Cystic fibrosis (CF), the most common inherited disease in the caucasian population, has been shown to be due to an abnormally low C1 permeability in epithelial cells. This low C1 permeability may be due to either (i) the inability of cAMP to activate C1 channels present in the plasma membrane, or (ii) the inability of cAMP to cause the translocation of C1 channels from the cytoplasm to the plasma membrane. The objective of this project is to directly assess whether the translocation of the CF transmembrane conductance regulator (CFTR) to the plasma membrane is required for agonist-mediated C1 secretion, and to determine if the location and translocation of CFTR is abnormal in cells expressing mutant forms of CFTR. The ability of cAMP to activate C1 channels in cells retrovirally transduced with either wild-type of mutant forms of CFTR will be assessed using the chloride-selective fluorophore, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). The functional responsiveness of cells expressing wild- type and mutant forms of CFTR will be correlated with CFTR's location under resting conditions and with the ability of cAMP to induce the translocation of CFTR from the cytoplasm to the plasma membrane as assessed by indirect immunofluorescence using antibodies directed against CFTR. Finally, the exact nature of the cytoplasmic organelles where CFTR is located at rest, and those involved in the cAMP-mediated trafficking of CFTR to the plasma membrane will be determined using immunogold electron microscopic techniques. This project will advance our knowledge of the mechanisms by which stimulus-secretion coupling occurs, and the role of wild-type and mutant variants of CFTR in this process. A more thorough understanding of these processes in CF is vital to attempts at clinical intervention into this lethal genetic disease.
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