The long term goals of this project are to understand the molecular mechanisms underlying the formation and function of the triad junction in muscle cells. This is a junction between plasma and sarcoplasmic reticulum (SR) membranes that is thought to have a critical role in coupling excitation and muscle contraction. This research will establish the developmental history of a key element of the triad junction, the "foot" protein in embryonic and neonatal chick skeletal muscle, in order to gain insight into the role of this protein in junction formation, muscle morphogenesis and the biogenesis of SR membranes. The plant alkaloid, ryanodine, binds specifically to the "foot" protein; this compound is, thus, a useful experimental probe of this protein. The developmental time course of the appearance of the "foot" or ryanodine-binding protein (RBP) and of the manifestation of its ability to bind ryanodine and to alter the calcium permeability of SR membranes will be examined. The appearance of the "foot" protein will be measured by specific tritiated ryanodine binding, and detection of the 400-500 kDa RBP using SDS-PAGE and RBP specific antibodies. The appearance of "foot" protein function will be assayed by measurement of intracellular calcium transients and contractile activity. These events will be correlated with the development of a mature muscle morphology and SR function in pectoral muscle from embryonic and neonatal chicks and primary cultures of embryonic muscle as visualized using electron microscopy and immunocytochemical techniques. The results of this research should contribute substantially to our understanding of the mechanism by which excitation of the surface membrane of a striated muscle cell triggers calcium release from an intracellular compartment, the sarcoplasmic reticulum, as the key step in initiation of muscle contraction.
