The Na,K-ATPase (a P-type ion pump) catalyzes active transport of Na+ and K+ in almost all animal cells. It is essential for transepithelial transport, such as in the kidney, and for the ion gradients that support electrical excitability in muscle and nerve. It is the receptor for inotropic cardiac glycosides in the heart, and many studies point to a role in hypertension. Its catalytic subunit belongs to a family of ion transport ATPases. The crystal structure of the first of these, the Ca2+ ATP of sarcoplasmic reticulum, has recently been determined, providing new insight into the possible mechanism of ion transport. Our theoretical analysis of Na,K-ATPase aligned with Ca2+-ATPase suggests that certain past assumptions about Na,K-ATPase structure were wrong, and that the Ca2+-ATPase structure provides a workable framework for testing specific hypotheses about Na,K-ATPase structure. This proposal will test the hypothesis that the Na,K-ATPase alpha subunit adopts the same fold as the Ca2+-ATPase, using monoclonal antibodies with mapped epitopes as structural probes. Antibodies against the Na,K-ATPase extracellular surface will be produced to facilitate purification and eventual crystallization of the Na,K ATPase. We will also focus our attention on the two Na,K-ATPase subunits that have no counterpart in the Ca2+ - ATPase: the beta and gamma subunits. We will test specific hypotheses about where and how these subunits associate with the alpha subunit, using cross-linking of native and mutagenized enzyme. We have found that the gamma subunit modulates the most physiologically significant properties of the Na,K-ATPase, its affinity for Na+ and K+, and we will exploit that to map the essential structural features of gamma by saturation mutagenesis. We will test the hypothesis that regulation by gamma, like the similar but distinct phospholamban protein that modulates the Ca2+-ATPase, entails reversible oligomerization in the membrane. In sum, a combination of protein chemistry, hybridoma technology, and molecular approaches will be used to investigate the structure of an essential plasma membrane protein.
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