The plasma membranes of polarized epithelial cells are divided into two distinct domains which manifest markedly different protein compositions. Among the proteins which are differentially distributed between these domains are members of the E1-E2 class of ion transporting ATPases. These ion pumps mediate numerous processes necessary for normal cell and tissue function, including the maintenance of osmotic balance, the generation of neural potentials, the renal and intestinal handling of solutes and the gastric secretion of acid. Alterations in the regulation of their activity have been implicated in the pathogenesis of numerous disease states. Two members of this family, the Na,K-ATPase and the H,K-ATPase, share a great deal of structural and mechanistic homology, yet differ dramatically in several important attributes. Thus, while the Na,K-ATPase is generally sorted to the epithelial basolateral membrane, the H,K-ATPase is directed to the apical surface. The two ion pumps also manifest distinct ion affinities, transport stoichiometries and inhibitor sensitivities. The research described in this proposal is designed to elucidate the molecular correlates of these differences. Our goal is to establish the structure-function relationships which determine the individual cell biologic and physiologic properties of these extremely important proteins. We will, therefore, undertake to 1) identify and characterize the sorting signals which are embedded in these proteins and are responsible for determining their subcellular distribution; 2) identify and characterize the cellular components which interpret and act upon these signals; and 3) map the protein determinants which bestow upon these ion transport ATPases their individual catalytic characteristic. These problems will be addressed using the cDNA clones encoding these pumps and the tools of genetic recombination and transfection. We will examine MDCK cells expressing the Na,K-ATPase, the H,K-ATPase or chimeric ion pumps created by genetically recombining portions of the two transport ATPases. The remarkable similarity relating the two parent proteins will greatly facilitate the construction of the relevant chimeras as well as the analysis of their properties. Observation of the subcellular distribution and ion transport reactions associated with the parent and chimeric pumps will allow us to assign sorting and transport functions to specific portions of the H,K-ATPase and Na,K-ATPase polypeptide chains. This system will be further exploited to identify protein components of MDCK cell which interact with these ion pumps in order to mediate their sorting. Use of the chimeras will allow us to establish the specificity of these associations and to analyze their role in membrane protein targeting.
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