During the next year of support we will be extending our studies on the 20,25-diazacholesterol (20,25-D) model of myotonia, and we plan to study calcium exchange in cultured muscle from patients with Duchenne muscular dystrophy. In order to further define the mechanism for the alteration of membrane physical properties in 20,25-D induced myotonia, we plan to analyze total membrane fatty acids by gas chromatography. If alterations in fatty acid chain length or saturation are found in the 20,25-D treated animals, we plan to look at the effects of cholesterol depletion on the appropriate metabolic pathways using cultured muscle and lipid depleted medium. To try to further define the mechanism of delayed relaxation in 20,25-D induced myotonia, we also plan to assay Ca2+ATPase activity in the T-tubule fractions of normal and 20,25-D treated muscle. T-tubule will be isolated as previously described and its purity assessed by nitrendipine binding. Ca2+ATPase will be measured using the same procedures we have used for red blood cell and sarcolemma Ca2+ATPase activity. Finally, preliminary to studying calcium exchange in cultured muscle from patients with Duchenne muscular dystrophy, we will be examining this in cultured chick muscle cells. Muscle will be grown on disks made of plastic with a scintillating material imbedded, and the cells will be profused with buffer containing 45Ca. Optimal flow rates and calcium concentrations will be determined as well as the effect of various buffers, pH, and other ion concentrations. When these conditions are worked out, we will go on to examine calcium exchange in cultured human muscle from normal individuals and patients suffering from Duchenne muscular dystrophy.