Understanding the transport mechanisms of the electrogenic transporters SLC26A3 andSLC26A6

了解生电转运蛋白 SLC26A3 和 SLC26A6 的转运机制

• 批准号: 441921356
• 负责人: Professor Dr. Dominik Oliver
• 金额: --
• 依托单位: Institut für Physiologie und Pathophysiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/441921356?language=en
• 关键词: Understanding; transport; mechanisms; electrogenic; transporters

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.

SLC26转运蛋白在介导化学计量偶联阴离子反向转运或非偶联渗透方面是不同的。这两种运输模式的机制尚不清楚。直到最近，实验高分辨率结构的细菌和哺乳动物SLC26亚型以及结构相关的转运蛋白出现，定义了一般的7TMIR架构，并使一个假设驱动的方法的分子机制。我们现在将使用功能策略来了解耦合哺乳动物SLC 26A3和A6作为重要的肠道亚型的阴离子易位机制。作为产电的，这些亚型是非常适合的模型，可以通过我们先前用非哺乳动物SLC 26 A5建立的实验方法来探测。虽然这两种亚型是密切相关的序列同源性，它们不同的运输化学计量，允许比较探测的机制细节。实验策略将结合联合收割机电生理学和离子比率法来全面表征转运活性，并结合定点突变来研究阴离子结合位点和机制。将通过半胱氨酸修饰、交联策略和荧光测定来探测阴离子易位的构象动力学。虽然SLC26转运蛋白的药理学仍然很大程度上缺乏，但已发现疏水阴离子可作为SLC26A5的竞争性抑制剂。我们将系统地探讨这类SLC26A3和A6抑制剂的结构要求和作用机制。最后，我们感兴趣的运输，这可能是由细胞质C-末端STAS域介导的调节。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。我们的方法是高度互补的项目在这个研究单位，旨在确定高分辨率的结构SLC26和分子动力学建模。