Mechanisms of CLC Transporters and Channels

这个项目的长期目标是发展一个详细的分子理解的CLC（“氯化物 通道”）膜蛋白家族。CLC包括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 另一半是次级活性转运蛋白，其化学计量地将氯离子交换为质子（利用 来自氯化物运动的能量泵送质子或反之亦然）。这两种类型的离子传输 在一个基因家族中，它们的机制可能是在一个中心主题上的微妙变化。 事实上，CLC通道似乎是通过“断裂的转运蛋白”机制起作用的。在这里，我们提出了一个高度 协调一致的方法，包括互补的计算和实验生物物理和 生物化学技术研究支持CLC-ec 1和CLC-0机制的分子细节， 分别为反向转运蛋白和通道的模型同系物。我们的主要目标是阐明反向转运蛋白 （“不间断”）机制，利用高分辨率CLC-ec 1结构和分子结构， 动力学模拟，他们允许，和反转运服从光谱分析。我们将应用 从“未断裂”转运蛋白CLC-ec 1的研究到CLC-0的电生理分析的见解 通道的“断裂”机制，研究通道和转运机制之间的保守性。要求1 将决定与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-ec 1上的定点自旋标记对之间的距离变化， 自旋标签的可访问性，并使用计算建模来开发向内和 外向型国家。AIM 2将确定CLC构象变化如何影响水动力学， 涉及质子运输的水线形成。这些研究将有助于揭示质子运输如何适应 整体CLC运输机制。AIM 3将描述氯离子/质子耦合机制- 结合动力学和光谱学，评估运输如何发生的详细模型 WT和非偶联突变体的测量，以及计算分析，以详细研究 离子的结合和移位如何与蛋白质构象变化相耦合。 整体影响：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，以及它们是如何相似和不同的，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 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.