Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

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Abstract

The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, ΔF508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperature-rescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 37°C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′bithiazolyl-2′-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature-rescued ΔF508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued ΔF508 CFTR for up to 12 hours at 37°C (P < 0.05). Using firefly luciferase-based reporters to investigate the mechanisms by which low temperature and corr-4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated ΔF508 CFTR response by 61% at 6 hours (P < 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented cAMP-stimulated currents, revealing that the biochemically detectable cell surface ΔF508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued ΔF508 CFTR was not sufficient to obtain maximal ΔF508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of ΔF508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.

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