Function and dysfunction of the CFTR chloride channel.
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Adenosine Triphosphate↗
Biomedical subjects
F Becq
Publications and source records attributed to F Becq.
Phosphatase inhibitors activate normal and defective CFTR chloride channels.
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.
1-Methyl-3-isobutylxanthine↗
The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels.
Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels.
Acetazolamide↗
Possible regulation of CFTR-chloride channels by membrane-bound phosphatases in pancreatic duct cells.
We have studied CFTR-Cl- channels in non-CF CAPAN-1 and in CFTR-transfected CFPAC-PLJ-CFTR-6 epithelial cells from human pancreas. Theophylline and IBMX induced the opening of cell-attached CFTR-Cl- channels. Theophylline, IBMX and the alkaline phosphatase (AP) inhibitor levamisole enhanced the activity of excised channels and reduced by 70-75% the apical membrane-associated APs activity. Okadaic acid had no effect on APs and channel activities. A polyclonal anti-alkaline phosphatase antibody (which detected apical APs) reduced APs activity and activated quiescent excised chloride channels. These results suggest that CFTR channels may be regulated by membrane-bound phosphatases.
1-Methyl-3-isobutylxanthine↗
Characterization of cAMP dependent CFTR-chloride channels in human tracheal gland cells.
Human tracheal gland cells are believed to be a major site at the origin of cystic fibrosis. Since this disease is due to mutations in a protein called CFTR, we looked for the activity of CFTR in human tracheal gland cells in culture. We have identified CFTR-like chloride-selective channels as having a linear current voltage relationship and unitary conductance of 7 pS in these cells. In cell-attached patches, theophylline (1 mM), IBMX (1 mM), or a cocktail of dibutyryl cAMP (1 mM) and IBMX (0.1 mM) promoted the opening of channels. The unitary current had a reversal potential close to the cell resting potential. Replacement of choline by K+ or Na+ in the pipette solution was without effect on the current-voltage relationship, the reversal potential or the unitary conductance, which is consistent with the chloride selectivity of the channel. Channels were always found clustered and their opening probability was not noticeably dependent on membrane potential. This work therefore represents the first observation of a CFTR-like channel activity in submucosal gland cells.
Base Sequence↗
Phosphorylation-regulated low-conductance Cl- channels in a human pancreatic duct cell line.
A low-conductance Cl- channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mumol/l), dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mumol/l) and forskolin (10 mumol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23 degrees C and 12 pS at 37 degrees C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl- > I- >> > HCO3- > gluconate. In inside-out excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg2+). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl- channel was 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 100 mumol/l) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 100 mumol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mumol/l). These results demonstrate that the apical low-conductance Cl- channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)
1-Methyl-3-isobutylxanthine↗
Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin.
Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl- channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl- channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl- over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.
Adenocarcinoma↗