Conformational changes in CIC chloride transporters

这是本项目的第五年，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。在这个项目中，我们正在使用多种方法来分析这些蛋白质的功能机制。之前，我们使用荧光方法证明了细菌ClC反转运蛋白中存在与转运相关的构象变化。目前，我们正在与加州大学戴维斯分校的Henning Stahlberg合作，在一系列条件下形成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，我们开始研究真核细胞ClC-4的转运机制。当在非洲爪蟾卵母细胞中异种表达时，这种内体ClC被发送到质膜，允许使用电生理学方法进行分析。我们寻找可能对探测转运过程有用的分子工具，发现Zn2+和Cd2+都抑制ClC-4电流。我们使用定点诱变技术定位了Zn2+结合位点，并发现了Zn2+结合与离子传输过程之间有趣的相互作用。这些发现揭示了先前未涉及运输相关构象变化的部分蛋白质的运动。

This was the fifth year for this project, 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. In this project, we are using a combination of methods to analyze the functional mechanisms of these proteins. Previously, we used fluorescence methods to demonstrate the existence a transport-related conformational change in a bacterial ClC antiporter. Currently, in collaboration with Henning Stahlberg at UC Davis, we are forming 2d crystals of this protein under a series of conditions to reveal the structural changes underlying this conformational change. In the past year we began to study the transport mechanism in a eukaryotic ClC antiporter, ClC-4. This endosomal ClC is sent to the plasma membrane when heterologously expressed in Xenopus oocytes allowing analysis using electrophyiological methods. We searched for molecular tools that might be useful in probing the transport process and found that Zn2+ and Cd2+ both inhibit ClC-4 currents. We used site-directed mutagenesis to locate the Zn2+ binding site and found intruiging interactions between Zn2+ binding and the ion transport process. These finding reveal movements in a part of the protein that has not been previously implicated in transport-related conformational changes.