STRUCTURE-FUNCTION OF PHOSPHOLAMBAN IN HEART

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Phospholamban (PLB) is the principal membrane protein of the heart phosphorylated in response to P-adrenergic stimulation. The protein is localized to cardiac sarcoplasmic reticulum (SR), where it exists as a pentamer of small, tightly associated, 52-amino acid subunits. When dephosphorylated, PLB inhibits the Ca pump and Ca transport by cardiac SR. Phosphorylation of PLB at Ser16 and Thr17 disinhibits the Ca pump, stimulates active Ca sequestration, and increases the rate of myocardial relaxation during beta adrenergic activation. Precisely how this detailed regulation occurs is currently unknown. In this application we propose structure/function studies on PLB to define a molecular mechanism of action. Aim 1 will address protein structure in native SR vesicles. The membrane protein topology, pentameric organization, and molar stoichiometry with the Ca pump will be determined. These studies will lay the framework for expression studies correlating protein structure with function. In Aim 2, PLB will be expressed in atrial tumor cells, which contain Ca pumps, but no PLB. Both wild-type and mutated subunits will be expressed. Using this cellular reconstitution system, we will determine which amino acids stabilize the pentamer, and if the pentameric structure is required for Ca pump regulation. Whether the Ca channel activity of PLB is involved in this process will be investigated, and the mechanism of regulation of active Ca transport by the phosphorylation sites will be determined. In Aim 3, mg quantities of PLB will be expressed and purified from insect cells. The goal here is to produce sufficient material for detailed biochemical studies, including protein crystallization to determine the three dimensional structure. Co- expression of PLB with the Ca pump in insect cells will also be attempted. In Aim 4, expression of wild-type and mutant PLB subunits will be targeted to mouse atrium and ventricle in transgenic animals. By overexpressing wild-type PLB, the role of the PLB/Ca pump stoichiometry in controlling the rate of myocardial relaxation will be assessed. By expressing mutant PLB subunits in mouse hearts, we hope to identify transdominant mutations that disrupt the function of the wild- type pentamer. Mutations that destabilize the pentamer and/or alter the phosphorylation sites will be investigated. Thus, the studies will address the cellular and molecular physiology of PLB, from the purified protein level to the whole animal. By correlating experimental results from Aims 1-4 a complete picture of PLB structure and function will emerge. The results will be important for a precise understanding of catecholamine regulation of the heart.