide, upon the addition of oxidized glutathione, the oxidation reac-tion is described by Equation 5.
and therefore is as follows.
The binding of ryanodine (Equation 2) is significantly slower than the oxidation reaction (Equation 5). Reconstitution data with an artificial bilayer lipid membrane has shown that the Ca 2ϩ release protein rapidly responds (less than 1 min) to changes in the local redox potential (19). It is therefore valid to assume that the oxidation reaction has reached equilibrium before the earliest time point at which ryanodine binding is measured (3 min). Using GSH and GSSG as a redox buffer, the redox potential of the solution is defined by, and the redox potential of the receptor is equal to the following, where the redox potential is referenced to a normal hydrogen electrode. E o is the standard potential of glutathione, Ϫ0.24 V, R is the gas constant (8.31 deg Ϫ1 mol Ϫ1 ), T is the absolute temperature (K), n is the number of electrons transferred (n ϭ 2), and F is the Faraday constant (96, 406 J/V) (20). Thiols that are more easily oxidized have larger values of K ox and more negative redox potentials. Combining Equations 6 -8 yields, where the minimum and maximum initial rates of binding (BЈ min and BЈ max )are obtained from the highly reduced and oxidized data shown at the limits of the redox titration (Fig.  1B). By plotting the left side of Equation 9 versus E sol , the redox potential of the receptor, E RyR is obtained from the x-intercept of Fig. 1C. Moreover, the fact that Fig. 1C is linear with a slope of 1 indicates that Equations 3 and 6 are valid. That is (BЈ Ϫ BЈ min )/(BЈ max Ϫ BЈ) is proportional to GSSG and is inversely proportional to GSH 2 , as is expected for the formation of a pure disulfide. It is not proportional to 1/GSH, which is what would be expected if oxidation to a mixed disulfide had been responsible for activation of the receptor. In Fig. 1B, the initial rate of ryanodine binding, BЈ, is plotted versus the solution redox potential, E sol . The solid line shown in Fig. 1B  where r ϭ 10 (E(sol)ϪE(RyR))nF/2.303RT . As shown in Fig. 1B, the redox potential of thiols associated with activation of ryanodine binding was dependent on the experimental conditions. The trace on the left was derived from SR that is incubated in 0.1-0.3 mM GSH for 10 min at room temperature prior to the measurement of the initial rate of binding at the indicated solution redox potential (E sol ). The trace on the right was derived from a redox titration carried out on SR vesicles without GSH pretreatment. Preincubation of the SR with GSH caused a 65 mV shift in the redox potential of the receptor to more negative values. In both the control and the pre-reduced samples, the maximum and minimum initial rates of binding (BЈ max and BЈ min ) were identical, indicating that the same group of thiols were being oxidized in the two redox titrations shown in Fig. 1B. Along with this shift in redox potential of the receptor, it was observed that the initial rates of ryanodine binding were highly reproducible only if the SR was first pre-reduced with GSH. As shown in Fig. 1D, going back and forth between Ϫ140 and Ϫ180 mV or between Ϫ105 and Ϫ198 mV (data not shown) does not alter time-dependent ryanodine binding as long as the protein is pretreated with GSH. Samples of SR vesicles that were not pre-reduced did not show reproducible binding characteristics at more positive redox potentials (data not shown). All data presented in subsequent figures are from samples that are pre-reduced at room temperature for 10 min with GSH as indicated. Furthermore, it should be noted that the initial rate of ryanodine binding is controlled by the solution redox potential, as defined by Equation 7, not by the absolute amount of GSH or GSSG in the buffer.
The Ca 2ϩ release channel is activated by Ca 2ϩ , sensitized to activation by Ca 2ϩ in the presence of caffeine and is inhibited by Mg 2ϩ . Redox titrations were carried out at various free Ca 2ϩ concentrations, and the calculated redox potential of RyR1 thiols was plotted versus Ca 2ϩ , in the presence and absence of caffeine ( Fig. 2A) or Mg 2ϩ concentration (Fig. 2B).
