Mechanisms of CLC Transporters and Channels

CLC转运蛋白和通道的机制

• 批准号: 10420639
• 负责人: Merritt C Maduke
• 金额: $ 3.84万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10420639
• 关键词: Behavior; Binding; Biological Assay; CLC Gene; Carrier Proteins; Chloride Channels; Coupling; Cryoelectron Microscopy; Electrons; Event; Family; Human; Hydration status; Hypertension; Ion Channel; Ion Transport; Ions; Kidney Diseases; Molecular; Molecular Conformation; Monitor; Muscle; Mutation; Organism; Pathway interactions; Process; Protein Conformation; Proteins; Pump; Resolution; Spectrum Analysis; Structure; Tissues; Variant; bone; nervous system disorder; public health relevance; simulation

The CLC (“Chloride Channel”) family encompasses two major ion-transport mechanisms: half of CLC homologs are electrodiffusive ion channels, and half are secondary active transporters that stoichiometrically exchange Cl– for H+. The occurrence of two mechanisms in one family suggests they operate by variations on a common theme. Indeed, experimental results support the hypothesis that CLC channels are “broken” transporters, in which tight coordination between inner and outer gates is lost. Thus, subtle differences in protein conformational dynamics and ion binding—and the interactions between them—can produce two different types of ion transport behavior in proteins with the same secondary structure. Understanding the molecular basis of these differences will inform our understanding of both CLC channels and transporters. As secondary active transporters, CLC transporters harness energy stored in one ion’s electrochemical gradient (Cl– or H+) to pump the other ion against its gradient. This occurs through tight coupling of protein conformational changes to ion binding and transport events. To develop a fully integrated structural description of ion coupling in the CLC transport mechanism, this project will combine complementary cutting-edge approaches, including cryo-electron microscopy to determine high-resolution structures, double electron-electron resonance spectroscopy to monitor the conformational state of the transporter under different conditions and with different mutations, MD simulations to determine hydration pathways under various conditions, and quantitative functional assays to connect structural dynamics to function.

CLC(“氯离子通道”)家族包括两种主要的离子传输机制： CLC同系物的一半是电扩散离子通道，一半是次级活性转运体 按化学计量比将氯离子交换为氢离子。两种机制在一个家族中的发生 表明他们在同一主题上以不同的方式运作。事实上，实验结果支持 CLC通道是“破碎的”转运体的假设，在这种假设中， 内门和外门丢失了。因此，蛋白质构象动力学和离子的细微差异 结合以及它们之间的相互作用可以产生两种不同类型的离子传输 具有相同二级结构的蛋白质中的行为。了解病毒的分子基础 这些差异将有助于我们对CLC通道和转运体的理解。 作为第二活性转运体，ClC转运体利用储存在一个离子中的能量 电化学梯度(Cl-或H+)，使另一个离子与其梯度相反。这通过以下方式发生 蛋白质构象变化与离子结合和转运事件的紧密耦合。发展，发展 CLC传输机制中离子耦合的全集成结构描述 该项目将结合互补的尖端方法，包括低温电子显微镜 为了确定高分辨率结构，双电子-电子共振光谱学 监测转运蛋白在不同条件和不同情况下的构象状态 突变，MD模拟以确定不同条件下的水合途径，以及 将结构动力学与功能联系起来的定量功能分析。