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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK037319-01A1
Application #
3236180
Study Section
General Medicine B Study Section (GMB)
Project Start
1987-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Weinman, E J; Minkoff, C; Shenolikar, S (2000) Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA. Am J Physiol Renal Physiol 279:F393-9
Weinman, E J; Steplock, D; Donowitz, M et al. (2000) NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3. Biochemistry 39:6123-9
Zizak, M; Lamprecht, G; Steplock, D et al. (1999) cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor. J Biol Chem 274:24753-8
Bernardo, A A; Kear, F T; Santos, A V et al. (1999) Basolateral Na(+)/HCO(3)(-) cotransport activity is regulated by the dissociable Na(+)/H(+) exchanger regulatory factor. J Clin Invest 104:195-201
Weinman, E J; Steplock, D; Lamprecht, G et al. (1999) Regulation of the Na/H exchanger regulatory factor in OK cells. Miner Electrolyte Metab 25:135-42
Minkoff, C; Shenolikar, S; Weinman, E J (1999) Assembly of signaling complexes by the sodium-hydrogen exchanger regulatory factor family of PDZ-containing proteins. Curr Opin Nephrol Hypertens 8:603-8
Weinman, E J; Steplock, D; Zhang, X et al. (1999) Molecular cloning of the cDNA and promoter sequences for the mouse sodium-hydrogen exchanger regulatory factor. Biochim Biophys Acta 1447:71-6
Weinman, E J; Steplock, D; Tate, K et al. (1998) Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF). J Clin Invest 101:2199-206
Lamprecht, G; Weinman, E J; Yun, C H (1998) The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J Biol Chem 273:29972-8
Weinman, E J; Chamras, H (1996) Reconstitution of human red blood cell Na/H and Na/Na exchange transport. Am J Med Sci 312:47-53

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