Physiological Significance of Na,K-pump Diversity

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The Na.K-pump (i.e., Na,K-ATPase) is the major pharmacological receptor for cardiac glycosides such as digitalis and ouabain. It is a plasma membrane-spanning protein complex that mediates the exchange of Na+ and K+ at the expense of metabolic energy. The resulting ionic gradients provide the basis for nutrient uptake, control of intracellular pH and Ca++, membrane excitability, and volume regulation. It follows that the Na.K-pump is central to cellular homeostasis. Its component subunits exist in multiple forms, but the physiological relevance of this subunit diversity remains unknown. The primary contributor to overall catalysis, the alpha subunit, exists in four isoforms, while an associated protein, the beta subunit, exists in three. Recent work suggests that the major functional distinction among the isoforms is their interaction with regulatory proteins such as protein kinase C (PKC), which also exist in multiple forms. The four specific aims of this project will evaluate explicitly the role played by this protein diversity in regulation. The first aim will use heterologous expression in mammalian cells to evaluate the isoform-specific differences in pump regulation initiated by stimulation of PKCs. The second specific aim will determine the mechanisms underlying the isoform-specific differences, again using the heterologous expression strategy. The third aim will identify the contribution played by isozymes of PKC in the regulation of pump isoforms, using specific inhibitors and genetic techniques to suppress kinase activity. Finally, the fourth specific aim will establish the regulatory consequences of subunit heterogeneity in cells that naturally express multiple forms by suppressing the endogenous isoforms. Takentogether, these studies will provide crucial data on the role played by protein diversity in regulation of the pump. More importantly, completion of these studies will increase our overall understanding of subunit diversity and its physiological importance in active Na+,K+ transport. These studies will take advantage of established cell lines in culture. Because of the fundamental role of the Na.K-pump in cellular homeostasis, it lends itself well to the use of these models. Indeed, this project expands the usefulness of existing nonmammalian model systems,consistent with the goals of the NCRR.