PHYSIOLOGICAL SIGNIFICANCE OF NA,K-PUMP ALPHA ISOFORMS

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DESCRIPTION: (Applicant's Abstract) 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 derived from ATP hydrolysis. Critical functions served by the pump include the maintenance of the electrochemical gradients for Na+ and K+ across the plasma membrane, the movement of Ca++, sugars, and amino acids via cotransport systems, and the transport of salts and water across epithelia. The pump consists of two dissimilar subunits, alpha and beta. The alpha subunit contains the binding sites for the substrates required by the pump and is phosphorylated transiently during ion transport. It exists in at least three distinct isoforms (alpha1, alpha2, and alpha3) with differences in enzyme kinetics and response to hormones. These differences must originate from structural diversity, yet the primary structures of the alpha isoforms are nearly identical. One exception to this similarity is the amino terminus, where structural alterations produce profound changes in enzyme kinetics and regulation by second messengers. Nevertheless, the structure-function relations underlying these changes are not known. Site-specific in vitro mutagenesis and DNA-mediated gene transfer techniques will be used to explore the consequences of amino terminal modification in all three isoforms. This will demonstrate the contributions of this region to differences in kinetics among the isoforms. To mediate its kinetic effects, the amino terminus must interact with another region within the alpha subunit. A promising candidate is another region of isoform dissimilarity near the center of the subunit, and this divergent region will also be altered to determine its effects on isoform-specific kinetics. Finally, the consequences of the changes in amino terminus and isoform-specific region on regulation by protein kinases will be examined. Taken together, these studies will provide crucial data on the role these regions play in functional differences among the isoforms. More importantly, completion of these studies will increase our understanding of alpha subunit diversity and its significance in active Na+,K+ transport.