The differential targeting of P-type ATPases in epithelial cells is essential for normal transport of nutrients, electrolytes, and fluid across all epithelia. In certain epithelial cells, two significantly homologous members of the P-type ATPases are simultaneously expressed but targeted to opposite epithelial cell plasma membrane domains. On the other hand, the loss of polarized targeting of the Na,K-ATPase is a feature of congenital polycystic kidney disease. The data generated from this project will be directly relevant to the mechanisms of both physiological and pathological targeting of P-type ATPases. A model epithelial cell culture system has been developed in which to study the mechanisms of differential assembly and targeting of P-type ATPases. The gastric H,K-ATPase beta-subunit (Hkbeta) has been stably transfected into MDCK cells, and, in this cell line, is apically targeted. A novel feature of this cell line is that Hkbeta, through its assembly of hybrid heterodimers with endogenous MDCK cell Na,K- ATPase alpha-subunit (NaKalpha), targets a fraction of the normally basolaterally-localized NaKalpha to the apical membrane at steady- state.
The specific aims of this proposed project are: 1) to characterize the molecular mechanisms of the regulation of the intracellular transport of heterodimeric P-type ATPases through the secretory pathway in mammalian epithelial cells; and, 2) by mutagenesis, to identify sorting signals within the cytoplasmic domain of Hkbeta that regulate its intracellular transport and plasma membrane targeting. The mechanism of the regulation of intracellular transport and plasma membrane targeting of hybrid NaKalpha-HKbeta heterodimers will be characterized by a combination of established molecular biological, cell biological, and biochemical techniques. Apical targeting of this hybrid membrane transporter may also provide data relevant to the mechanisms of targeting of other clinically and therepeutically important transporters such as the cystic fibrosis transmembrane regulator, the oligopeptide transporter, and Na-dependent nutrient transporters. If successful, this preliminary study should support more elaborate future integrated investigation into the mechanisms of the regulation of the differential assembly, targeting, and activity of P-type ATPases in epithelial cells and of other apical membrane transporters.
