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WS06.5 SLC26A9 chloride channels: Generation and functional characterization of stably-overexpressing FRT epithelial cells
S12
Workshop 6. Fixing ion transport
Oral Presentations
WS06.5 SLC26A9 chloride channels: Generation and functional characterization of stably-overexpressing FRT epithelial cells
WS06.6 Mucociliary transport is impaired in cystic fibrosis pig airways
J.J. Salomon1 , S. Spahn1 , X. Wang2 , J. F¨ullekrug3 , C.A. Bertrand2 , M.A. Mall1 . 1 University of Heidelberg, Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), Heidelberg, Germany; 2 University of Pittsburgh, Department of Cell Biology, Pittsburgh, United States; 3 University of Heidelberg, Molecular Cell Biology Laboratory, Internal Medicine IV, Heidelberg, Germany
A. Ermund1 , L.N. Meiss1 , A. B¨ahr2 , N. Klymiuk2 , G.C. Hansson1 . 1 Medical Biochemistry, Gothenburg, Sweden; 2 Institute of Molecular Animal Breeding and Biotechnology, Munich, Germany
Objectives: Previous findings in mice revealed that SLC26A9, a recently identified epithelial chloride channel, is essential for preventing airway obstruction associated with IL13-induced airway inflammation. These results suggest that SLC26A9 may be a promising alternative Cl− channel in mucoobstructive lung diseases [e.g. cystic fibrosis (CF)]. Thus, we functionally characterized a newly generated epithelial cell line with stable expression of SLC26A9 to investigate molecular regulation. Methods: Fisher rat thyroid (FRT) cells were stably transduced with a HA-tagged SLC26A9 construct. Transepithelial Cl− currents were measured by applying a Cl− gradient in Ussing chambers. Whole-cell patch clamp experiments were performed. Results: SLC26A9 expression in transduced FRT (FRT-SLC) cells was found at high levels. Transepithelial measurements revealed that the basal short circuit current (Isc ) was significantly increased in FRT-SLC (12.3±2.0 mA/cm2 , P < 0.01) compared to control-transfected FRT (FRT-CTL; 3.9±0.5 mA/cm2 ) cell monolayers. CAMP (IBMX/forskolin)-stimulated Cl− secretion was significantly increased in FRT-SLC compared to FRT-CTL cells (DIsc = 4.9±0.5 vs. DIsc = 0.7±0.2 mA/cm2 , P < 0.01). SLC26A9-mediated currents were inhibited by GlyH-101 and niflumic acid. Whole-cell patch clamp recordings confirmed a constitutive Cl− current in FRT-SLC cells and a similar inhibitor selectivity. Conclusion: In FRT-SLC cells, SLC26A9 contributes to constitutive and cAMPstimulated Cl− currents. This established epithelial cell model overexpressing SLC26A9 may be useful for further studies of the regulation and pharmacological activation of SLC26A9 as an alternative Cl− channel in CF.
Objectives: The basic defect in cystic fibrosis (CF) is well characterized, but the link between defects in the CF transmembrane conductance regulator (CFTR) and the phenomenon of stagnant mucus is not well understood. We have shown that the ileal mucus in CFTR mutant mice adheres to the epithelium, is denser, and is less penetrable than that of wildtype mice and that addition of 115 mM NaHCO3 to mucus partially normalizes this mucus phenotype. Using this knowledge of effects on ileal mucus, we are developing an airway experimental set-up. Methods: Excised airways from CFTR knockout (CF) and wildtype (WT) pigs are pinned to a chamber, alcian blue mucosal stain is added to visualize the mucus and the transport is recorded. During the experiment, mucus is collected for proteomics using masspectrometry. Scanning electron microscopy is used to visualize the properties of the mucus, and transmission electron microscopy is used to study mucin secretion in detail. Results: Average clearance rates of strands in CF pig airways are lower than in WT pig airways. Preliminary results suggest that the initial defect in newborn pigs involves clearance of Muc5b from the glands. Furthermore, we show that removal of bicarbonate from WT airways reduces the average clearance rate to the level of CF airways. Conclusion: Evidence suggests the importance of bicarbonate in the proper unpacking and secretion of mucins. Thus, restoration of bicarbonate to the apical surface of the epithelium in combination with osmolytes may induce proper mucin unpacking in CF epithelia, and therefore could relieve the mucus obstruction that causes clinical problems for cystic fibrosis patients.