Not only does the Ca 2ϩ concentration affect the redox poten-FIG. 1. The ryanodine receptor has a well defined redox potential. Time-dependent ryanodine binding was measured as described under "Experimental Procedures." The initial rate of binding BЈ (pmol/mg/min) was calculated from a linear regression fit of binding measurements at 3, 6, 9, and 12 min. The solution redox potential was calculated from Equation 7 using different mixtures of GSH and GSSG. In A, ryanodine binding as a function of time is shown at Ϫ230 mV (q), Ϫ180 mV (‚), Ϫ130 mV (OE), and Ϫ100 mV (E). In B, initial binding rate, BЈ, was plotted as a function of the solution redox potential and was fit to Equation 10 (solid lines). SR was either pretreated with GSH (q, E RyR ϭ Ϫ165.5 mV) or was not pretreated with GSH (E, E RyR ϭ Ϫ100 mV). In C, the data set derived from B following pretreatment with GSH was plotted as 30.8 ϫ log 10 ((BЈ-BЈ min )/(BЈ max Ϫ BЈ)) (mV) versus E sol (mV). From a linear regression fit, the slope ϭ 1.023, the x-intercept ϭ Ϫ165.5, and r 2 ϭ 0.996. In D, following pretreatment of SR vesicles with GSH for 10 min at room temperature, the redox potential of the solutions were set to Ϫ140 mV (q, ‚) or Ϫ180 mV (OE,E), and samples were then incubated at 37°C for 40 min. The final redox potential of the binding buffer was adjusted to either Ϫ140 mV (q,E) or Ϫ180 mV (OE, ‚). Ryanodine binding was then initiated at t ϭ 0 by addition of 4.0 nM [ 3 H]ryanodine. All binding assays were carried out at 37°C at 50 M free Ca 2ϩ and were repeated at least three times with almost identical results. Redox titrations, shown in A and B were repeated at least ten times on three different SR preparations. tial of sulfhydryl groups involved in activation of the ryanodine receptor ( Fig. 2A) but also the solution redox potential effects the Ca 2ϩ dependence of the initial rate of ryanodine binding (Fig. 3). As is evident in Table I, at highly oxidizing redox potentials (more positive than Ϫ140 mV), the receptor becomes more sensitive to activation by Ca 2ϩ (K d Ca 2ϩ and the EC 50 decreases). The Hill coefficient n H is unaffected by the redox potential of the solution. The data indicate a strong coupling between the Ca 2ϩ binding site and the redox-sensitive thiols associated with RyR1.
The hyperreactive thiols on the Ca 2ϩ release mechanism of SR sense the state of the release channel (15). They are specifically labeled by nanomolar concentrations of the alkylating reagent CPM, when the channel is in a closed configuration. In Fig. 4A, a redox titration is shown following pretreatment of the SR with 20 nM CPM, under conditions in which the Ca 2ϩ release channel is in its closed state (1.0 mM CaCl 2 ). Under these conditions, no increase in the rate of ryanodine binding is observed as the solution redox potential increases. The receptor no longer demonstrates redox sensitivity. In control experiments in which the Ca 2ϩ release channel is in its open state during CPM pretreatment (50 M Ca 2ϩ ), a subsequent redox titration was identical to that shown in Fig. 1B (data not  shown). A normal redox titration is observed when CPM binds to non-hyperreactive thiols. Although the ryanodine receptor still shows a biphasic Ca 2ϩ concentration dependence, following CPM treatment the enhanced rate of ryanodine binding observed at more positive redox potential (Fig. 3) is not present (Fig. 4). CPM eliminates redox control of the ryanodine receptor by blocking hyperreactive thiols. DISCUSSION In this paper, we demonstrate for the first time that hyperreactive thiols on the skeletal muscle