Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a plasma membrane C1-channel regulated by phosphorylation. Phosphorylation of four serine residues in the R domain by CAMP-dependent protein kinase activates the channels. Phosphorylation-dependent activation is reversible through the action of an as yet unidentified protein phosphatase. Protein kinase C also activates the CFTR C1- channel, but stimulates less current than does PKA. In this study, we will use electrophysiological and biochemical tools to understand how phosphorylation and dephosphorylation regulate CFTR. First, to learn how CAMP-dependent protein kinase activates the CFTR C1- channel, we will use the single-channel patch-clamp technique to compare the activity of wild- type and mutant CFTR C1- channels. We will study the effect of mutating each of the four phosphorylatable serine residues on the single channel open probability and on channel open and closed lifetimes. This work will help us determine how each serine residue contributes to CFTR C1- channel regulation. Second, to learn how phosphorylation by protein kinase C activates the CFTR C1-channel, and why it has an effect different from PKA, we will use th single channel patch-clamp technique and phosphopeptide mapping to determine which PKC phosphorylation sites are responsible for channel activation. Finally, we will identify the epithelial phosphatase that dephosphorylates and inactivates CFTR C1- channels. We will purify the enzyme from epithelial cells, and study its function and regulation. The results of these studies will provide us with new knowledge, at the molecular level, of how CFTR C1- channels are controlled, and hence, how transepithelial C1-secretion is regulated.
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