The overall goal of this project is the structural and functional characterization of the molecular components of the dihydropyridine receptor of the cardiac Ca2+ channel. In order to achieve this objective, we are developing a library of monoclonal antibodies to the dihydropyridine receptor of the cardiac Ca2+ channel. Identification and structural characterization of the protein components of the dihydropyridine receptor of the cardiac Ca2+ channel will be performed by indirect immunoperoxidase staining of protein blots of sarcolemma and by immunoprecipitation of 125I-labeled sarcolemma proteins. Structural characterization of the dihydropyridine receptor will involve proteolytic and glycosidase treatments of right-side-out and inside-out sarcolemma vesicles.
The aim of these studies will be to determine the molecular weight and subunit composition of dihydropyridine receptor and the arrangement and orientation of the protein components of the dihydropyridine receptor of the cardiac Ca2+ channel. Ultrastructural localization of the dihydropyridine receptor of the cardiac Ca2+ channel using indirect immunogold labeling with monoclonal antibodies will be performed on canine ventricular muscle and isolated cardiac sarcolemma membranes.
The aim of the ultrastructural studies will be to determine the distribution of the dihydropyridine receptor and to examine the arrangement and orientation of the molecular components of the dihydropyridine receptor in the sarcolemma membrane of canine cardiac muscle. Purification of the dihydropyridine receptor of the cardiac Ca2+ channel will be performed using monoclonal antibody affinity columns and/or nifedipine-Sepharose affinity columns. Subunits of the dihydropyridine receptor will be prepared by gel filtration in the presence of sodium dodecyl sulfate. The amino acid, carbohydrate and N-terminal amino acid sequence analysis will be performed on the purified receptor and/or the purified subunits. The isolation and characterization of cDNA clones for the dihydropyridine receptor will be performed using monoclonal antibodies and a library of cardiac cDNA cloned in Lambda gt11 expression vector. Functional characterization of the purified dihydropyridine receptor will involve determination of the binding properties of the various radioactive Ca2+ channel blockers to the purified dihydropyridine receptor, reconstitution of the purified dihydropyridine receptor in phospholipid vesicles and phosphorylation of the purified dihydropyridine receptor. The proposed studies will lead to an understanding of the structure and function of the cardiac Ca2+ channel which initiates and modulates the transmembrane influx of extracellular calcium during excitation-contraction coupling in cardiac muscle.
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