Functional analysis of nonsynonymous single nucleotide polymorphisms in human SLC26A9

Abstract

Slc26 anion transporters play crucial roles in transepithelial Cl− absorption and HCO3− secretion; Slc26 protein mutations lead to several diseases. Slc26a9 functions as a Cl− channel and electrogenic Cl−-HCO3− exchanger, and can interact with CFTR. Slc26a9(−/−) mice have reduced gastric acid secretion, yet no human disease is currently associated with SLC26A9 coding mutations. Therefore, we tested the function of non-synonymous, coding, single nucleotide polymorphisms (cSNPs) of SLC26A9. Presently, eight cSNPs are NCBI-documented: Y70N, T127N, I384T, R575W, P606L, V622L, V744M and H748R. Using two-electrode voltage-clamp and anion selective electrodes, we measured the biophysical consequences of these cSNPs. Y70N (cytoplasmic N-terminus) displays higher channel activity and enhanced Cl−-HCO3− exchange. T127N (transmembrane) results in smaller halide currents but not for SCN−. V622L (STAS domain) and V744M (STAS adjacent) decreased plasma membrane expression which partially accounts for decreased whole cell currents. Nevertheless, V622L transport is reduced to ~50%. SLC26A9 polymorphisms lead to several function modifications (increased activity, decreased activity, altered protein expression) which could lead to a spectrum of pathophysiologies. Thus, knowing an individual’s SLC26A9 genetics becomes important for understanding disease potentially caused by SLC26A9 mutations or modifying diseases, e.g., cystic fibrosis. Our results also provide a framework to understand SLC26A9 transport modalities and structure-function relationships.

Characterization of SLC26A9 in Patients with CF-Like Lung Disease

“…SLC26A9 has been recently identified in humans as a Cl − channel present at the apical membrane of lung epithelium [Bertrand et al., ; Demitrack et al., ; Dortwart et al., ; Lohi et al., ; Loriol et al., ]. Controversy exists about Cl − /HCO 3 − exchange activity of SLC26A9 [Chen et al., ; Dorwart et al., ]. In the present study, results obtained from intracellular pH measurements upon Cl − removal (CO 2 /HCO 3 ‐equilibrated solutions) did not support a significant HCO 3 − transport mediated by SLC26A9 (Supplemental Information).…”

“…p.Arg575Trp was one of the SNPs reported by Chen et al. (). This group did not evidence any functional modification of the Cl − transport by expressing SLC26A9–p.Arg575Trp in X. laevis oocytes contrary to the present data.…”

“…Indeed, they may induce a loss of ion transport activity of the protein itself as well as alteration of the positive modulation on CFTR activity exerted by endogenous SLC26A9. So far, variants in SLC26A9 were either designed in silico [Avella et al., ] or stemmed from the public SNP data base for human polymorphisms [Chen et al., ]. In the present study, we discuss the pathogenic role of 2 SLC26A9 missense mutations (p.Val486Ile and p.Arg575Trp) evidenced in patients presenting with diffuse bronchiectasis.…”

Targeting ion channels in cystic fibrosis

“…These results support a role of SLC26A9 as an alternative chloride channel that may contribute to the regulation of airway surface liquid (ASL) essential for mucus clearance under pathophysiological conditions. Third, data emerging from genetic studies in humans provide independent evidence supporting the role of SLC26A9 as a disease modifier in CF and potentially other chronic obstructive lung diseases [83][84][85][86][87]. Specifically, consistent with a protective function of SLC26A9 in Th2-mediated airway inflammation, it was shown that a functional SNP (rs2282430) is associated with asthma in children [83].…”

“…Binding of this micro-RNA to the 3′ UTR results in reduced SLC26A9 protein expression, which may result in reduced chloride channel function in vivo. In the meantime, several additional polymorphisms were identified that alter SLC26A9 function by a variety of molecular mechanisms ranging from altered protein expression to decreased or increased chloride channel activity in vitro [84]. Interestingly, some of these variants were recently detected in patients with CF-like lung disease [85].…”