The mechanisms of chloride reabsorption in the kidney proximal tubule are poorly understood. In red blood cells, the major mechanism for transport of chloride across the membrane is via the anion exchanger band 3 which mediates the exchange of one chloride for one bicarbonate. Based on the transport activity and inhibitors sensitivity, different isoforms of anion exchangers have been identified in the kidney proximal tubule. These, among others, include chloride/formate, chloride/oxalate, chloride/hydroxyl, and possibly chloride/bicarbonate exchangers. There is little information on the molecular properties of the anion exchanger protein(s) in the kidney proximal tubule. Anion exchange transport systems in general share several properties of the band 3 protein including sensitivity to inhibition by disulfonic stilbenes, probenecid, furosemide, and arginyl amino group modifier phenyglyoxal. Using disulfonic stilbene (DNS) affinity chromatography, we have identified and partially purified a protein with anion exchange characteristics in brush border membranes isolated from rabbit kidney proximal tubules. This protein has a molecular weight of 162 kD. The 162 kD protein is sensitive to the known inhibitors of the band 3 and displays anion exchanger activity in reconstituted vesicles.Incubation of the DNDS affinity-purified proteins with protein kinase A and ATP resulted in decreased Cl-Cl exchange activity in reconstituted liposomes. The 162 kD protein has been purified to homogeneity utilizing anion exchange (Pac Q) and size exclusion chromatography. The liposomes reconstituted with the purified 162 kDa protein displayed anion exchange activity as assayed by 36Cl-Cl exchange. The major objectives of the current proposal are characterization of functional, cellular, and molecular properties of the 162 kD protein. Specifically, the 162 kD protein will be evaluated with regard to 1. Functional identity. 2. Signal transduction. 3. Tissue distribution, and 4. Molecular structure and the encoding cDNA. The purified 162 kD protein will be reconstituted into liposomes. The functional identity of the 162 kD protein will then be determined by assaying the influx of different radiolabeled anions into liposomes reconstituted with the 162 kD protein. The signal transduction pathways involved in regulating the proximal tubule anion exchanger will be studied by incubating the partially or completely purified protein with ATP and various kinases followed by reconstitution. The liposomes will then be assayed for anion exchange activity. Polyclonal antibodies have been raised against the 162 kD protein in guinea pigs with the 162 kD band cut out of the gel. The antibody will be used to study the tissue distribution of the 162 kD protein by Western blot analysis and probe a kidney cDNA library for the clones encoding the 162 kD protein. The 162 kD protein was cut out, digested, purified, and subjected to gas phase sequencing machine. Two novel partial amino acid sequences have been obtained. Oligonucleotides of 23 and 21 bases in length have been synthesized according to the peptide sequences and will be used to probe a kidney cDNA library for the clones encoding the 162 kD protein. The encoding full length cDNA will be cloned into an expression vector and will be used to transfect COS cells. The characteristics and functional properties of the 162 kD protein will be studied in the transfected cells by measuring the influx of radiolabeled anions in these cells. The results of the proposed studies should significantly enhance our understanding about the cellular and molecular properties of the anion exchanger(s) in the kidney proximal tubule.
Showing the most recent 10 out of 39 publications
