The overall goal of my research is to understand the mechanism of Ca2+ release from the junctional sarcoplasmic reticulum of cardiac muscle. The specific objective of this proposal is the structural and functional characterization of the ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel of cardiac muscle. In order to accomplish our objectives, we are developing a library of monoclonal antibodies to the ryanodine receptor of the Ca2+ release channel from canine cardiac sarcoplasmic reticulum. Identification of the ryanodine receptor of the Ca2+ release channel will be performed by indirect immunoperoxidase staining of protein blots of cardiac sarcoplasmic reticulum proteins. Structural characterization of the ryanodine receptor will involve immunoblotting of cardiac sarcoplasmic reticulum following proteolytic and glycosidase treatments. Ultrastructural localization of the ryanodine receptor of the Ca2+ release channel using indirect immunogold labeling with monoclonal antibodies will be performed on canine ventricular muscle. Purification of the ryanodine receptor of the Ca2+ release channel from digitonin solubilized sarcoplasmic reticulum will be performed using monoclonal antibody affinity columns. The amino acid, carbohydrate and N-terminal amino acid sequence analysis will be performed on the purified receptor. The isolation and characterization of cDNA clones for the ryanodine receptor of the Ca2+ release channel will be performed using monoclonal antibodies and a library of cardiac cDNA cloned in lambda gt11 expression vector. Our final objective will be the functional characterization of the purified receptor. The dissociation constant and maximal binding of (3H) ryanodine and 45Ca2+ to the purified ryanodine receptor will be determine. Reconstitution of the purified receptor into phospholipid vesicles and planar lipid bilayers will be performed in order to demonstrate the identical nature of the ryanodine receptor and the Ca2+ release channel of the junctional sarcoplasmic reticulum. The proposed studies will lead to an understanding of the structure and function of the ryanodine receptor of the Ca2+ channel which initiates and modulates Ca2+ release from the junctional sarcoplasmic reticulum during excitation-contraction coupling in cardiac muscle.
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