Deletion of the phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most prevalent mutation in cystic fibrosis (CF). This mutation (ΔF508CFTR) leads to a reduced cAMP- sensitive Cl- conductance in epithelial cells. While the mutant protein can function as a Cl- channel, it seems to be misprocessed and unable to accumulate at normal levels in the plasma membrane. Under conditions where the biosynthetic block of ΔF508CFTR is not complete, the residence time of ΔF508CFTR in the plasma membrane is a critical determinant of the cAMP- sensitive Cl- conductance. To assess the stability of the mutant and wild- type CFTR, we compared their functional half-lives at the plasma membrane of transfected Chinese hamster ovary cells. The plasma membrane Cl- conductance was assessed by patch-clamp recordings and/or by fluorimetric determinations of the membrane potential. Accumulation of ΔF508CFTR in the plasma membrane was promoted by growing the transfected cells at reduced temperature (24-28 °C), and was verified by immunoblotting and by detecting the appearance of a plasmalemmal cAMP-activated Cl- conductance. Subsequently increasing the temperature to 37 °C inhibited further delivery of newly synthesized ΔF508CFTR to the surface membrane. By studying the time dependence of the disappearance of the Cl- conductance, the functional half-life of the mutant protein at the plasma membrane was determined to be <4 h, which is considerably shorter than the half-life of wild-type CFTR (>24 h). The latter was estimated by terminating protein synthesis or secretion with cycloheximide or brefeldin A, respectively. Inhibition of protein synthesis did not alter the rate of disappearance of ΔF508CFTR at 37 °C, validating the difference in turnover between mutant and wild-type CFTR. These results indicate that the structural abnormality of ΔF508CFTR affects not only the delivery of the protein to the plasma membrane, but also its stability therein. Moreover, they suggest that overcoming the processing block at the endoplasmic reticulum may not suffice to restore normal Cl- conductance in CF.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology