Our overall hypothesis is that the mutations in RyR1 responsible for human Malignant Hyperthermia (MH) and Central Core Disease (CCD) alter the stability of the EC coupling macromolecular complex and the dynamics of Ca2+ signaling, These changes are responsible for susceptibility to agents that trigger MH and to create the weakness associated with CCD. Data from our dysgenic (mdg) and dyspedic mouse models have defined the critical regions of the slow voltage gated Ca2+ channel in the surface membrane (y is-DHPR) and RyR1 of the sarcoplasmic reticulum (SR) that are responsible for bi-directional signaling between these two proteins. We have also recently demonstrated a three-way interaction between RyR1, the vis-DHPR and a Ca2+-entry channel in the surface membrane. In vitro studies of RyRs and Y is-DHPRs with MH mutations expressed in null cell lines have demonstrated that they have a phenotype similar to muscle from MH susceptible humans and pigs. Three mouse models have been created expressing RyR1 with MH/CCD nutations. Additional mice will be created with other RyR1 mutations to allow us to study the relationship between severity of the phenotype and the site of a mutation. The themes of Projects 1-4 are closely interrelated, and the expertise from each of the project PI's is unique and essential to the interdisciplinary goals of the proposed program. The administrative, tissue culture transgenic animal, human tissue and morphology cores will provide a unifying resource for common reagents and morphology studies for the four project investigators. The scope of investigations will range from creation and extensive phenotyping of MH and CCD mice (Projects 1 and 3), studies of protein function and interactions in isolated muscle triads (Project 2), influence of mutations on the RyR1 structure and biochemistry (Project 3), and their influence on the cellular physiology of muscle and dendritic cells (Projects 1, 2, & 4). The expected outcome of the work proposed by this Program Project is a better understanding how MH and CCD mutations of RyR1 differentially perturb the dynamics of cytoplasmic Ca2+ at rest and during activity, how they promote subtle to catastrophic cellular dysfunctions and their relationship to genetic background.
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