The long-term goal of this research is to understand the molecular basis of calcium channel function and of excitation-contraction (E-C) coupling in skeletal muscle. Many of the experiments make use of cultured skeletal myotubes from mice with the muscular dysgenesis mutation. In mutant mice, the gene encoding the skeletal muscle dihydropyridine (DHP) receptor is altered, and skeletal muscle lacks slow L-type calcium current and E-C coupling. Complementary DNAs encoding different kinds of DHP receptor constructs will by expressed in cultured dysgenic myotubes, and three different physiological parameters of this expression will be measured. Intramembrane charge movement and L-type calcium conductance will be quantified with the patch clamp technique and calcium release from the sarcoplasmic reticulum will be assessed by microspectrofluorometry with the indicator dye Indo-1. The cDNA constructs to be examined will be chimaeras in which one or more of the internal repeats of the skeletal muscle DHP receptor are replaced by the equivalent portion of the cardiac DHP receptor. By establishing correlations between changes in the identity of repeats and changes in one or more of the measured physiological parameters, it will be possible to begin assigning these physiological important signals to specific structural components of the DHP receptor. To enable biochemical analysis of the expressed DHP receptor cDNAs, new methods appropriate for efficient transfection of primary myotube cultures will be established. As a test of whether junctional tetrads that are seen in freeze fracture of normal muscle represent DHP receptors, it will be determined whether expression of DHP receptor cDNAs in dysgenic myotubes leads to the appearance of these structures. To establish the relationship between junctional tetrads, E-C coupling and L-type calcium channels, focal stimulation and current recording techniques will be applied to developing muscle fibers. To gain insight into the reason for the generalized over-expression of receptors for calcium channel antagonists in mutant, cardiomyopathic hamsters, whole cell calcium currents will be measured in neurons, cardiac cells and skeletal muscle. Finally, it will be determined whether cDNAs encoding two new representatives of calcium channel proteins, the carp skeletal muscle DHP receptor and a rabbit brain calcium channel, produce calcium currents and E-C coupling in dysgenic myotubes.
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