MOLECULAR MECHANISMS OF PROXIMAL TUBULE ION TRANSPLANT

这个项目的总体重点仍然是结构、功能

The general focus of this project continues to be the structure, function and physiologic roles of the protein(s) mediating Na+-H+ exchange across the plasma membranes of proximal tubule cells. cDNAs encoding four Na+-H+ exchanger isoforms (NHE1-4) have been cloned from mammalian cells. Transcripts for all four isoforms are expressed in the kidney. During the past project period we generated isoform-specific antisera to demonstrate that NHE1 is expressed on the basolateral membrane whereas NHE3 is expressed on the brush border membrane of proximal tubule cells. Based on this initial identification of NHE isoforms expressed in proximal tubule cells, we will pursue three interrelated aims to characterize the structure and function of NHE proteins in the proximal tubule and elsewhere along the nephron. The first general aim is to map the cellular and subcellular sites of expression of NHE isoforms. For this purpose we will continue to generate isoform-specific polyclonal and monoclonal antibodies. These antibodies will be used to determine the cell and membrane sites of expression of each isoform in the proximal tubule and along the nephron by use of immunofluorescence and immunoelectron microscopy, and by Western blot analysis of membrane fractions. We will also assess the temporal and spatial sites of expression of NHE isoforms in the developing nephron of the neonatal kidney. The second general aim is to characterize NHE isoforms with respect to important functional properties. Transport will be studied in stably transfected lines of LAP1 cells that express each isoform (NHE1-4), and in Sf9 cells infected with recombinant baculovirus encoding each isoform. The third general aim is to assess the relationship of structure to function of NHE isoforms. We will confirm that NHE1 and NHE3 are each components of oligomeric complexes, and will characterize the composition of these complexes and the steps involved in their biosynthesis and assembly. The topology of NHE3 will be assessed by localization of anti-peptide antibodies and by vectorial proteolysis of brush border membrane vesicles. Structure- function relationships within the amphipathic domains of NHE proteins will be studied by use of chimeric constructs to identify subdomains that determine functional properties found to differ between isoforms, and by mutation of specific amino acid residues likely to participate in binding and/or transport of cations and H+. Information about both the molecular properties of NHE isoforms and their sites of expression along the nephron will provide insight into the physiologic roles of NHE isoforms in such integrated kidney functions as HCO3 - reabsorption and acid secretion.