The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.

BACKGROUND AND AIMS Deletion of the codon for phenylalanine at position 508 (DeltaF508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In heterologous cells, defective processing of the DeltaF508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent plasma membrane Cl(-) conductance. However, data with respect to the processing block of DeltaF508 protein in native epithelia are limited and conflicting. METHODS To characterize both the fate and function of DeltaF508 protein in a native epithelium, we measured CFTR-mediated Cl(-) secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and DeltaF508 homozygous patients with cystic fibrosis (CF). RESULTS Ussing chamber studies showed that cAMP-dependent and cholinergic Cl(-) secretion was absent from rectal tissues freshly excised from DeltaF508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not DeltaF508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in DeltaF508 homozygous tissues. CONCLUSIONS Collectively, these data show that there is insufficient maturation and transport of DeltaF508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl(-) secretion in the CF colon.

[1]  D. Porteous,et al.  Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild-type Cftr gene expression , 1994, Mammalian Genome.

[2]  J. Riordan,et al.  The PDZ-binding Chloride Channel ClC-3B Localizes to the Golgi and Associates with Cystic Fibrosis Transmembrane Conductance Regulator-interacting PDZ Proteins* , 2003, The Journal of Biological Chemistry.

[3]  Richard C. Boucher,et al.  Regulation of Murine Airway Surface Liquid Volume by CFTR and Ca2+-activated Cl− Conductances , 2002, The Journal of general physiology.

[4]  A. Aleksandrov,et al.  Nucleoside triphosphate pentose ring impact on CFTR gating and hydrolysis , 2002, FEBS letters.

[5]  K. Kunzelmann,et al.  Electrolyte transport in the mammalian colon: mechanisms and implications for disease. , 2002, Physiological reviews.

[6]  B. Tümmler,et al.  Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings. , 2001, The Journal of clinical investigation.

[7]  R. Boucher,et al.  Expression and localization of epithelial aquaporins in the adult human lung. , 2001, American journal of respiratory cell and molecular biology.

[8]  B. Tümmler,et al.  Residual chloride secretion in intestinal tissue of ΔF508 homozygous twins and siblings with cystic fibrosis , 2000 .

[9]  J. Isenberg,et al.  Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis. , 2000, Gastroenterology.

[10]  A. Wissner,et al.  Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients. , 2000, American journal of physiology. Gastrointestinal and liver physiology.

[11]  K. Kunzelmann,et al.  CFTR-mediated inhibition of epithelial Na+ conductance in human colon is defective in cystic fibrosis. , 1999, American journal of physiology. Gastrointestinal and liver physiology.

[12]  B. Tümmler,et al.  ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis , 1999 .

[13]  B D Schultz,et al.  Pharmacology of CFTR chloride channel activity. , 1999, Physiological reviews.

[14]  J. Wine,et al.  Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation (*) , 1996, The Journal of Biological Chemistry.

[15]  S Grinstein,et al.  Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP. , 1994, The EMBO journal.

[16]  R. Kopito,et al.  Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. , 1994, The Journal of biological chemistry.

[17]  F. Collins,et al.  Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization. , 1994, The Journal of clinical investigation.

[18]  Johanna M. Rommens,et al.  The cystic fibrosis mutation (ΔF508) does not influence the chloride channel activity of CFTR , 1993, Nature Genetics.

[19]  Wendy L. Kimber,et al.  Cystic fibrosis in the mouse by targeted insertional mutagenesis , 1992, Nature.

[20]  Matthew P. Anderson,et al.  Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive , 1992, Nature.

[21]  J. Riordan,et al.  Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR) , 1992, Cell.

[22]  M. Welsh,et al.  Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. , 1992, The Journal of clinical investigation.

[23]  James M. Wilson,et al.  Submucosal glands are the predominant site of CFTR expression in the human bronchus , 1992, Nature Genetics.

[24]  J. Riordan,et al.  Mislocalization of ΔF508 CFTR in cystic fibrosis sweat gland , 1992, Nature Genetics.

[25]  Pascal Barbry,et al.  Altered chloride ion channel kinetics associated with the ΔF508 cystic fibrosis mutation , 1991, Nature.

[26]  H. D. de Jonge,et al.  Ion transport abnormalities in rectal suction biopsies from children with cystic fibrosis. , 1991, Gastroenterology.

[27]  M. Welsh,et al.  Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. , 1991, Science.

[28]  L. Tsui,et al.  Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance , 1991, Cell.

[29]  J. Marshall,et al.  Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis , 1990, Cell.

[30]  Matthew P. Anderson,et al.  Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells , 1990, Nature.

[31]  M. Knowles,et al.  Chloride secretory response of cystic fibrosis human airway epithelia. Preservation of calcium but not protein kinase C- and A-dependent mechanisms. , 1989, The Journal of clinical investigation.

[32]  R. Azizkhan,et al.  Altered intestinal chloride transport in cystic fibrosis , 1988, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.