No abstract

Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.congenital myopathy ͉ disease model ͉ intracellular calcium release

Skeletal muscle sarcoplasmic reticulum (SR) is shown to contain an NADH-dependent oxidase (NOX) that reduces molecular oxygen to generate superoxide. Its activity is coupled to an activation of the Ca2+ release mechanism, as evident by stimulation in the rate of high-affinity ryanodine binding. NOX activity, coupled to the production of superoxide, is not derived from the mitochondria but is SR in origin. The SR preparation also contains a significant NADH oxidase activity, which is not coupled to the production of superoxide and appears to be mitochondrial in origin. This mitochondrial component is preferentially associated with the terminal cisternae region of the SR. Its activity is inhibited by diphenylene iodonium (10 microM), antimycin A (200 nM), and rotenone (40 nM) but is not coupled to the generation of superoxide or the stimulation of the ryanodine receptor. The rate of superoxide production per milligram of protein is larger in SR than in mitochondria. This NOX may be a major source of oxidative stress in muscle.

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...

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. In muscle cells, cytosolic Ca 2ϩ levels are regulated by the intramuscular organelle, the sarcoplasmic reticulum (SR) 1 (1-3). Following the arrival of an action potential at the surface membrane and subsequent depolarization of the transverse tubule, the SR releases its lumenal store of Ca 2ϩ through the Ca 2ϩ release channel (CRC)/ryanodine receptor (RyR), thus triggering the contraction process. The interaction between the action potential at the transverse tubule and the release of Ca 2ϩ from the SR has been termed excitation-contraction coupling (ECC). In skeletal muscle, the molecular mechanism underlying ECC has remained unclear. Following excitation, resting Ca 2ϩ levels are reestablished through the active transport of the Ca 2ϩ back into the lumen of the SR by the Ca release by a large class of non-thiol channel stimulators, a high molecular weight complex was formed. The addition of channel inhibitors resulted in the reduction of the disulfides formed, the dissociation of key SR proteins and the exposure of hyperreactive thiols (14,15). Based on the results derived from fluorescence assays, ion flux measurements, single-channel experiments, and SDS-gel electrophoresis, the authors concluded that thiol oxidation-reduction chemistry plays a critical role in the channel gating of the SR CRC⅐RyR complex.Endogenous and exogenous redox agents have been observed to have profound effects on a wide range of ion channel systems. In addition to the SR Ca 2ϩ release channel, ion channels as varied as excitatory amino acid receptors, inositol 1,4,5-triphosphate (IP 3 )-gated Ca 2ϩ release channels, and even certain K ϩ channels have been demonstrated to be modulated by redox agents. For example, the N-methyl-D-aspartate-sensitive excitatory amino acid receptor has been shown to be modulated by both thiol oxidants and reductants (16).Glutathione is one of the most abundant low molecular weight peptides in eukaryotic cells and the most prevalent intracellular thiol. Depending on the cell type, glutathione levels have been estimated to range from 1 to 10 mM (17, 18). In the cell, glutathione acts as both a reducing agent and an antioxidant. Among its many physiological roles, glutathione * This work was supported by a grant from the American Heart Association Oregon Affiliate (to J. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom all correspondence should be addressed: Physics Dept., Portland State University, P. O. Box 751, Portland, 1 The abbreviations used are: SR, sarcoplasmic reticulum; CRC, Ca 2ϩ release channels; RyR, ryanodine receptor; RyR 1 , ryanodine receptor (skeletal isoform); ECC, excitation-contraction coupling; CPM, 7-diethylamino-3-(4Ј-maleimidylpheny...