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

Abstract

Hyperreactive sulfhydryl groups associated with the Ca 2؉ release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca 2؉ channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the Ca 2؉ release protein. 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) wit...

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