The Na,K-ATPase is an important membrane-associated enzyme responsible for maintaining the high internal potassium concentration and low internal sodium concentration characteristic of most animal cells. The ion gradients created by the Na,K-ATPase are fundamental to such diverse cellular functions as the regulation of cell volume and pH, the uptake of nutrients and membrane excitability. In the kidney the ion gradients generated by the Na,K-ATPase are essential for normal renal function. Changes in renal Na,K-ATPase activity have been implicated in the chronic adaptation of the kidney to alterations in Na reabsorptive or K secretory load, the renal hypertrophy associated with diabetes, and tubular cyst formation found in polycystic kidney disease. The purpose of this project is to better understand the role of the Na,K- ATPase in directing renal function. This will involve the characterization: 1) To verify the expression of the Na,K-ATPase alpha subunit isoforms in the developing and mature kidney. To ascertain if Na,K-ATPase oligomers composed of different alpha subunit isoforms are present in the kidney and if these heteromers have different inhibitor sensitivities or functional properties than oligomers consisting of alpha single isoform. 2) To characterize the biochemical and enzymatic properties of the Na,K-ATPase alpha and beta subunits when assembled with the gamma subunit. 3) To characterize the effect of regulatory phosphorylation of the alpha subunit isoforms by protein kinases on function. To determine if phosphorylation of the Na,K-ATPase alpha subunit isoforms by protein kinases is regulated during renal development. 4) To determine if growth factors produced by the metanephroi and developing kidney regulate Na,K-ATPase expression. Relatively little is known concerning the role of alpha subunit oligomerization and the gamma subunit in directing Na,K-ATPase function in the kidney. Moreover, the role of phosphorylation in regulating alpha isoform activity may provide important insights in understanding the function of the Na,K-ATPase isoforms during renal development. The characterization of the changes in Na,K-ATPase expression and activity associated with the fetal and adult kidney may also provide clues to the role of the Na,K-ATPase in the nephropathy associated with several diseased states.
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