Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

The skeletal muscle Ca 2؉ -release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H 2 O 2 treatment of C3635A-RyR1 channels or wildtype RyR1, following their expression in human embryonic kidney cells, enhances [ 3 H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120 -4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides). Ca 2ϩ

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91 phox membrane subunit and the cytoplasmic regulatory p47 phox subunit co-localized in transverse tubules of adult mice fibers with the ␣ 1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91 phox and p22 phox , which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47 phox and p67 phox . NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [ 3 H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca 2؉ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.The NADPH oxidases (NOX) 3 are flavoprotein enzymes that use NADPH as electron donor to mediate the univalent reduction of molecular oxygen to superoxide anion (1), a free radical that by spontaneous or enzymatically catalyzed dismutation is readily converted into H 2 O 2 . The phagocytic NOX isoform (NOX2) was first discovered as a pivotal component of the neutrophil respiratory burst (2, 3). The functional NOX2 enzyme is composed of two integral plasma membrane subunits, gp91 phox and p22phox , which make up cytochrome b 558 , plus three cytosolic regulatory subunits: p40 phox , p47 phox , and p67 phox (2, 4). A variety of tissues, including endothelial cells (5), smooth muscle cells (6), neurons (7-9), and astrocytes (7, 10), possess nonphagocytic NOX homologues (11,12). Several reports indicate that NOX2 and its homologues have a central role in the generation of reactive oxygen species (ROS) in response to diverse physiological extracellular stimuli (13-17). Moreover, membrane depolarization stimulates NOX activity in phagocytes (18) and endothelial cells (19,20). NOX stimulation is also apparent following agonist-induced stimulation of N-methyl-D-aspartate receptors in hippocampal neurons (21). Some N...

Calmodulin, a highly versatile and ubiquitously expressed Ca2+ sensor, regulates the function of many enzymes and ion channels. Both Ca2+-dependent inactivation and Ca2+-dependent facilitation of the voltage-gated Ca2+ channels Cav1.2 and Cav2.1 are regulated through an interaction with Ca2+-bound calmodulin. This review addresses the functional regulation of Cav1.2 and Cav2.1 by calmodulin and discusses how Ca2+ binding to a single calmodulin molecule can regulate opposing functions of the voltage-gated Ca2+ channels.

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