SLC26A9 stimulates CFTR expression and function in human bronchial cell lines

Abstract

We investigated the possible functional- and physical protein-interactions between two airway Cl(-) channels, SLC26A9 and CFTR. Bronchial CFBE41o- cell lines expressing CFTR(WT) or CFTR(ΔF508) were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole-cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR(WT) and CFTR(ΔF508) cell lines. In CFBE41o- cells and CFBE41o-/CFTR(WT) cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)-stimulated currents were enhanced in SLC26A9-transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR((inh)-172) or GlyH-101 inhibition in both groups). In CFBE41o-/CFTR(ΔF508) cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK-stimulated currents and level of CFTR((inh)-172) inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK-stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK-stimulated currents was not found in oocytes co-expressing SLC26A9 and CFTR(ΔF508). In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR(ΔF508). Our co-immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9-STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents.

SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation

“…In wild-type bronchi, cAMP-mediated stimulation induced a sustained Cl -secretory response that was significantly inhibited by CFTR inh -172 ( Figure 1H). In contrast to previous results in cultured HBE cells transduced with SLC26A9 (14), our studies in native bronchial epithelia detected no differences in either cAMPinduced I sc or inhibition by CFTR inh -172 in tissues from Slc26a9 -/-compared with wild-type mice ( Figure 1H). The CFTR inh -172- insensitive I sc was completely inhibited by bumetanide in tissues from both wild-type and Slc26a9 -/-mice ( Figure 1H).…”

“…The CFTR inh -172- insensitive I sc was completely inhibited by bumetanide in tissues from both wild-type and Slc26a9 -/-mice ( Figure 1H). To determine, whether SLC26A9-mediated Cl -transport and functional interaction with CFTR required the presence of HCO 3 - (10,11,14), we compared bioelectric properties in wild-type and Slc26a9 -/-bronchi in the absence and presence of HCO 3 -. HCO 3 -had no effect on basal I sc , constitutive Cl -secretion (amiloride-insensitive I sc ), or cAMP-induced Cl -secretion, and, as in HCO 3 --free conditions, constitutive and cAMP-dependent Cl -secretion were not different in wild-type compared with Slc26a9 -/-tissues (Supplemental Figure 2).…”

“…HCO 3 -had no effect on basal I sc , constitutive Cl -secretion (amiloride-insensitive I sc ), or cAMP-induced Cl -secretion, and, as in HCO 3 --free conditions, constitutive and cAMP-dependent Cl -secretion were not different in wild-type compared with Slc26a9 -/-tissues (Supplemental Figure 2). This lack of functional interaction between SLC26A9 and CFTR in native mouse airway epithelia may reflect species-dependent differences or differences in regulation of SLC26A9 and CFTR function in native airway epithelia versus transduced cell lines (14). Finally, we tested a potential contribution of SLC26A9 to Ca 2+ -activated Cl -conductance (CaCC) by comparing UTPinduced Cl -secretory responses in bronchi from wild-type and Slc26a9 -/-mice.…”

“…Recent studies showed that the SLC26A9 protein functions as a Cl -channel with minimal HCO 3 -conductance that is regulated by CFTR and WNK kinases in heterologous cells (10)(11)(12)(13). Further, SLC26A9 contributes to constitutive and cAMP-dependent Cl -secretion in human bronchial epithelial (HBE) cells, where it has been suggested to interact functionally with CFTR in vitro (11,14). Based on its functional properties in transduced cells and expression pattern in human and mouse airways (8,15), we hypothesized that SLC26A9 may function as an alternative Cl -channel that may contribute to ASL homeostasis in health and allergic airway disease.…”

Primers on Molecular Pathways: Bicarbonate Transport by the Pancreas

“…This interaction also increases CFTR-Cl – channel activity [16,21], although Slc26a9-sulfate transporter anti-σ interaction activation requires cAMP [25,28]. Not surprisingly, Slc26a9 does not stimulate ΔF508-CFTR activity [29], the most common mutation in cystic fibrosis, implying that Slc26a9 may be associated with the severity of cystic fibrosis phenotypes.…”