A relationship between specific states of the actomyosin cross-bridge cycle and their conformation is not yet established. Using photolysis of caged compounds and rapid freezing we will examine the structure of cross-bridges in various states by electron microscopy and image analysis. In the process of excitation-contraction coupling, depolarization of the surface membrane is translated into a signal for release of calcium from the sarcoplasmic reticulum (SR). The SR calcium release channel has been identified: it is the foot protein, whose cytoplasmic domain spans the gap between the SR surface membrane and/or its invaginations, the transverse (T) tubules. We seek identification of a component of the surface membrane/T tubules (the junctional tetrads) with the L type calcium channel (or dihydropyridine receptor) which is thought to be the voltage sensor of E-C coupling. Using electron microscopy of muscle fibers from the E-C coupling defective mutation muscular dysgenesis, of dysgenic muscle fibers rescued by transfection with the cDNA for the alpha 1 subunit of DHPRs, and of transfected chinese hamster ovary cells, we will establish if a correlation exists between junctional tetrads and DHPRs. Development of the membrane systems in skeletal muscle fibers in vivo proceeds through several well coordinated steps. We will explore whether a relationship exists between specific events in membrane development and the appearance and rearrangement of the intermediate filament system.
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