title: Maintaining low Ca2+ level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells.
abstract: The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.
authors.affiliation: Institut de Physiologie et Biologie Cellulaires CNRS UMR 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Norez
authors.forename: Fabrice
authors.initials: F
authors.lastname: Antigny
authors.forename: Frédéric
authors.initials: F
authors.lastname: Becq
authors.forename: Clarisse
authors.initials: C
authors.lastname: Vandebrouck
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D010649:Phenylalanine / Q000235:genetics; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010649:Phenylalanine; D002118:Calcium