Understanding the transport mechanisms of the electrogenic transporters SLC26A3 andSLC26A6

SLC26转运体在介导化学计量偶联阴离子反转运或非偶联渗透方面存在差异。这两种传输模式的机制尚不清楚。直到最近，细菌和哺乳动物SLC26同种异构体以及结构相关转运体的实验高分辨率结构才出现，定义了一般的7TMIR结构，并使假设驱动的分子机制方法成为可能。我们现在将使用功能策略来了解哺乳动物SLC26A3和A6作为重要肠道异构体的阴离子易位机制。由于是电致的，这些同工异构体是非常合适的模型，可以通过我们之前用非哺乳动物SLC26A5建立的实验方法来探索。虽然这两种异构体根据序列同源性密切相关，但它们在运输化学计量上有所不同，从而允许对机制细节进行比较探测。实验策略将结合电生理学和离子配比来全面表征转运活性，并结合位点定向诱变来研究阴离子结合位点和机制。阴离子易位的构象动力学将通过半胱氨酸修饰、交联策略和荧光测定来探测。虽然SLC26转运体的药理学仍然很大程度上缺乏，但疏水阴离子已被发现作为SLC26A5的竞争性抑制剂。我们将系统探索SLC26A3和A6抑制剂的结构要求和作用机制。最后，我们对转运的调控感兴趣，这可能是由细胞质c端stas结构域介导的。因此，我们将研究STAS结构域和介导离子易位的跨膜结构域之间的相互作用。我们的方法与该研究单元内旨在确定slc26的高分辨率结构和分子动力学建模的项目高度互补。

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.