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°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 τ1 ≃4 s and τ2 ≃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 μM 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.
Original language | English (US) |
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Journal | American Journal of Physiology - Lung Cellular and Molecular Physiology |
Volume | 266 |
Issue number | 5 10-5 |
State | Published - 1994 |
Externally published | Yes |
Keywords
- cell culture
- cystic fibrosis
- epithelia
- patch clamp
- submucosal gland
ASJC Scopus subject areas
- Pulmonary and Respiratory Medicine
- Cell Biology
- Physiology