The Na,K-pump (ie., Na,K-ATPase) is thought to be the major pharmacological receptor for cardiac glycosides such as digitalis and ouabain. It is a membrane-spanning protein complex that mediates the transmembrane exchange of Na+ and K+ at the expense of metabolic energy. Critical functions served by the pump include the generation and maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane, the absorption of Ca++, sugars and amino acids via cotransport systems, and the transport of salts and water across epithelia. The Na,K-pump is inhibited by cardiac glycosides, and within a given species, there is variation in ouabain affinity between tissues that may be as great as 500-fold. The basis for this variability is not well understood. There is also tissue variability in the expression of different isoforms of the component subunits of the Na,K-pump. This project will test the hypothesis that the basis of the variation in ouabain affinity is the cell-specific expression of different isoforms of the alpha and beta 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 various isoforms into recipient cell lines to observe directly their effects on ouabain affinity. Expression of the mRNAs that encode the native subunits will be monitored by hybridization analysis after transfer of a foreign isoform to determine its effects on the regulation of the native isoforms. From these studies, the molecular basis of the tissue variability in ouabain affinity will be elucidated.
