STRUCTURAL ANALYSIS OF THE CA++ RELEASE CHANNEL

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DESCRIPTION: The overall goal of the proposal is to define the molecular events involved in the regulation of Ca release from ryanodine sensitive intracellular stores and to determine the mechanisms by which defects in these pathways give rise to Malignant Hypothermia and Central Core Disease. The general hypothesis on which the proposal is based is that changes in the activity of the Ca-release channel are accompanied by global conformation changes that can be characterized biochemically and with cryoelectron microscopy and image reconstruction. In support of this hypothesis preliminary data is included showing the images of closed and open Ca-release channels and Ca-release channels treated with rapamycin to remove FKBP12. Extensive biochemical evidence is also presented for peptide mapping and immunochemical identification and characterization of the ryanodine receptor (RyR) in intact rightside out SR and in 28S and 14S proteolytic complexes. These studies will be extended in the five specific aims of the current proposal. The first aim is to generate different functional states of the Ca release channel (opened by caffeine and AMP-PCP, inactivated by mM Ca and inhibited by ruthenium red or neomycin) and to characterize these states by 3-H ryanodine binding and single channel analysis. The structures of these functional states will then be determined by electron cryomicroscopy and angular reconstruction. The second is to use different proteolytic mapping approaches combined with surface and membrane interior labeling, immunological and peptide binding (high affinity peptides identified using a phage display library) to map the topology of Ca release channel domains and structural changes that relate to different functional states. The 3-dimensional structure of a 14S proteolytic fragment of the protein which contains the channel and the ryanodine binding site will also be determined. The third aim is to map the binding site for FKBP12 and determine its effects on the structure of the Ca release channel. The 3-dimensional structure of the channel will be determined before and after the removal of FKBP12 by rapamycin treatment and upon readdition of purified FKBP12 to the depleted channel. The FKBP12 binding site will be mapped on the Ca release channel by examining label transfer from 125-I-APDP or 125-I-SASD and peptide mapping and sequencing of the 28S complex. The fourth and fifth aims both use electron cryomicroscopy to compare Ca release channel structures. In aim four the 3-dimensional structures of normal pig and malignant hypothermic pig Ca2+ release channels will be compared under conditions where (1) affinity differences for ryanodine are greatest, (2) in the absence of Ca, where both channels should be closed, and (3) in the presence of Ca and ryanodine where both channels should be open. In the final aim, the three dimensional structures of the skeletal and cardiac Ca release channels will be compared in the open and closed conformations. This may help provide a structural basis for the differences between cardiac and skeletal muscle EC coupling. The studies outlined in this proposal may lead to important information on Ca channel structure, topology and mechanism as well as identifying structural or mechanistic alterations found in disease states such as Malignant Hyperthermia and Central Core Disease.