Regulation of intracellular and extracellular pH (acid-base transport), as well as other ionic concentrations, such as Cl- and Na+, are key to maintaining ion gradients across membranes. Moreover, normal cell function is a balance between inward and outward movement of these ions, often varying in response to intracellular pH. This is especially true in the central nervous system, digestive tract, respiratory tract, and urinary system. Several ion-transporter protein cDNAs have been cloned in the last few years. Recently, we used expression cloning to clone and characterize the renal electrogenic Na/HCO3 cotransporter (NBC). We have now cloned and expressed another novel acid-base transporter, the Na+ driven Cl-HCO3 exchanger from Drosophila. This transporter has been physiologically identified in neurons, muscle, fibroblasts, and certain epithelia. In the kidney, this Na+ driven Cl-HCO3 exchanger functions in fibroblasts, mesangial cells, and the proximal tubule. So far the role of the Na+ driven Cl-HCO3 exchanger appears to be regulation of intracellular pH. Our expression data indicate that the Na+ driven Cl-HCO3 exchanger is actually a Na+ driven anion exchanger (NDAE1). We have isolated several mammalian clones, the human NDAE1-gene, and developed a NDAE1-antibody. Preliminary immunolocalization in rat kidney shows pronounced staining in the glomerulus, distal and collecting ducts, and tubules of the inner medulla. We hypothesize the NDAE1 plays a key role in the regulation of intracellular pH, Cl-, and Na+ in the kidney. To test this hypothesis, we propose three aims: First, to determine in which tissues NDAE1 exists, we will clone the mammalian renal NDAE1 homologue and generate antibodies against both the Drosophila and mammalian proteins. Using a combination of Northern analysis, Western analysis, and immunochemistry, we will determine which cells and tissues express NDAE1. Comparing both organisms will help us determine if Drosophila might be a useful model for renal ion transport. Second, we will study the molecular physiology of NDAE1 by expression in Xenopus oocytes. Using a combination of microelectrode techniques, we will measure intracellular ions (H+, Cl-, and Na+), voltage clamp, and iontophoretically control cellular ions. Third, we will use functional complementation of NDAE1-fragments to determine structurally important domains of the NDAE1 protein. Since we have a candidate region for interaction with cations, we will augment our domain survey by exchanging this homologous domain of NBC and the anion exchangers. This combination of approaches, applied to the newly cloned Na+ driven anion exchanger, will allow us to determine how and where NDAE1 regulates intracellular pH, Cl-, and Na+ in the kidney.
|Effective start/end date||5/1/00 → 4/30/07|