The goal of this project is to determine the molecular mechanisms involved in the assembly of the triad junction between T-tubules and sarcoplasmic reticulum during the development of excitation-contraction (E-C) coupling in skeletal muscle. An immunofluorescence study of the distribution of the alpha subunit of the skeletal muscle dihydropyridine (DHP) receptor (the putative voltage sensor in E-C coupling) in developing normal muscle and dysgenic (mdg) myotubes in culture suggested that a specific protein-protein interaction of the alpha subunits of the DHP receptor in involved in the normal organization of the receptor complex in the junctional T-tubules. The coordinated expression of several myofibrillar and membrane markers was compared between normal and dysgenic muscle developing in vivo and in vitro. Proteins of the I-Z-I complex (alpha-actin, titin T12) and the sarcoplasmic reticulum (Ca2+- ATPase) organize early while components of the A-band (myosin, titin T30) and the T-tubules assume a cross-striated orientation later in myogenesis. Although dysgenic muscle lags behind in the later phase of sarcomere formation, mutant myotubes can achieve the mature organization in the myofibrils and E-C coupling membranes. De novo expression of the DHP receptor alpha1 subunit from normal nonmuscle nuclei fused with dysgenic myotubes restored normal functions as well as normal organization of myofibrils and E-C coupling membranes.
