This is a competing renewal application for a grant investigating the role of Na-coupled anion transport processes in the absorption and secretion of electrolytes by intestine and other epithelia (kidney, trachea). Two cotransport systems will be studied: The first is the Na-K-Cl cotransproter, the functions and regulation of which were well described in our prior 2 grant periods. We propose now to continue studies already begun on the purification of the cotransport protein, to reconstitute the cotransporter by adding solubilized membrane proteins to liposomes and to clone the cDNA for the cotransporter using the Xenopus oocyte expression system as the cloning assay. RNA will be extracted form tissue sources of the cotransporter and purified for poly (A)+RNA. This will be microinjected into oocytes. The second contransport system to be investigated is the electrogenic Na-HCo3 cotransporter, previously identified in the renal proximal tubule. There is reason to believe it exists in ileal brush border membranes and is central to ileal HCo3 secretion. We will study both intact ileal epithelium and ileal brush border membrane vesicles to demonstrate its presence. We will also inject mRNA from ileum and other intestinal and renal sources into Xenopus oocyts to express the cotransporter there. In addition to its importance in elucidating normal epithelial physiology, the studies outlined may provide insights into the pathophysiology of diarrheal and bronchial diseases and how the latter can be ameliorated with specific drugs.
