This project is devoted to analysis of avian Ca2+ -ATPase molecular forms: their structure, function, and regulation in heart and brain and in tissue cultured cells. The regulatory protein, phospholamban, that depresses calcium ion transport in the heart, is also a focus of study. The encoding DNAs for these molecules have been cloned and sequenced and expressed in tissue cultured cells. Monoclonal and polyclonal antibodies to each of these proteins are being used to asses expression, to localize the molecules within cells, and to isolate the molecules in pure form. Altered forms of the Ca2+ ATPase and of phospholamban are being generated with molecular biological techniques and are being used to study the topologies of the molecules vis a vis the membrane and to study structure/function and structure/regulation relationships. High priorities in this endeavor are (a) to determine the sites on the Ca2+ ATPase that are involved in interactions with modulators of calcium ion transport: the endogenous modulator phospholamban and pharmacological agents such as thapsigargin, (b) to determine what aspects of phospholamban structure are involved in interactions with the Ca2+ -ATPase, (c) to determine what structural features of the Ca2+ -ATPase and phospholamban are necessary and sufficient localization in the sarcoplasmic reticulum or endoplasmic reticulum. Initial steps towards determining the three-dimensional structure of the Ca2+ -ATPase are also underway through production of its hydrophilic domains in quantity for structural analysis.
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