This proposal will study the molecular events that couple activation and intracellular calcium release in muscle. The specific goal is to understand in both normal and diseased muscle how reciprocal interactions between the DHPR and RyR control muscle activation. The project will use two unique muscle preparations: dysgenic mice which lack DHPRs and dyspedic mice which lack RyRs. Cells from these animals will allow the properties of the DHPR and RyR to be studied both in isolation and in the presence of chimeric constructs of the reciprocal protein expressed after cDNA injection.
The first aim will investigate the biophysical mechanisms underlying RyR1 control of DHPR channel activity. Dyspedic myotubes will allow the single channel properties of DHPR to be determined while the protein is isolated from any interaction with the RyR. Comparison to results from normal myotubes will indicate how RyR enhances macroscopic currents through the DHPR.
The second aim will determine the structural features of the DHPR protein that mediate interactions with the RyR. Chimeric DHPR made up of sections of the skeletal and cardiac isoforms of the protein will be expressed in dysgenic myotubes. Since RyR is known to affect only skeletal DHPR function systematic substitutions of skeletal sequences for those from cardiac channels should reveal the critical areas involved in DHPR-RyR interactions. In the third aim, naturally occurring mutations in RyR known to be expressed in patients with central core disease will be investigated. RyR function will be assayed in myotubes using optical recording of calcium transients and caffeine and voltage to stimulate release. Once the properties of normal and dysgenic myotubes are studied, RyR receptors with engineered substitutions will be expressed to assess the functional consequences of these clinically important mutations.
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