Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

Min-Hwang Chang,Consuelo Plata,Aleksandra Sindic,Wasantha K Ranatunga,An-Ping Chen,Kambiz Zandi-Nejad,Kim W Chan,James Thompson,David B Mount,Michael F Romero

doi:10.1074/jbc.m109.001669

Abstract

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl(-)-HCO(3)(-) exchanger and Cl(-) channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl(-)-HCO(3)(-) exchange and Cl(-) currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-DeltaSTAS) virtually eliminated the Cl(-) currents with only a modest affect on nCl(-)-HCO(3)(-) exchange activity. Co-expression of a9-DeltaSTAS and the (R)CFTR fragment did not alter the residual a9-DeltaSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.

Highlights

Slc26 genes and proteins have attracted the attention of physiologists and geneticists
Mouse studies have revealed Slc26a3 and Slc26a6 have been shown to functionally interadditional roles for these Slc26 proteins in mammalian physi- act with the R-region of the cystic fibrosis ClϪ channel (CFTR)
Most importance for this study is that these previous studies in expression systems have shown that Slc26a3 or Slc26a6 interacting with CFTR results in mutual activation [6, 7, 15, 59]

Summary

Animal Health and Welfare

Xenopus laevis were housed and cared for in accordance and approval of the Institutional Care and Use Committees of Case Western Reserve University (Xenopus) and the Mayo Clinic (Xenopus). CFTR Constructs—CFTR construct boundaries were according to the previous definitions of Chan and co-workers [17]: nucleotide-binding domain (NBD1)CFTR (433– 633 aa); R-region (R)CFTR (634 – 835 aa); (NBD1ϩR)CFTR (433– 835 aa); and (NBD1ϩR⌬3)CFTR (433–707 aa) These regions were subcloned into a Xenopus oocyte expression plasmid, cRNA were made and injected into oocytes. (R)CFTR was purified using denaturing conditions and suspended in buffer (50 mM Tris, pH 8.0, 400 mM NaCl, 6 M guanidine HCl, 2 mM dithiothreitol) [20] For these experiments, this (R)CFTR solution was dialyzed in 20 mM Tris, pH 8.0, 200 mM KCl, 10% glycerol to fold into the native protein form [20], the polypeptide likely remains largely unstructured. Protein fractions were analyzed by Coomassie-stained SDS-PAGE

Oocyte Experiments

RESULTS

DISCUSSION

Final pHi