sarcoplasmic reticulum ryanodine receptor (RyR1) have a well defined redox potential that is sensitive to the concentrations of channel modulators such as Ca 2ϩ , Mg 2ϩ , and caffeine. Conditions that favored the opening of the Ca 2ϩ release channel (micromolar Ca 2ϩ and millimolar caffeine) caused the redox potential of the receptor to become more negative, which favored the oxidation of critical thiols to a disulfide, and an enhancement of the rate of ryanodine binding (Fig. 1B). Conditions that close down the Ca 2ϩ release channel (submicromolar Ca 2ϩ , millimolar Ca 2ϩ , or Mg 2ϩ (Fig. 2B)), increase the receptor redox potential, which favors the reduced form of critical sulfhydryls and a decrease in the rate of ryanodine binding. Just as caffeine sensitizes the ryanodine receptor to activation by Ca 2ϩ , caffeine also main-  (18). At each Ca 2ϩ concentration and solution redox potential, samples were incubated in ryanodine binding buffer as described under "Experimental Procedures," and at a redox potential, E sol , as determined by Equation 7. The initial rate of ryanodine binding was measured as a function of the free Ca 2ϩ concentration at 37°C at the following solution redox potentials: Ϫ230 mV (E), Ϫ220 mV (), Ϫ160 mV (ƒ), Ϫ140 mV (‚), Ϫ120 mV (OE), Ϫ90 mV (Ⅺ), Ϫ60 mV (f), and control (untreated with GSSG or GSH, q). All experiments were repeated at least three times with almost identical results.
tained the redox potential of the receptor at more negative values, even at low Ca 2ϩ concentrations.
This control of the redox potential of hyperreactive thiols by physiologically relevant channel modulators suggests that these thiols play a role in controlling channel gating during excitation-contraction coupling. However, even under conditions which strongly favor the activation of the Ca 2ϩ release protein, 50 M Ca 2ϩ and 10 mM caffeine, the redox potential does not get more negative than Ϫ175 mV. The normal cytoplasmic redox potential is approximately Ϫ230 mV (22), as measured in cultured pancreatic cells. This is 55 mV more negative than the redox potential of the receptor under maximum activating conditions. Under these conditions, only 1.6% of the total receptors are oxidized and activated by thiol oxidation (calculated from Equations 6 and 9). Under less optimal conditions for channel activation, the receptor redox potential is more positive, and the open probability of the channel should be extremely small. The only condition in which a significant fraction of these thiols are oxidized is when the cell is oxidatively stressed and the cellular redox potential increases, or if the redox potential in the myoplasm is more positive than the estimated value of Ϫ230 mV (22). Under these conditions, one would expect that small changes in the cellular redox potential to have a major influence on excitation-contraction coupling.
In perhaps a related mode of operations, the OxyR transcription factor of Escherichia coli activates antioxidant genes in response to elevated H 2 O 2 levels (23). The redox potential of OxyR is Ϫ185 mV, which is significantly higher than that of the cellular redox potential under normal non-oxidatively stressed conditions (Ϫ270 mV). However, an oxidatively induced drop in the GSH/GSSG ratio leads to an increase in the cellular redox potential and an activation of OxyR. In a similar manner, an increase in the cellular redox potential during oxidative stress (induced by fatigue, aging, or ischemia) should result in oxidation of the ryanodine receptor/Ca 2ϩ release protein and the opening of the Ca 2ϩ release pathway.
