The brush border membrane (BBM) of the kidney proximal tubule contains a transport protein which mediates the reabsorption of sodium from the lumen in exchange for hydrogen ions in the cell. Studies using intact proximal convoluted tubule cells, isolated BBM vesicles, and detergent solubilized BBM proteins assayed after reconstitution into artificial lipid vesicles indicate that the activity of the Na+-H+ exchanger is regulated directly by processes involving protein phosphorylation mediated by specific protein kinases such as cAMP dependent protein kinase (PKA) , calcium phospholipid dependent protein kinase, and calcium calmodulin dependent multifunctional protein kinase II. The long term goals of the laboratory are to define the mechanisms by which the exchanger is regulated by isolating and characterizing the components of the Na+-H+ exchanger. Toward this end, the present grant proposes to define a polypeptide suggested to be a regulatory co-factor of the BBM Na+-H+ exchanger. Studies using detergent solubilized BBM proteins subjected to limited trypsin digestion or fractionated by column chromatography indicate that the activity of the exchanger can be dissociated from its regulation by PKA. Co-reconstitution experiments suggest that PKA mediated inhibition of the Na+-H+ exchanger requires a 42 kDa phosphoprotein that is distinct from the transporter itself. To characterize this putative regulatory cofactor, its partial amino acid sequence will be determined in conjunction with the development of monospecific antibodies. The primary structure will be obtained by cloning the cDNA from a rabbit proximal tubule library using oligonucleotide probes obtained from the peptide sequences. Assays of PKA regulation of Na+-H+ exchanger in native BBM vesicles and detergent solubilized membrane proteins (or fractions thereof) will be performed in the presence or absence of selected antibodies and/or synthetic peptides. Immunohistochemical, Western immunoblot, and Northern RNA hybridization analyses will be employed that determine the regional expression of the 42 kDa protein in the nephron.
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