Mechanisms of CLC Transporters and Channels

CLC转运蛋白和通道的机制

• 批准号: 10540388
• 负责人: Merritt C Maduke
• 金额: $ 47.76万
• 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/10540388
• 关键词: Active Biological Transport; Affect; Anions; Behavior; Binding; Biological Assay; Carrier Proteins; Cations; Chloride Channels; Coupled; Coupling; Cryoelectron Microscopy; Crystallography; Data; Electrons; Event; Family; Homologous Gene; Human; Hydration status; Hypertension; Ion Channel; Ion Transport; Ions; Kidney Diseases; Kinetics; Knowledge; Measurement; Methodology; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Muscle; Mutation; Organism; Pathway interactions; Play; Process; Protein Conformation; Protein Region; Proteins; Protons; Pump; Resolution; Role; Secondary to; Side; Spectrum Analysis; Structure; Testing; Tissues; Variant; Water; antiporter; biophysical techniques; bone; crosslink; design; extracellular; mutant; nervous system disorder; public health relevance; simulation; water channel

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转运机制： 的CLC同系物是电扩散离子通道，一半是次级活性转运蛋白 化学计量地将Cl-交换为H+。一个家庭中两种机制的发生 表明它们是通过一个共同主题的变化来运作的。事实上，实验结果支持 假设CLC通道是“破碎的”转运蛋白，其中CLC通道之间的紧密协调 内外门都失去了。因此，蛋白质构象动力学和离子动力学的细微差异 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的行为。了解的分子基础 这些差异将告知我们对CLC通道和转运体的理解。 作为次级活性转运蛋白，CLC转运蛋白利用储存在一个离子的 电化学梯度（Cl-或H+）以逆着其梯度泵送另一离子。这是通过 蛋白质构象变化与离子结合和转运事件的紧密耦合。发展 CLC运输机制中离子耦合的完全集成的结构描述， 该项目将结合联合收割机互补的尖端方法，包括低温电子显微镜 为了确定高分辨率结构，双电子-电子共振光谱法， 监测不同条件下和不同浓度下转运蛋白的构象状态， 突变，MD模拟以确定各种条件下的水合途径，以及 定量功能分析，以连接结构动力学功能。