We do basic research on the cellular and molecular mechanisms involved in the differentiation of excitable membranes in: 1) the postsynaptic membrane of the skeletal neuromuscular junction 2) the membranes of the excitation-contraction coupling system of skeletal muscle (the transverse tubules, sarcoplasmic reticulum and the triad junctions they form). In 16 day embryos of dysgenic (mdg/mdg) mice, which die at birth due to failure of excitation contraction coupling caused by a mutation in the gene for the voltage-sensitive calcium channel (dihyropyridine receptor) alpha 1 subunit, the development of excitation-contraction coupling membranes and myofibrils (as seen by immunofluorescence) is normal, while in 18 day embryos, the transverse tubules are sparse and fragmentary, and a minority of muscle fibers show normal differentiation of myofibrils and sarcoplasmic reticulum. In cultured myotubes, the sarcoplasmic reticulum becomes organized with respect to the I-band early in myofibrillar development, while transverse tubules take on their mature orientation along the interface between A- and I-bands at a later stage, after the maturation of the A-band. Qualitatively, the development of these systems is the same in normal and dysgenic muscle cultures, although only half as many dysgenic myotubes reach the later stage of development as do normals. We conclude that the dihydropyridine receptor alpha 1 subunit is not constitutively required for myofibrillar and membrane system development but may affect them indirectly. The components of the excitation-contraction coupling membrane system are arranged in highly ordered fashion with respect to the sarcomere, (the contractile unit of the striated muscle fiber and repeating subunit of the myofibril). We have found that a fraction of the myofibrils isolated from adult rat muscle retain associated sarcoplasmic reticulum and transverse tubule membranes in the normal periodic arrangement. We have also found that ankyrin and spectrin, proteins previously implicated in plasma membrane - cytoskeletal interactions, are associated with all isolated myofibrils in the regions of the sarcomere where membrane retention most often occurs. Ankyrin and spectrin may thus be involved in linking the triad membranes to the muscle cytoskeleton at specific sites along the sarcomere.
