Mechanisms of CLC Transporters and Channels

该项目的长期目标是对 CLC（“氯化物 通道”）膜蛋白家族。 CLC 包含两大类离子传输机制： 一半的 CLC 同系物是电扩散离子通道（催化氯离子的下坡运动），而 另一半是次级主动转运蛋白，它们以化学计量将氯离子交换为质子（利用 氯化物运动到泵浦质子的能量，反之亦然）。这两种类型的离子传输都是 在一个基因家族中，表明它们的机制可能是单一中心主题的微妙变化。 事实上，CLC 通道似乎通过“损坏的转运蛋白”机制发挥作用。在这里我们提出一个高度 由互补的计算和实验生物物理和 生化技术研究支持 CLC-ec1 和 CLC-0 机制的分子细节， 分别是反转运蛋白和通道的模型同源物。我们的主要目标是阐明反向转运蛋白 （“完整”）机制，利用高分辨率 CLC-ec1 结构和分子 它们允许进行动力学模拟，以及反转运蛋白对光谱分析的适应性。我们将申请 从“完整”转运蛋白 CLC-ec1 的研究到 CLC-0 的电生理分析的见解 通道的“破碎”机制来研究通道和转运蛋白机制之间的守恒。目标1 将决定与 CLC 运输周期相关的全球结构变化。这里我们将使用 EPR 来 测量 CLC-ec1 上定点自旋标签对之间的距离变化，评估 自旋标签的可访问性，并使用计算模型来开发内向和自旋标签的结构模型 外向型国家。 AIM 2 将确定 CLC 构象变化如何影响水动力学和 水线的形成涉及质子传输。这些研究将有助于揭示质子传输如何适应 整体 CLC 传输机制。 AIM 3 将表征氯/质子耦合机制 – 结合动力学和光谱来评估运输如何发生的详细模型 对 WT 和解偶联突变体进行测量，并结合计算分析进行详细研究 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“断裂”和反向转运蛋白“未断裂”的分子细节 机制以及它们的相同点和不同点，将有助于揭示 CLC 功能如何出错 对神经系统疾病、高血压以及肾脏、肌肉和骨骼疾病的影响。我们的 该方法将适用于其他具有医学重要性的大膜蛋白 分子机制同样受到晶体学限制的阻碍。

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.