(400) A novel SLC26A9-inhibitor reveals SLC26A9 as not the constitutive airway chloride conductance

Background: The solute carrier family 26 member 9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to basal airway chloride conductance and airway surface hydration by operating as an anion exchanger or as a Cl^- channel. SLC26A9 was shown to be a genetic modifier in cystic fibrosis (CF) and therefore may serve as a possible alternative Cl^- secretory pathway in CF airways. However, whether SLC26A9 or the cystic fibrosis transmembrane conductance regulator (CFTR) is really responsible for airway Cl^- transport under basal, i.e. non-stimulated conditions, is still unclear, in part due to the regulatory relationship between both proteins. In addition, there is still a large uncertainty regarding the airway epithelial localization of SLC26A9.

Methods: Iodide-sensitive YFP-fluorescence quenching was used for cell-based high throughput screening of chemical libraries in cells overexpressing SLC26A9. Patch-Clamp and open/short-circuited Ussing Chamber studies were performed in overexpressing and endogenous systems to analyze the contribution of SLC26A9 to resting and stimulated Cl^- currents, in the presence or absence of CFTR. Intracellular and airway surface liquid (ASL)-pH measurements were accomplished to determine the contribution of SLC26A9 to HCO₃^- secretion and pH regulation in the airways. SLC26A9 expression was confirmed in all systems by RT-PCR and immunoblot analysis. SLC26A9 localization was determined by immunostaining with a highly specific antibody. Animals were chronically treated via intratracheal instillation before the collection of tissues.

Results: A potent inhibitor of SLC26A9 was identified by screening a drug-like small molecule library and subsequent chemical modifications. This compound inhibited SLC26A9-mediated I^- quenching with an IC₅₀ of 90.9 ± 13.4 nM, did not inhibit other members of the SLC26-family (SLC26A3,4,6), and had insignificant effects on other Cl^- channels such as CFTR, TMEM16A, and VRAC. The SLC26A9-inhibitor very clearly inhibited overexpressed SLC26A9-mediated Cl^- currents in HEK293 cells but showed little effect on the ion transport in highly differentiated human airway epithelial cells or mouse trachea under resting conditions, and this was slightly enhanced upon chronic treatment of the tissues with IL-13. In both airway systems, SLC26A9 showed a clear apical expression in ciliated epithelial cells. CFTRInh-172 strongly inhibited basal transport in the non-stimulated airways. The SLC26A9-inhibitor did inhibit transport in airways stimulated with cAMP-enhancing agonists. SLC26A9 did also not seem to contribute to airway HCO₃^- secretion and ASL-pH regulation in the presence or absence of CFTR, as measured in highly differentiated primary airway cultures.

Conclusions: The novel identified, highly potent SLC26A9-specific inhibitor used in this study suggests that SLC26A9 does not contribute significantly to the basal, non-stimulated airway Cl^- transport as a direct conductor, even when enhanced in inflammatory conditions. Rather CFTR seems to be in charge of this function. SLC26A9 appears to contribute to activated Cl^- secretion, possibly in a CFTR-dependent manner. Given our data, we conclude that stimulation of SLC26A9 in CF airways might not be an effective strategy to enhance apical Cl^- secretion. The novel inhibitor could be useful to inhibit gastric acid secretion to treat gastritis or gastric ulcers, as SLC26A9 was proposed as a Cl^--secretory counter-channel for gastric HCl secretion.

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