title: CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance.
abstract: Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.
authors.affiliation: Cystic Fibrosis Research Laboratory, Stanford University, California 94305.
authors.forename: C
authors.initials: C
authors.lastname: Haws
authors.forename: W E
authors.initials: WE
authors.lastname: Finkbeiner
authors.forename: J H
authors.initials: JH
authors.lastname: Widdicombe
authors.forename: J J
authors.initials: JJ
authors.lastname: Wine
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008175:Lung Neoplasms; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D009579:Nitrobenzoates / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D009579:Nitrobenzoates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin; D007545:Isoproterenol