The overall objective of this research is to determine how the sodium and potassium-dependent adenosine triphosphatase (Na,K- ATPase) is genetically regulated by ions and hormones. Na,K- ATPase transports K+ into cells in exchange for Na+, energized by the hydrolysis of ATP. In the distal tubule and collecting duct of the kidney, Na,K-ATPase maintains the ion gradients that energize the reabsorption or secretion of salts and water and is transcriptionally regulated by steroid hormones and ion concentrations. Na,K-ATPase regulation will be studied in the Madin-Darby canine kidney (MDCK) continuous cell line. Use of the MDCK cell line allows genetic and biochemical analyses that would otherwise be impossible. Mutant cell lines have been isolated that have differences in Na,K-ATPase regulation and express immunologically different Na,K-ATPase. the hypothesis to be tested is that the mutant cells have genetic alterations which affect essential features of Na,K-ATPase function and regulation. Cell lines expressing different Na,K-ATPase or responding differently to Na,K-ATPase regulators will be studied to determine if there are differences in Na,K-ATPase alpha and beta subunit mRNA, rate of synthesis and degradation, subunit abundance, ouabain binding, enzyme activity, or active cation transport. Differences in the peptide coding regions and transcription regulating regions of the alpha and beta subunit DNA will be examined by restriction mapping and/or sequencing DNA fragments isolated from cDNA or genomic libraries prepared from the cell lines. These studies will define the alterations in structure that affect Na,K-ATPase biosynthesis, maturation, and ion transport. Investigation of regulatory mutants will identify factors important for Na,K-ATPase genetic control. Delineation of the salient features of Na,K-ATPase function and regulation will improve our understanding of how Na,K-ATPase can be modulated in normal or disease states to produce long-term changes in the cellular mechanisms of electrolyte homeostasis.
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