Mechanisms of CLC Transporters and Channels

CLC转运蛋白和通道的机制

• 批准号: 10328564
• 负责人: Merritt C Maduke
• 金额: $ 48.07万
• 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/10328564
• 关键词: Active Biological Transport; Affect; Anions; Behavior; Binding; Biological Assay; CLC Gene; Carrier Proteins; Cations; Chloride Channels; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Data; Electrons; Event; Extracellular Protein; Family; Human; Hydration status; Hypertension; Ion Channel; Ion Transport; Ions; Kidney Diseases; Kinetics; Knowledge; Measurement; Methodology; 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; base; biophysical techniques; bone; crosslink; design; 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（“氯化频道”）家族包括两个主要的离子传输机制：一半 CLC同源物的是电缩合离子通道，一半是次级活动转运蛋白 该化学计量法将Cl-交换为H+。一个家庭中的两个机制发生 暗示它们是通过共同主题的变化来运作的。确实，实验结果支持 CLC通道是“破裂”转运蛋白的假设，其中紧密的协调 内门和外门丢失。那是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的行为。了解分子基础 这些差异将为我们对CLC渠道和转运蛋白的理解提供信息。 作为二级活性转运蛋白，CLC转运蛋白利用存储在一个离子中的能量 电化学梯度（Cl–或H+），将另一离子泵送至其梯度。这是通过 蛋白质构象与离子结合和转运事件的紧密耦合。发展 CLC传输机构中离子耦合的完全整合的结构描述， 项目将结合完整的尖端方法，包括冷冻电子显微镜 为了确定高分辨率结构，双电子电子共振光谱 在不同条件下和不同的条件下监视转运蛋白的构象状态 突变，MD模拟以在各种条件下确定水合途径，并且 定量功能测定，将结构动力学连接到功能。