This is the first amendment of a new submission designed to explore the mechanisms through which protein kinase C regulates the sodium pump in a proximal tubule model. There are three Specific Aims. The first is to determine the mechanism whereby phosphorylation of the alpha subunit serine-11 and/or serine-18 residue by protein kinase C alters the activity of the sodium pump. These studies will involve assessing the phosphorylation of transfected wild-type and amino-terminus mutants of the rodent alpha subunit expressed in OK cells and will involve measurements of rubidium, ATPase activities and changes in intracellular sodium concentrations.
The second aim has two parts. The first is to determine which amino acids at the amino-terminus interact with other cytosolic domains of the Alpha subunit. The second is to determine whether the aminoterminus specifically interacts with other cytosolic domains of the alpha subunit through the phosphates linked to serine 11 and/or serine 18. Those studies will require methods outlined under Specific Aim #1.
Specific Aim #3 is to determine the cytosolic domains of the alpha subunit that interact with the amino terminus. These studies will use a variety of techniques to determine the interaction or closeness of polypeptide segments of the sodium pump. These will involve affinity purification of the peptides, use of the yeast two-hybrid system and chemical cross linking studies.
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