The long term objectives of the proposed research are the identification and characterization of the proteins involved in signal transmission at the sarcoplasmic reticulum/transverse tubule junction in skeletal muscle. For muscle to contract, it must release calcium from internal stores known as the sarcoplasmic reticulum (SR). It is not known at this point how a muscle fiber accomplishes this. The molecular mechanism of SR calcium release is not completely understood yet basic to a functional understanding of both normal and diseased muscle. This proposal is unique because the model systems for studying excitation-contraction coupling are two specialized fish muscles, the heater organ of billfish, and the sonic muscles of toadfish, that have unusual properties. The heater organ is a muscle that generates heat but does not produce force; the muscle cells lacks contractile proteins yet have retained the two internal membrane networks found in normal skeletal muscle cells. In the heater organ, the SR, T tubules and mitochondria are involved in a controlled futile cycle that involves ATP-dependent calcium cycling at the SR. Understanding how the muscle cell organelles are working together to generate heat instead of force should provide information on the regulation of calcium translocation in muscle cells. This is important for diseases associated with fatal levels of muscle thermogenesis due to a breakdown in calcium regulation in muscle, which occurs in malignant hyperthermia and thyroid dysfunction. The sound-generating muscles of toadfish, have superfast contraction speeds, and are enriched in the proteins required for calcium release and reuptake. The extensive junctional membranes of these cells provide an unparalleled system for structural studies of the membrane proteins involved in signal transmission. The major aim of the proposal is to determine if the modified thermogenic cells have retained a normal molecular pathway for """"""""excitation-thermogenic"""""""" coupling. To establish this, the SR of the heater cells will be purified and junctional proteins involved in the calcium release process identified. Antibodies that cross-react with heater tissue will be produced and immunolocalization studies will be used to identify the morphological correlate to the junction in the modified muscle cells. Structural studies in the superfast contracting toadfish muscles will focus on the architecture of the junction and the proteins which span the SR-T gap. By comparing the evolutionarily modified systems to normal skeletal muscle basic information about the molecules involved in calcium regulation will be obtained .
