Enhanced cell-surface stability of rescued ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones

Abstract

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR ΔF508, and analysed its cell-surface trafficking after rescue [rΔF508 (rescued ΔF508) CFTR]. We show that rΔF508 CFTR endocytosis is 6-fold more rapid (∼30% per 2.5 min) than WT (wild-type, ∼5% per 2.5 min) CFTR at 37 °C in polarized airway epithelial cells (CFBE41o−). We also investigated rΔF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 °C) and following treatment with pharmacological chaperones. At low temperature, rΔF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rΔF508 CFTR is TS. Furthermore, rΔF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 °C to >8 h at 27 °C. Pharmacological chaperone treatment at 37 °C corrected the rΔF508 CFTR internalization defect, slowing endocytosis from ∼30% per 2.5 min to ∼5% per 2.5 min, and doubled ΔF508 surface half-life from 2 to 4 h. These effects are ΔF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing ΔF508 CFTR from ERAD, but also by enhancing its cell-surface stability.