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 (SR) during the development of excitation- contraction (E-C) coupling in skeletal muscle. Immunofluorescence studies of the distribution of the skeletal muscle dihydropyridine receptor (DHPR) (the voltage sensor in E-C coupling), the ryanodine receptor (RyR) (the calcium release channel of the sarcoplasmic reticulum) and triadin - both in developing normal muscle and dysgenic (mdg) myotubes in culture - showed that protein-protein interactions mediated by the DHPR plays a role in the normal organization of the triad. Recordings of cytoplasmic free calcium with fluorescent indicators revealed that only action potential-induced calcium transients are eliminated in the dysgenic mutant. Calcium-induced calcium release events were essentially unaltered in the DHPR null mutant, demonstrating that these events reflect a fundamental and independent function of the RyR. The last studies of this project now show that both of these membrane channels are expressed early in development and appear colocalized in clusters in T-tubules and SR, respectively. Interactions between dihydropyridine and ryanodine receptors are likely to be involved in the clustering process, which presumably represents an early step in triad formation. Parallel investigation of the molecular assembly, the ultrastructure and the developing function of the E-C coupling apparatus during myogenesis indicate that the molecular specialization of junctional T-tubules and SR occurs concomitantly with the initial formation of junctions. Whereas these early junctions exist in a variety of configurations, they are capable of sustaining action potential- induced calcium transients. The maturation of E-C coupling properties can be accounted for by a dramatic increase in junction density that occurs when the junctions associate with the myofibrils.
