The overall goal of this study is to elucidate the mechanism by which binding of certain modulators to the cytoplasmic domains of the skeletal muscle sarcoplasmic reticulum Ca release channel((RYR1) regulates the activity of the channel. The central working hypothesis is that channel opening is initiated by a change in the interaction between an N-terminal domain on one subunit and a central cytoplasmic domain of a second subunit of the RYR1 tetramer. It is also proposed that calmdulin binding sites and the mutations in malignant hyperthermia and central core disease are close to or within these domains. The application has 3 specific aims: 1. The sites of interaction between the N-terminus and the central cytoplasmic domains of RYR1 will be defined using selective sulfhydryl labeling combined with protease digestion, sequence specific antibodies and N-terminal sequencing. Crosslinking reagents will be used to identify the regions close to reactive cysteines in the N-terminal region of RYR1. Interactions between expressed fragments of RYR1 will also be assessed using a biosensor system and GSH-sepharose beads. 2. Binding sites for calmodulin (CaM) will be identified and the effects of Ca concentration and mutations in the CaM molecule on CaM and ryanodine binding and channel activity will be assessed. 3. Using the techniques and approaches employed in the first two specific aims, the properties of RYR1 from pigs with malignant hyperthermia and genetically matched controls will be compared with respect to binding to CaM and sensitivity to sulfhydryl reagents.
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