The long-term goal of this proposal is to determine the three-dimensional architecture and composition of the triad junction in striated muscle by cryo-electron microscopy and computer image processing, including three- dimensional reconstruction. Triad junctions are specialized regions in muscle where invaginations of the plasmalemma known as t-tubules and the intracellular sarcoplasmic reticulum (SR) membrane system are in close apposition. Excitation-contraction (E-C) coupling refers to the process by which neuronally induced depolarization of the plasmalemma/t-tubules causes release of Ca2+ (the immediate stimulus of contraction) from the internal Ca2+ stores of the SR. The signal transducing events associated with E-C coupling occur at the triad junction by mechanism(s) that are incompletely understood. Knowledge of the structure of the triad is essential to fully understanding E-C coupling and the for the rational search for therapies to diseases of skeletal muscle and heart. In the main strategy that will be taken, for which substantial progress has already been made, the purified solubilized ryanodine receptor/calcium release channel (RyR), the major component (by mass) of the triad junction, will be studied in isolation and complexed with site-specific probes (e.g. amino-acid-sequence specific antibodies) and macromolecular ligands known or suspected to be constituents (e.g. FK-506 binding protein) or modulators (e.g. calmodulin) of the triad junction. Comparative studies of the rabbit skeletal muscle RyR with that from heart will be done to determine whether structural differences can be correlated with the apparently different mechanisms of E-C coupling in the two muscle types. We will refine the methods we have developed to reconstitute the solubilized RyR into lipid bilayers for structural studies; structural characterization of defined structural states of the RyR (e.g. open and closed) should then be feasible. We will explore a second strategy for determining the structure of the triad in which electron tomographic reconstruction techniques will be applied to tilt-series images of isolated (fixed, sectioned, and embedded) triad junctions. Isolated triads will also be imaged in the frozen- hydrated state to maintain structural integrity, and gold cluster- containing ligands will be used to identify and map components of the triad junction with much higher precision than has been possible by conventional immunoelectron microscopy.
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