Mechanisms of CLC Transporters and Channels

CLC转运蛋白和通道的机制

• 批准号: 9174309
• 负责人: Merritt C Maduke
• 金额: $ 46.21万
• 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 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9174309
• 关键词: Active Biological Transport; Affect; Binding; Biochemical; Carrier Proteins; Chloride Channels; Chloride Ion; Chlorides; Chronic; Computer Analysis; Computer Simulation; Constipation; Coupled; Coupling; Crystallization; Crystallography; Development; Disease; Electron Spin Resonance Spectroscopy; Electrons; Electrophysiology (science); Elements; Event; Failure; Family; Gene Family; Goals; Homologous Gene; Human; Hypertension; Hyponatremia; Ion Channel; Ion Transport; Ions; Kidney Diseases; Kinetics; Measurement; Measures; Medical; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Movement; Muscle; Organism; Osteoporosis; Pathway interactions; Process; Proteins; Proton Pump; Protons; Resolution; Side; Site; Spin Labels; Structural Models; Structure; Techniques; Testing; Therapeutic; Tissues; Validation; Variant; Water; Work; antiporter; biophysical techniques; bone; inhibitor/antagonist; innovation; insight; leukodystrophy; molecular dynamics; mutant; nervous system disorder; public health relevance; reconstitution; simulation; stoichiometry; tool

The long-term goal of this project is to develop a detailed molecular understanding of the CLC ("Chloride Channel") family of membrane proteins. The CLCs comprise two major classes of ion-transport mechanisms: half of CLC homologs are electrodiffusive ion channels (catalyzing downhill movement of chloride), while the other half are secondary active transporters that stoichiometrically exchange chloride for protons (harnessing the energy from movement of chloride to pump protons or vice versa). That both types of ion-transport are within one gene family suggests their mechanisms may be subtle variations on a single central theme. Indeed, CLC channels appear to act by a "broken transporter" mechanism. Here we propose a highly concerted approach composed of complementary computational and experimental biophysical and biochemical techniques to study the molecular details underpinning the mechanism of CLC-ec1 and CLC-0, model homologs for antiporters and channels, respectively. Our main goal is to elucidate the antiporter ("unbroken") mechanism, taking advantage of high-resolution CLC-ec1 structures and the molecular dynamics simulations they allow, and of antiporter amenability to spectroscopic analysis. We will apply insights from studies of the "unbroken" transporter CLC-ec1 to electrophysiological analysis of the CLC-0 channel's "broken" mechanism to study conservation between channel and transporter mechanisms. AIM 1 will determine global structural changes associated with the CLC transport cycle. Here we will use EPR to measure distance changes between pairs of site-directed spin labels on CLC-ec1, evaluate changes in accessibility of spin labels, and use computational modeling to develop structural models for the inward- and outward-facing states. AIM 2 will determine how CLC conformational change affects water dynamics and water-wire formation involved in proton transport. These studies will help reveal how proton transport fits into the overall CLC transport mechanism. AIM 3 will characterize the chloride/proton coupling mechanism – evaluating detailed models of how transport occurs, using a combination of kinetic and spectroscopic measurements on WT and uncoupled mutants, together with computational analysis to investigate in detail how binding and translocation of ions are coupled to protein conformational changes. Overall Impact: Revealing molecular details of CLC ion channel "broken" and antiporter "unbroken" mechanisms, and how they are alike and different, will help reveal how CLC function can go wrong, with implications for neurological diseases, hypertension, and diseases of kidney, muscle, and bone. Our methodology will be applicable to other large membrane proteins of medical importance where unraveling molecular mechanisms has similarly been stymied by limitations of crystallography.

该项目的长期目标是对CLC建立详细的分子理解（“氯化物” 渠道”）膜蛋白家族。CLCs包括两个主要类别 - 传输机制： 一半的CLC同源物是电缩合离子通道（催化氯化物的下坡运动），而 另一半是二级活性转运蛋白，可将氯化物化为质子（施加氯化物） 从氯化物到泵送质子的能量，反之亦然）。两种类型的离子传输都是 在一个基因家族中，他们的机制可能是单个中心主题的微妙变化。 实际上，CLC通道似乎是通过“破碎的转运蛋白”机制作用的。在这里，我们提出了一个高度的建议 协同的方法由互补的计算和实验生物物理和 研究分子细节的生化技术是CLC-EC1和CLC-0的机理的基础 分别为抗植物和通道建模。我们的主要目标是阐明抗客 （“不间断”）机制，利用高分辨率CLC-EC1结构和分子 他们允许的动力学模拟，以及抗流剂的合理性进行光谱分析。我们将申请 从“不间断”转运蛋白CLC-EC1的研究到CLC-0的电生理分析的见解 通道的“破裂”机制，用于研究通道和转运蛋白机制之间的保护。目标1 将确定与CLC运输周期相关的全球结构变化。在这里，我们将使用EPR 测量CLC-EC1上的位置定向自旋标签对之间的距离变化，评估 自旋标签的可访问性，并使用计算建模为内向和 向外的国家。 AIM 2将决定CLC构象变化如何影响水动力学和 质子运输涉及的水线形成。这些研究将有助于揭示质子运输如何适应 总体CLC运输机制。 AIM 3将表征氯化/质子耦合机制 - 使用动力学和光谱镜的组合评估运输方式的详细模型 对WT和未耦合突变体的测量以及计算分析以详细研究 离子的结合和易位如何与蛋白质构象变化耦合。 总体影响：揭示CLC离子通道的分子细节“破碎”和抗胞生物“不间断” 机制以及它们的相似之处和不同的方式，将有助于揭示CLC功能如何出错，并使用 对肾脏，肌肉和骨骼的神经系统疾病，高血压和疾病的影响。我们的 方法学将适用于其他重要重要性的大型膜蛋白 分子机制也被晶体学的局限性所困扰。