Generation and functional characterization of epithelial cells with stable expression of SLC26A9 Cl⁻ channels

Abstract

Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis

“…Subsequently, a genome-wide study identified variants 5′ of and within noncoding regions of SLC26A9 that associate with age at onset of CFRD (8). SLC26A9 is a member of the SLC26 family of anion transporters that functions as a WNK kinase-regulated Cl -/HCO 3 exchanger and Clchannel (and possibly as a Na + -anion cotransporter) (10)(11)(12)(13)(14). Cryoelectron microscopy paired with electrophysiologic studies show that murine SLC26A9 forms homodimers that operate as rapid transporters of Clas opposed to forming ion channels (15).…”

Influenza virus infection alters ion channel function of airway and alveolar cells: mechanisms and physiological sequelae

“…In the distal respiratory tract, epithelial cells are covered by the alveolar lining fluid (ALF), with a volume of~1 l in mice and 1-10 ml in humans, and plays an important role in governing surfactant homeosta-sis (112). Maintenance of the PCL volume and composition requires the careful coordination of Na ϩ absorption, mainly through the amiloride-sensitive epithelial Na ϩ channel (ENaC), and Cl Ϫ and bicarbonate (HCO 3 Ϫ ) secretion, through the cystic fibrosis transmembrane conductance regulator (CFTR), Ca 2ϩ -activated Cl Ϫ (5,26,40,53), and SLC26A9 Cl Ϫ channels, present in the apical membranes of epithelial cells (82,90). Cl Ϫ ions enter the basolateral membranes through Na ϩ /K ϩ /2 Cl Ϫ (NKCC) transporters (27).…”

Extracellular Cl− regulates human SO4 2−/anion exchanger SLC26A1 by altering pH sensitivity of anion transport

“…Although K + substitution of bath Na + did not reduce Cl − uptake, its DIDS-sensitive component was abolished, suggesting that high bath K + activated a DIDS-insensitive, SLC26A1-dependent Cl − uptake. The reported Na + -dependent component of SLC26A9-mediated Cl − transport was DIDS-sensitive [46,67] and represented (at least in part) coupled transport of Na + with Cl − [13], albeit insensitive to inhibitors of cation chloride cotransport. The previously reported lack of Cl − transport by mouse SLC26A1 [82] was based on experiments carried out in Na + -free, NMDG + solutions.…”