This project probes the arrangement of proteins and the biogenesis of membranes composing the macromolecular complexes responsible for regulated calcium release in skeletal and cardiac muscle. The basic approach is to establish molecular-structural and structural- functional correlations, under the premise that structure can be best understood by observing its changes following specific molecular perturbations, and that function cannot be fully modeled without knowledge of the underlying structure. Regarding the arrangement of proteins, the key hypotheses to be tested are: 1. calsequestrin's disposition depends on a combination of links to the SR membrane and intermolecular bonding in the presence of bivalent cations; 2. junctophilin 1 and 2 are essential for the docking of SR to surface membranes. In the absence of junctophilin the permanent association between surface and internal membrane systems may not be possible; 3. skeletal type excitation-contraction coupling, based on a molecular link between proteins of two different membrane systems, evolved at the transition between low chordates and vertebrates and it required the evolution of a new type of ryanodine receptor; 4. caveolin-3 is necessary for the development of T tubules. The experimental strategies involve perturbation of the molecular composition and developmental events by selected addition, removal and substitution of key components and perturbation of the functional state by changes in ionic composition. The structural approaches to be used center on techniques of transmission and freeze-fracture electron microscopy, correlated with immunofluorescence and confocal light microscopy. ? ?
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