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~con~traction (E~C) coupling in skeletal muscle. Immunofluorescence studies of the distribution of the skeletal muscle dihydropyridine receptor (DHPR)(the putative voltage sensor in E~C coupling), the ryanodine receptor (RyR) (the calcium release channel of the sarcoplasmic reticulum) and triadin in developing normal muscle and dysgenic (mdg) myotubes in culture showed that a protein~protein interaction mediated by the DHPR play a role in the normal organization of the triad proteins. The alpha1 subunit of the DHPR is essential for the normal targeting of the alpha2 subunit; it also facilitates the normal organization of the RyR and triadin although it is not absolutely required. De novo expression of the DHPR alpha1 subunit from normal nonmuscle nuclei fused with dysgenic myotubes restored normal functions and normal molecular organization of the E~C coupling membranes. Recordings of cytoplasmic free calcium with fluorescent indicators revealed three types of calcium transients in developing myotubes action potential~induced transients, fast localized transients, and propagated calcium waves with at least two underlying mechanisms: E~C coupling and calcium~induced calcium release. Only action potential~induced transients are eliminated in the dysgenic mutant, suggesting that fast localized transient and calcium waves represent properties of the RyR independent from interactions with the DHPR.
