Pneumologie 2015; 69 - A7
DOI: 10.1055/s-0035-1556599

Alternative chloride channels in cystic fibrosis (CF): Functional characterization of SLC26A9 in stably-overexpressing FRT epithelial cells

JJ Salomon 1, S Spahn 1, X Wang 2, J Füllekrug 3, CA Bertrand 2, MA Mall 1
  • 1Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), University of Heidelberg, Germany
  • 2Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, USA
  • 3Molecular Cell Biology Laboratory, Internal Medicine IV, University of Heidelberg, Germany

Introduction: In CF patients, an impaired Cl-/fluid secretion across airway epithelia causing airway surface dehydration and airway mucus obstruction is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previous in vivo results revealed that the epithelial chloride channel SLC26A9 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 CF. We therefore generated and characterized a novel epithelial cell line with stable expression of SLC26A9 to study its pharmacological activation.

Methods: As an in vitro epithelial cell model, Fisher rat thyroid (FRT) cells were used and 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. The effect of knockdown of osmotic-stress sensing kinases (e.g. WNKs) on Cl- currents was studied.

Results: SLC26A9 expression in transduced FRT (FRT-SLC) cells was found at high levels on mRNA and protein level. Transepithelial measurements revealed that the basal short circuit current (Isc) was significantly increased in FRT-SLC (12.3 ± 2.0µA/cm2, P < 0.01) compared to control-transfected FRT (FRT-CTL; 3.9 ± 0.5µA/cm2) cell monolayers. CAMP (IBMX/forskolin)-stimulated Cl- secretion was significantly increased in FRT-SLC compared to FRT-CTL cells (ΔIsc = 4.9 ± 0.5 vs. ΔIsc = 0.7 ± 0.2µA/cm2, P < 0.01). Whole-cell patch clamp recordings confirmed a constitutive Cl- current in FRT-SLC cells. In RNAi studies, knock-down of the kinase WNK1 in FRT-SLC epithelial monolayers diminished the basal Isc to 81.6 ± 7.4% compared to control-treated FRT-SLC cells.

Discussion: We established a novel stable HA-tagged SLC26A9-overexpressing FRT epithelial cell line which provides a valuable cell model for further SLC26A9 functional assays. SLC26A9 contributes to constitutive and cAMP-stimulated Cl- currents that are regulated by osmotic stress-sensing kinases. This model may facilitate exploration of SLC26A9 as a novel therapeutic target in CF.

*Presenting author