The goal is to understand, at the molecular level, how Ca is accumulated and released by sarcoplasmic reticulum (SR) in cardiac muscle. Junctional SR vesicles and free SR vesicles will be isolated from heart, and their different Ca uptake and Ca release properties analyzed. Particular emphasis will be placed on characterization of the Ca-induced Ca release channel localized to junctional SR vesicles, which appears to be an intracellular Ca channel primarily responsible for elevating intracellular Ca concentration during muscle contraction. Three key cardiac SR proteins, calsequestrin, high molecular weight (HMW) proteins (putative components of the SR feet), and phospholamban will be purified to homogeneity and characterized in detail. Calsequestrin and HMW proteins are localized predominantly to junctional SR in heart, whereas phospholamban is uniformly distributed throughout the SR. All three proteins are strongly implicated in SR Ca homeostasis, but their precise functions remain poorly defined. Emphasis is placed on primary structure determination of these three proteins, ultrastructural characterization, and determination of phosphorylation effects on protein conformation and function. We will determine whether purified phospholamban behaves as a channel and/or interacts directly with the SR Ca pump to modulate Ca transport, and whether the HMW proteins and calsequestrin (and copurifying 110- to 160-kDa proteins) interact with each other, other proteins, and/or the lipid bilayer to alter SR ionic permeability. We will also determine whether the HMW proteins are integral protein components of the SR feet. Experiments will focus on defining potential protein-protein and protein-lipid interactions in purified-reconstituted systems. In this way the functions of the three proteins may be better understood at the molecular level. Important techniques to be utilized in the studies include fusion of the cardiac SR vesicles with planar lipid bilayers to measure single Ca channel activity; gas-phase protein sequencing and recombinant DNA methods to determine primary structure; spectrophotometric, covalent probe, monoclonal antibody and hydrodynamic techniques to examine higher order structure and membrane-protein topology; and membrane-protein reconstitution to examine protein function. Completion of the studies described will increase our understanding of the role of the SR in basic mechanisms of excitation-contraction coupling in cardiac muscle.
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