ANALYSIS OF CARDIAC GLYCOSIDE AFFINITY IN CULTURED CELLS

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The Na, K-pump (ie., Na, K-ATPase) is thought to be the major pharmacological receptor for digitalis such as ouabain. It is an intrinsic membrane protein complex that extrudes Na+ from the cell and absorbs K+ from the exterior at the expense of metabolic energy. Its turnover and the resulting electrochemical gradients are responsible for the potential difference across the plasma membrane and indirectly mediate ionic balance, cellular volume, and epithelial transport of salts and water. The Na, K-pump is inhibited by cardiac glycosides, and there is variation in the ouabain affinity between tissues that may be as great as 500-fold. The basis for this variability is not well understood, This project will test the hypothesis that the variation in ouabain affinity of the Na, K-pump is caused by the cell-specific expression of different isoforms of its constituent subunits. The subunit composition and the expressed ouabain affinity will be examined in a number of cultured cell lines by RNA hybridization analysis and enzymatic assays to correlate the presence of specific isoforms with a given binding affinity. DNA-mediated gene transfer will be used to introduce the isoforms into recipient cell lines to observe directly their effects on ouabain affinity. From these studies, the molecular basis of the tissue variability in ouabain affinity will be elucidated. To carry out this project, the candidate will take advantage of a broad background that has included comparative biochemistry, transport physiology, and molecular biology. An RCDA will ensure that the candidate can continue this multidisciplinary approach. The department in which the work will be conducted is internationally recognized in the field of transport physiology and provides an ideal environment in which to develop an independent research career. Its location within a major health science center should further stimulate the development of innovative research in the function and regulation of the Na, K-pump.