Proton-potassium adenosine triphosphatases (H+, K+-ATPases) located in the renal collecting duct are involved in maintenance of blood K+ balance. Although aldosterone is known to be one of the principal regulators of K+ homeostasis and urinary acidification, no firm linkage has been established between aldosterone and the regulation of genes encoding the subunits of the H+, K+-ATPase or the activity of the pump in the kidney. The long term goal of our research is to gain an understanding of the regulation of H+, K+-ATPase activity in the kidney. At least three H+, K+-ATPases containing distinct isoforms of the alpha subunit populate the collecting duct. These H+, K+-ATPases contain the """"""""gastric"""""""" 1, the """"""""colonic"""""""" 2a or the novel 2b subunit. The 2b H+, K+- ATPase appears to be a candidate for the aldosterone-responsive form of the pump. We propose to study the expression of the 2a and 2b H+, K+- ATPases following two parallel lines of investigation. First, aldosterone-responsive transcriptional regulation of the 2b subunit will be investigated in vivo in a rabbit cortical collecting tubule cell line (RCCT-28A). This will be accomplished by characterizing the response of the 2a and 2b mRNAs, protein levels and H+, K+-ATPase activity following application of aldosterone to RCCT-28A cells. The elements governing transcription from the 2 gene will be located by DNase I hypersensitivity and promoter deletion analysis. The response elements will them be mapped to single-nucleotide resolution by in vivo footprinting studies. Second, the activity of the two 2 H+, K+-ATPases will be studied by expression of the cloned cDNAs in mammalian cells. A direct comparision will be made between the activities and pharmacological properties of the expressed 2a and 2b H+, K+-ATPases. Finally, phosphorylation of the 2 subunits will be investigated as a possible mechanism for differential regulation of the 2a and 2b H+, K+- ATPases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054721-04
Application #
6498136
Study Section
General Medicine B Study Section (GMB)
Program Officer
Ketchum, Christian J
Project Start
1999-02-15
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2004-01-31
Support Year
4
Fiscal Year
2002
Total Cost
$193,166
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Zies, Deborah L; Gumz, Michelle L; Wingo, Charles S et al. (2006) Characterization of the rabbit HKalpha2 gene promoter. Biochim Biophys Acta 1759:443-50
Zies, Deborah L; Wingo, Charles S; Cain, Brian D (2002) Molecular regulation of the HKalpha2 subunit of the H+,K(+)-ATPases. J Nephrol 15 Suppl 5:S54-60
Caviston, T L; Campbell, W G; Wingo, C S et al. (1999) Molecular identification of the renal H+,K+-ATPases. Semin Nephrol 19:431-7