All ETDs from UAB

Advisory Committee Chair

James F Collawn

Advisory Committee Members

Zsuzsanna Bebok

Helen Kim

Kevin L Kirk

Elizabeth S Sztul

Document Type

Dissertation

Date of Award

2008

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Cystic fibrosis (CF) is a genetic disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel that functions at the apical surface of epithelial cells and plays a critical role in mucociliary clearance. The most common mutation in the CFTR gene involves a deletion of phenylalanine at position 508 in the protein (ΔF508 CFTR). ΔF508 CFTR is recognized as misfolded in the endoplasmic reticulum (ER) and is degraded. In vitro manipulations such as culturing cells at 27°C can “rescue” ΔF508 CFTR to the plasma membrane (rΔF508 CFTR), but no pharmacological agent has produced comparable results. Compared to the wild type (WT) protein, rΔF508 CFTR is very rapidly cleared from the plasma membrane and has channel-gating defects. Some other disease-causing CFTR mutations also exhibit altered cell surface properties. Herein, we characterized how two small molecules, which help with proper protein folding (pharmacological chaperones corr-4a and VRT-325) improved the cell surface stability and function of rΔF508CFTR, and we examined the cell surface trafficking and function of two previously uncharacterized CFTR mutations, R31C and R31L. Corr-4a ([2-(5-Chloro-2- methoxy-phenylamino)-4’-methyl-[4,5’]bithiazolyl-2’-yl]-phenyl-methanone) promotes CFTR rescue and Vertex-532 (4-Methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol) affects its channel gating. Our analysis of ΔF508 CFTR-expressing polarized epithelial cells treated iv with corr-4a revealed that rΔF508 CFTR was stabilized at the cell surface, but the cAMP responsiveness was still abnormal. Cell surface biotinylation and Ussing chamber analyses revealed that in the presence of corr-4a, there was enhanced channel activity at 6 h. The addition of the VRT-532 dramatically increased the cAMP responsiveness of the channel, thus the combination of compounds was necessary to correct two major defects associated with this mutation. We also determined that, like ΔF508, the R31C and the R31L mutations compromised the biogenesis and enhanced the clearance of CFTR from the cell surface, which synergistically decreased surface expression to cause disease. In summary, our studies on the trafficking and functional defects of 3 naturally occurring mutations in CFTR contribute to understanding the molecular basis for the altered trafficking of the CFTR protein; this is an important step in developing therapies for CF.

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