Structurally disordered region in the SLC26A9 STAS-domain contains subregions that control SLC26A9 Cl^-conductance

SLC26A9 is a Cl - channel and electrogenic Cl - -HCO 3 ‑ exchanger found in several human epithelia, including the lung, intestine / GI tract, and kidney. SLC26 proteins are defined by a C-terminal, cytoplasmic sulfate transporter / anti-σ-domain (STAS). In mammals, several STAS domains have protein-protein interactions with the regulatory-region of CFTR (R-CFTR), the Cl - channel associated with cystic fibrosis. However, the functional consequences, i.e., regulatory effects (up/downregulation), of STAS-to-R-CFTR binding seem to differ by SLC26 member. Our prior work showed that the SLC26A9:CFTR interaction decreases SLC26A9 channel and exchange activity. If SLC26A9-STAS was replaced with SLC26A6-STAS, no inhibition of SLC26A9 conductance occurred when interacting with R-CFTR (PMID 19643730). Complete deletion of SLC26A9-STAS removed Cl - conductance. Thus, we hypothesized that a disordered loop within STAS is the likely interaction area, and therefore deleted this STAS-disordered loop (ΔLoop) to test this region’s functionality. We used a voltage clamp to measure Cl - currents (IV; conductance G calculated) in Xenopus laevis oocytes (n=10 / construct) injected with cRNA of ΔLoop and subcomponent areas of SLC26A9. Each construct contained a 3xFLAG tag such that stained oocytes from these constructs allowed determination of membrane expression. ΔLoop has three subregions: K/R rich, Middle, S/T rich. Both ΔLoop and ΔLoop(K/R) increase Cl - currents (and conductance) by 5x greater comparted to wild-type SLC26A9. Five amino acid extension of ΔLoop (N- or C-terminal) produces Cl - currents equivalent to ΔLoop deletion. However, 10 amino acid extension of ΔLoop (N- or C- direction) produces currents equivalent to WT-SLC26A9. Membrane localization indicates that ΔLoop and regional deletion constructs have similar membrane abundance to WT, while boundary deletions inhibit channel trafficking to the membrane. As ΔLoop(S/T) slightly increased conductance, the ordered S/T-rich motif [T 620 SVSYITFS 628 ] was examined for possible phosphorylation effects by serial alanine substitution. Each alanine substitution in the S/T-rich motif decrease SLC26A9 Cl - conductance vs WT-SLC26A9. However, for S623A the conductance decrease was due to minimal plasma membrane expression rather than apparent change of channel activity. Together these data suggest that the disordered loop is multifaceted and can maintain low conductance (autoinhibition) or by potential phosphorylation. As cystic fibrosis (CF) results from loss-of-function, trafficking or degradation mutations, the robust Cl - conductance from elimination of SLC26A9 autoinhibitory, disordered loop, presents an opportunity to supplement or replace Cl - conductance in CF tissues and potentially patients. R25-DK101405 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.