Understanding the transport mechanisms of the electrogenic transporters SLC26A3 andSLC26A6

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SLC26 transporters are divergent in mediating either stoichiometrically coupled anion antiport or uncoupled permeation. The mechanisms underlying both transport modes are unknown. Only recently, experimental high-resolution structures of a bacterial and a mammalian SLC26 isoform as well as of structurally related transporters emerged, defining the general 7TMIR architecture and enabling a hypothesis-driven approach to molecular mechanisms. We will now use a functional strategy to understand the anion translocation mechanisms in coupled mammalian SLC26A3 and A6 as important intestinal isoforms. Being electrogenic, these isoforms are well-suited models that can be probed by experimental approaches established by us previously with non-mammalian SLC26A5. While both isoforms are closely related according to sequence homology, they differ in transport stoichiometry, allowing for comparative probing of mechanistic details. The experimental strategy will combine electrophysiology and ion ratioing to characterize transport activity comprehensively and site-directed mutagenesis to examine anion binding sites and mechanisms. The conformational dynamics underlying anion translocation will be probed by cysteine-modification, crosslinking strategies, and fluorometric assays. While pharmacology of SLC26 transporters is still largely lacking, hydrophobic anions have been found to act as competitive inhibitors of SLC26A5. We will systematically explore structural requirements and mechanisms of such inhibitors of SLC26A3 and A6. Lastly, we are interested in regulation of transport, which may be mediated by the cytoplasmic C-terminal STAS-domain. We will therefore examine the interaction between the STAS domain and the transmembrane domain that mediates ion translocation. Our approach is highly complementary to the projects within this Research Unit aiming at determination of high-resolution structures of SLC26s and at molecular dynamics modeling.