Conformational changes in CIC chloride transporters

本项目继续研究clc型氯离子通道蛋白的构象变化。氯离子传导离子通道ClC家族参与了一系列生物过程；这些通道维持骨骼肌的静息膜电位，调节中枢神经元的兴奋性，并参与多种细胞内区室的pH稳态。尽管这些通道具有重要的生理意义，但人们对其作用机制知之甚少。我们正试图通过检查几个家族成员，包括真核和原核同源物来了解这些蛋白质的功能特性。目前，我们与瑞士的Henning Stahlberg合作，在一系列条件下形成了这种蛋白质的二维晶体，以揭示这种构象变化背后的结构变化。在过去的一年里，我们发现了一种稳定的、低pH值形式的二维晶体，它揭示了与以前观察到的蛋白质不同的构象。我们现在有了这种晶体形式的三维地图，并且正在完成它的分析。这是第一个直接的证据，表明构象变化是中CLC转运过程的一部分。我们目前正集中研究溶酶体CLC -7的机制。我们使用最近发表的一种方法将ClC-7从溶酶体重定向到质膜上，并正在使用电生理学研究其运输特性。我们的初步表征，几乎准备发表，检查了CLC -7转运体的外部pH依赖性，并探讨了在CLC中观察到的一种新型门控形式的运输周期之间的关系。

This project continued studying conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels is involved in a host of biological processes; these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these channels function are poorly understood. We are attempting to understand the functional properties of these proteins by examining several family members, including both eukaryotic and prokaryotic homologs. Currently, in collaboration with Henning Stahlberg in Switzerland, we formed 2d crystals of this protein under a series of conditions to reveal the structural changes underlying this conformational change. In the past year we have found a stable, low pH form of the 2d crystal which reveals a different conformation of the protein than those previously observed. We now have a 3 dimensional map of this crystal form and are completing its analysis. This is the first direct evidence of a conformational change that is part of the CLC transport process. We are currently focusing our mechanistic efforts on the lysosomal CLC, ClC-7. We have used a recently published method to retarget ClC-7 from lysosomes to the plasma membrane and are studying its transport properties using electrophysiology. Our initial characterization, nearly ready for publication, examines the external pH dependence of the ClC-7 transporter and probes the relationship between the transport cycle in a novel form of gating observed in the CLC's.