This application describes studies on characterization of key proteins regulating Ca release from junctional sarcoplasmic reticulum (JSR) in cardiac muscle. Four SR proteins will be studied, which are abundantly expressed in heart, co-localize to the junctional membrane, and form a complex. These four proteins are junctin, triadin, calsequestrin, and the ryanodine receptor (RyR). The interactions stabilizing this junctional protein complex will be characterized and the role of the complex in regulating Ca release elucidated. Junctin is the major calsequestrin-binding protein in JSR. It is an integral membrane protein which, along with triadin, appears to anchor calsequestrin to the JSR membrane. Recombinant junctin will be expressed and purified from Sf21 insect cells and its binding interactions with calsequestrin, triadin, and the RyR investigated. Antibodies to junctin will be produced for ultrastructural localization. The junctin gene will be cloned and the intron-exon boundaries defined. Triadin is a second calsequestrin-binding protein in cardiac JSR, which is homologous to junctin, and also binds to the RyR. Three triadin isoforms exist in heart which will be expressed and purified. Triadin interactions with junctin, calsequestrin, and the RyR will be characterized. Site-specific antibodies will be used for ultrastructural localization of each triadin isoform in heart. Calsequestrin is the major intraluminal Ca-binding protein in JSR. It appears to be anchored to the ryanodine receptor via interactions with triadin and junctin. The studies of calsequestrin binding to triadin and junctin will be localized. The RyR is the Ca release channel, which associates with the complex above from the lumenal face of the junctional membrane. The subdomain of the RyR interacting with the complex will be identified. Protein complex function will be investigated by biochemical co-reconstruction of junctin, triadin, and calsequestrin with the RyR in liposomes; by co-expression of the proteins with the RyR in Sf21 insect cells; and by targeted overexpression of the proteins in transgenic mouse hearts. Studies assessing Ca release will be performed using each of these systems. Finally, three other categories of RyR regulatory sites, positioned on the cytoplasmic side of the membrane, will be localized. These are the calmodulin-binding site(s), the PK-A phosphorylation site, and the calpain-cleavage sites. Completion of these studies will increase our understanding of the molecular architecture and protein interactions occurring at the junctional membrane. The structural and functional roles of several fundamental proteins controlling Ca release in heart will be defined.
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