Workshop 6. Fixing ion transport
Oral Presentations
WS06.5 SLC26A9 chloride channels: Generation and functional characterization of stably-overexpressing FRT epithelial cells
WS06.6 Mucociliary transport is impaired in cystic fibrosis pig airways
J.J. Salomon1 , S. Spahn1 , X. Wang2 , J. F¨ullekrug3 , C.A. Bertrand2 , M.A. Mall1 . 1 University of Heidelberg, Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), Heidelberg, Germany; 2 University of Pittsburgh, Department of Cell Biology, Pittsburgh, United States; 3 University of Heidelberg, Molecular Cell Biology Laboratory, Internal Medicine IV, Heidelberg, Germany
A. Ermund1 , L.N. Meiss1 , A. B¨ahr2 , N. Klymiuk2 , G.C. Hansson1 . 1 Medical Biochemistry, Gothenburg, Sweden; 2 Institute of Molecular Animal Breeding and Biotechnology, Munich, Germany
Objectives: Previous findings in mice revealed that SLC26A9, a recently identified epithelial chloride channel, is essential for preventing airway obstruction associated with IL13-induced airway inflammation. These results suggest that SLC26A9 may be a promising alternative Cl− channel in mucoobstructive lung diseases [e.g. cystic fibrosis (CF)]. Thus, we functionally characterized a newly generated epithelial cell line with stable expression of SLC26A9 to investigate molecular regulation. Methods: Fisher rat thyroid (FRT) cells were stably transduced with a HA-tagged SLC26A9 construct. Transepithelial Cl− currents were measured by applying a Cl− gradient in Ussing chambers. Whole-cell patch clamp experiments were performed. Results: SLC26A9 expression in transduced FRT (FRT-SLC) cells was found at high levels. Transepithelial measurements revealed that the basal short circuit current (Isc ) was significantly increased in FRT-SLC (12.3±2.0 mA/cm2 , P < 0.01) compared to control-transfected FRT (FRT-CTL; 3.9±0.5 mA/cm2 ) cell monolayers. CAMP (IBMX/forskolin)-stimulated Cl− secretion was significantly increased in FRT-SLC compared to FRT-CTL cells (DIsc = 4.9±0.5 vs. DIsc = 0.7±0.2 mA/cm2 , P < 0.01). SLC26A9-mediated currents were inhibited by GlyH-101 and niflumic acid. Whole-cell patch clamp recordings confirmed a constitutive Cl− current in FRT-SLC cells and a similar inhibitor selectivity. Conclusion: In FRT-SLC cells, SLC26A9 contributes to constitutive and cAMPstimulated Cl− currents. This established epithelial cell model overexpressing SLC26A9 may be useful for further studies of the regulation and pharmacological activation of SLC26A9 as an alternative Cl− channel in CF.
Objectives: The basic defect in cystic fibrosis (CF) is well characterized, but the link between defects in the CF transmembrane conductance regulator (CFTR) and the phenomenon of stagnant mucus is not well understood. We have shown that the ileal mucus in CFTR mutant mice adheres to the epithelium, is denser, and is less penetrable than that of wildtype mice and that addition of 115 mM NaHCO3 to mucus partially normalizes this mucus phenotype. Using this knowledge of effects on ileal mucus, we are developing an airway experimental set-up. Methods: Excised airways from CFTR knockout (CF) and wildtype (WT) pigs are pinned to a chamber, alcian blue mucosal stain is added to visualize the mucus and the transport is recorded. During the experiment, mucus is collected for proteomics using masspectrometry. Scanning electron microscopy is used to visualize the properties of the mucus, and transmission electron microscopy is used to study mucin secretion in detail. Results: Average clearance rates of strands in CF pig airways are lower than in WT pig airways. Preliminary results suggest that the initial defect in newborn pigs involves clearance of Muc5b from the glands. Furthermore, we show that removal of bicarbonate from WT airways reduces the average clearance rate to the level of CF airways. Conclusion: Evidence suggests the importance of bicarbonate in the proper unpacking and secretion of mucins. Thus, restoration of bicarbonate to the apical surface of the epithelium in combination with osmolytes may induce proper mucin unpacking in CF epithelia, and therefore could relieve the mucus obstruction that causes clinical problems for cystic fibrosis patients.