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

Abstract

The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, DF508, 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 378C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxyphenylamino)-49-methyl-[4,59bithiazolyl-29-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature-rescued DF508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued DF508 CFTR for up to 12 hours at 378C (P , 0.05). Using firefly luciferase-based reporters to investigate the mechanisms by which low temperature and corr4a 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 DF508 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 cAMPstimulated currents, revealing that the biochemically detectable cell surface DF508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued DF508 CFTR was not sufficient to obtain maximal DF508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of DF508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

“…For this, we developed an assay in which airway epithelial cells carrying ΔF508/ΔF508 or ΔF508/W1282X CFTR mutations (parental CFBE41o-and IB3-1 cells, respectively), 7,29,38 which we refer to as "CF cells," were first incubated with PRs or the CFTR correctors Corr-4a or Vrx-325 26 at 37°C for 18 h and then washed and re-cultured for up to 12 h in the presence of cycloheximide (CHX, 100 μg ml -1 , refreshed every 6 h) to inhibit protein neosynthesis. 29 CHX toxicity in this system was excluded by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) cell viability assay.…”

Regulated recycling of mutant CFTR partially restored by pharmacological treatment

“…Correctors C4 and C18 act on the biosynthetic pathway of F508del because they are capable of promoting the escape of the mutant from ERAD and enable its trafficking to the PM (Holleran et al, 2012;Pedemonte et al, 2005). Prior studies have also demonstrated peripheral activity of C4 by acute treatment which slowed removal of thermally destabilized F508del from the surface, perhaps by reducing its ubiquitination (Jurkuvenaite et al, 2010;Pedemonte et al, 2005). Here, we report that acute addition of C4 or C18 but not CFFT-002, coincident with thermal destabilization, partially blocks targeting to the lysosomes and restores trafficking to the recycling pathway.…”

“…Low temperature incubation (,30˚C for 24 hours) or more recently, treatment with pharmacological small molecules called correctors, can partially rescue CFTR F508del trafficking to the cell surface (Pedemonte et al, 2005;Van Goor et al, 2006). However, despite partial trafficking rescue by corrector treatment or low temperature incubation, rescued CFTR F508del still exhibits severely impaired anion transport function and significantly reduced PM density and recycling efficiency (Cholon et al, 2010;Jurkuvenaite et al, 2010;Sharma et al, 2004).…”

Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

“…Although small-molecule correctors allow ⌬F508 CFTR to escape the ER and the rescued ⌬F508 CFTR can reach the cell surface and function as chloride channel (29), the mutant CFTR is unstable and readily degraded at the surface (30). Therefore, great attention has been given to identify a novel compound that enhances CFTR stability at cell membrane.…”