title: Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.
abstract: The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
authors.affiliation: Hospital for Sick Children, Research Institute, Toronto, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Mohamed
authors.forename: D
authors.initials: D
authors.lastname: Ferguson
authors.forename: F S
authors.initials: FS
authors.lastname: Seibert
authors.forename: H M
authors.initials: HM
authors.lastname: Cai
authors.forename: N
authors.initials: N
authors.lastname: Kartner
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
authors.forename: J R
authors.initials: JR
authors.lastname: Riordan
authors.forename: G L
authors.initials: GL
authors.lastname: Lukacs
mesh_terms: D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D016764:Cell Polarity; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004285:Dogs; D007454:Iodides / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D007454:Iodides; D007473:Ion Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP