∆F508-CFTR stability at the apical membrane of human nasal epithelial cells
LE3 .A278 2008
2008
Chappe, Valerie
Acadia University
Bachelor of Science
Honours
Biology
F508 is the most common mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel that causes the genetic disease Cystic Fibrosis (CF). F508 leads to a severe CF phenotype because the mutated protein is degraded before reaching the apical membrane of epithelial cells. Successfully rescued F508-CFTR, accomplished by chemical chaperones or low temperature, are less stable than non-mutated CFTR and result in lower than normal activity and half-life. Evidence has shown that stimulating protein kinase C (PKC) may increase the stability of CFTR at the membrane of epithelial cells. This study investigated F508-CFTR stability at the membrane of the human nasal epithelial cell line JME/CF15, derived from a F508 homozygous patient, with a focus on the effect of stimulating PKC. Cells were stimulated with Vasoactive Intestinal Peptide (VIP), a G protein coupled receptor agonist. Western blots, iodide efflux, and immunostaining followed by confocal microscopy were used to explore the effect of VIP-dependent PKC stimulation on F508-CFTR trafficking and activity. Experiments were also performed on recombinant BHK cells stably expressing F508-CFTR, using phorbol 12-myristate 13-actate (PMA) to stimulate PKC. Our results show that VIP and PMA have a significant effect on F508-CFTR in CF15 and BHK cells respectively at physiological temperature by rescuing its trafficking and activity. Results also indicate that the observed effects were due to stimulating a PKC pathway. A combination of strategies, including improving trafficking, function, and stability, will be necessary to minimize the effects of the F508 mutation to significantly improve the condition of CF patients; this study demonstrates that stimulating PKC potentially affects each of the three important parameters to correct the F508-CFTR severe phenotype.
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https://scholar.acadiau.ca/islandora/object/theses:535