Channel inhibitory region within the STAS domain of human SLC26A9

Abstract

Slc26 gene family encodes multifunctional anion transport proteins: sensor, exchangers and channels. Structurally, Slc26 members have 10–14 predicted transmembrane spans followed by a cytoplasmic domain. This carboxy‐terminus contains a STAS (sulfate transporter anti‐sigma) domain whose function we have studied in mouse Slc26a9 (Chang et al, JBC 284: 28306, 2009). Mutations in SLC26 proteins lead to a number of diseases including diastrophic dysplasia, congenital chloride diarrhea, deafness and potentially Ca‐oxalate, kidney stones. While several groups have addressed the exchanger vs. channel nature of Slc26 proteins, elements within each Slc26 protein contributing to (or controlling) the anion transport character have not been probed. Interestingly, mapping the STAS domain conserved motifs reveals a hypervariable region. This hypervariable region ranges between 30 and 130 amino acids; no similarities exist between different Slc26 members. We made a SLC26A9 construct (Δloop), lacking this STAS subregion of SLC26A9. We expressed Δloop in Xenopus oocytes and monitored transport using ion selective microelectrodes and voltage clamping. SLC26A9 Δloop showed >5‐fold increased Cl− currents compared with the wild type SLC26A9 without obvious change in Cl−‐HCO3− exchange activity. We also made several SLC26A9 mutants to remove putative phosphorylation sites located in this loop: T620A, S621A, S623A, T626A and S628A(replacing T or S with A). All of these mutants show decreased (10–50%) Cl− currents compared with the wild type SLC26A9. These results suggest this variable loop contains a channel‐inhibitory motif and also phosphorylation sites maintaining basic SLC26A9 function. We hypothesize that this variable loop is a regulatory element collaborating with conserved STAS domain regions and that this structural interaction may confer specific transport modes for SLC26A9 and possibly other SLC26 proteins.