Disorder Within Order: Identification of the Disordered Loop of STAS Domain As the Inhibitory Domain in SLC26A9 Chloride Channel

The chloride transporter-channel SLC26A9 is mediated by a reciprocal regulatory mechanism through the interaction between its cytoplasmic sulfate transporter and anti-sigma (STAS) domain and the R domain of cystic fibrosis (CF) transmembrane regulator. In vertebrate Slc26a9s, the STAS domain structures are interrupted by a disordered loop which is conserved in mammals but is variable in nonmammals. Despite the numerous studies involving the STAS domains in SLC26 proteins, the role of the disordered loop region has not been identified. Deletion of the entire Slc26a9-STAS domain results in loss of Cl channel function. Surprisingly, we found that partial or full deletion of the STAS-disordered loop substantially increases the SLC26A9 chloride transport-channel activity. Bioinformatics analysis reveals that the disordered loops there are three subregions: a K/R-rich region, a "middle" region, and an ordered S/T-rich motif. In this study, the role of this STAS-disordered loop is investigated by using serial deletions and the ordered S/T-rich motif is examined by serial alanine substitution. Substitutions of alanine for serine or threonine in the 620 to 628 S/T-rich motif decrease SLC26A9 chloride channel activity. These experiments parse the functional roles of SLC26A9-STAS-disordered loop and its subdivisions modifying overall SLC26A9 activities. Recently, SLC26A9 has emerged as one of the potential substitutes for abnormal CF transmembrane regulator in CF. Our findings suggest that deletion of variable loop of human SLC26A9 may provide a new gene therapy strategy in the treatment of CF disease.