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) acetylcholine receptor aggregates, which form on muscle fibers in culture, in response to embryonic brain extract. These receptor aggregates are a model for the developing postsynaptic membrane 3) the membranes of the excitation-contraction coupling system of skeletal muscle (the transverse tubules, sarcoplasmic reticulum and the triad junctions they form). Immunofluorescence and immunogold labeling at the electron microscope level shows that the peripheral membrane protein, ankyrin is associated with the triad junction in rat skeletal muscle, where it may be involved in organizing membrane channels or ion pumps, or in linking the triads to the muscle cytoskeleton. In 18 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 Ca++ channel alpha-1 subunit) the expression of the alpha-1 subunit is sharply reduced or absent. However, spatial organization of the sarcoplasmic reticulum, transverse tubules and myofibrils (as seen by immunofluorescence) is normal, although these components are less prominently displayed than in normal embryos. It appears that the alpha-1 subunit is not required for the assembly of the excitation-contraction coupling system, but its absence may indirectly inhibit maturation. In rat myotubes grown in culture, transverse tubule membranes form an extensive system which is distinct in molecular composition from the plasma membrane, despite the direct continuity of the two systems. The precursors of the transverse tubules appear to be intracellular (not connected to the plasma membrane) vesicles or tubules that first appear in myoblasts. Intracellular tubular membranes with markers for both transverse tubules and sarcoplasmic reticulum are associated with the Z lines of immature myofibrils in newly formed myotubes.