Our previous studies have shown that oxidation induced by quinones, porphyrins and H 2 O 2 is strongly Ca 2ϩ dependent (8,10,24). A likely explanation for the Ca 2ϩ dependence of oxidation-induced Ca 2ϩ release from SR vesicles is directly related to the measurements of redox potential described in this manuscript. Ca 2ϩ lowers the redox potential of the ryanodine receptor, which then allows these thiols to be oxidized more easily. We propose that conformational changes induced by channel activators stabilize one or both members of a redox pair, which in an appropriate redox environment results in thiol oxidation. It is well known that the SR Ca 2ϩ release channel can be activated by non-thiol reagents in the absence of GSH and GSSG. An oxidation reaction is not required to open the Ca 2ϩ release channel in vitro. However, as shown in Fig. 3, the degree of activation of the receptor is strongly dependent on the solution redox potential. In a more oxidized environment, there is a large stimulation of the receptor. Whereas in a more reduced environment, there is a relatively small degree of channel activation by Ca 2ϩ . The gain of the system appears to be set by the cellular redox potential. The labeling of hyperreactive thiols by CPM eliminates redox potential control of FIG. 4. CPM eliminates redox control by blocking hyperreactive thiols. SR vesicles were treated with 20 nM CPM as described under "Experimental Procedures." The reaction mixture was quenched at room temperature for 10 min in 0.1-0.3 mM GSH. In A the Ca 2ϩ concentration was decreased from 1.0 mM to 50 M by addition of EGTA, the solution redox potential was adjusted to the value shown, and the rate of ryanodine binding was measured at 3, 6, 9, and 12 min at 37°C as described under "Experimental Procedures." The dashed line is that of a redox titration carried out in the absence of CPM, from Fig. 1. In B, the Ca 2ϩ concentration was adjusted to the value shown, and the redox potential of the solution was set at Ϫ120 mV (Ⅺ,f), or Ϫ220 mV (OE, ‚). Samples (f,OE) were not pretreated with CPM, whereas samples (‚, Ⅺ) were pretreated with 20 nM CPM. The control (q) was not treated with CPM and was not placed in a redox buffer. All experiments were repeated at least three times with almost identical results. RyR1. It does not eliminate Ca 2ϩ -dependent activation and inhibition of RyR1. Studies that ignore the cellular redox environment are ignoring an important effector of Ca 2ϩ channel function.
A number of recent studies have shown that some thiol oxidizing reagents induce a biphasic concentration-and timedependent activation and subsequent inactivation of RyR1 (10,25). At low concentrations, the receptor is activated in a timedependent manner. At higher concentrations, a time-dependent inactivation of ryanodine binding is observed after a delay of 30 -50 min. The inactivation is likely to be caused by an oxidation of a second set of less reactive thiols. In this study, time-dependent inactivation was not observed. Experiments were designed to look at relatively short exposures to less oxidizing environments. The sulfhydryls examined are hyperreactive thiols whose oxidation is responsible for activation of the ryanodine receptor, not those responsible for subsequent inactivation.
As shown in Fig. 1B, pre-incubation of the SR with GSH caused a 65 mV shift in the redox potential of the receptor to more negative values. Two possible explanations explain this large shift in the redox potential of RyR1 upon pre-reduction with GSH come to mind. Despite the fact that most of the SR preparation was isolated in the presence of 50 M dithiothreitol, oxidation during the last stages of the isolation procedure may have caused a shift in the redox potential of RyR1 to a more positive value. The redox potential of those hyperreactive thiols that control ryanodine binding may be altered by the oxidation of a second class of "regulatory thiols." Aghdasi et al. (9) has previously shown that SR vesicle preparations are sensitive to oxidation by dissolved oxygen. Alternatively, the 10min incubation with GSH may be required to transport GSH across the SR membrane and generate a suitable redox environment on the luminal side of the SR (21). In other words, the luminal redox potential may influence the redox potential of hyperreactive thiols on the cytoplasmic face of the SR.
The carboxyl-terminal domain of the IP 3 receptor from the endoplasmic reticulum has a similar amino acid sequence to that of the ryanodine receptor. It has been shown that increased ratios of GSSG/GSH activate IP 3 binding (26), and it has been suggested that there are conserved sequences, which are strong candidates for sites of redox reactions common to both the IP 3 and RyR (27). The RyR1, RyR2, and the IP 3 receptors may contain similar or related molecular mechanisms for dealing with oxidative stress.