The major clinical manifestations of cystic fibrosis have been attributed to reduced C1 permeability across luminal membranes of selected epithelia. This defect does not appear to be in the C1 channel itself, but rather in the response of CF cells to cAMP, a response that in normal cells leads to the activation of this channel. Utilizing an antibody specific for the DIDS binding peptide which by all criteria recognizes the C1 channel affected in cystic fibrosis, an immunofluorescent study in the 184 epithelial cell line was begun. Our findings to date suggest that the activation of the C1 channel that occurs in response to the elevation of cAMP may result from the movement of the channel from a vesicular and clearly intracellular localization to the plasma membrane. One of our goals is to determine if this striking result has parallels in the tissues affected by cystic fibrosis. If this is so, our investigations will then determine if this movement is imperfectly carried out in cells from afflicted patients. Our proposed investigations are grouped into three Specific Aims: 1. To carry our further morphological studies of vesicular trafficking in established cell lines and primary cultures of cells with normal and CF phenotypes.2. To determine if the C1 channel from plasma membranes differs from that in cytoplasmic vesicles with regard to phosphorylation or functional characteristic. 3. To develop a permeabilized or perforated cell model for vesicular trafficking and exocytosis so as to determine if aspects of the intracellular message cascade could be defective in cells from patients having cystic fibrosis.
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