The apical membrane of the proximal convoluted tubule of the kidney contains a Na+-H+ exchange transport system. This transporter is important in reclamation of filter bicarbonate and, hence, the acid base status of the organism. A Na+-H+ exchanger is also present in other cells and activation of this transporter has been implicated in a number of cell processes including growth, tumor formation, and defense of cell volume. At the present time, the transporter has been defined functionally and is known to be regulated. Little is known, however, about the nature of the transport protein(s) itself. The present application proposes studies of the Na+-H+ exchanger from the brush border membrane of the proximal tubule of the rabbit. There are three specific aims. 1) Solubilization and reconstitution of renal brush border Na+-H+ exchanger. Characterization of the Na+-H+ would be facilitated by development of the methodology to solubilize the transporter from renal brush border membranes and to assay its activity in a reconstituted system using artificial phospholipid vesicles. Detergent extracts of brush border membrane proteins will be reconstituted into proteoliposomes and tested for the presence of proton gradient stimulated, amiloride inhibitable sodium uptake. The transport studies will determine if the transport characteristics in the proteoliposomes are similar to those in natural membranes. 2) Isolation and characterization of renal brush border Na+-H+ exchanger. Protein separation techniques will be used to fractionate, purify, and characterize the Na+-H+ exchanger. Protein fractions will be reconstituted into phospholipid vesicles and Na+-H+ exchange activity assayed. The nature of the proteins will be determined by SDS-PAGE. 3) Regulation of renal brush border Na+-H+ exchanger by protein kinases. The regulation of the Na+-H+ exchanger by cAMP dependent protein kinase, protein kinase C, and calcium-calmodulin dependent protein kinase will be determined by phosphorylating the solubilized membrane proteins and purified protein fractions with the specific protein kinases. The effect of phosphorylation of selected proteins on Na+-H+ exchange transport will be determined in reconstituted proteoliposomes. SDS-PAGE and autoradiography will be used to determine the substrates of the protein kinases.
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