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的CLC家族参与许多生物过程;这些通道维持骨骼肌中的静息膜电位，调节中枢神经元的兴奋性，并参与各种细胞内隔室中pH的稳态。尽管它们的生理重要性，这些通道的功能机制知之甚少。我们正试图了解这些蛋白质的功能特性，通过检查几个家庭成员，包括真核和原核同源物。在这个项目中，我们使用荧光方法来证明细菌CLC反向转运蛋白中存在与转运相关的构象变化。目前，我们正在与瑞士的Henning斯塔尔贝格合作，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。在过去的一年中，我们发现了在高pH和低pH下稳定的晶体形式，该项目向前推进。这些晶体的初步投影图表明，脂质双层膜中蛋白质的高pH和低pH形式之间的构象存在差异。我们目前正集中我们的机制努力在溶酶体CLC，CLC-7。最近的报道表明，另一种哺乳动物ClC，ClC-7，虽然通常靶向溶酶体，但可以通过突变双亮氨酸分选基序重新靶向质膜。我们已经使用这种方法重新定位ClC-7，并正在使用电生理学研究其转运特性。我们的初步表征，几乎准备出版，检查外部pH值依赖的CLC-7转运蛋白和探针之间的关系，在一种新的形式的门控中观察到的CLC的运输周期。

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. In this project, we are using a the fluorescence methods to demonstrate the existence a transport-related conformational change in a bacterial ClC antiporter. Currently, in collaboration with Henning Stahlberg in Switzerland, 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's project has moved forward with our discovery of the crystal form stable at both high and low pH. Preliminary projection maps from these crystals suggest differences in conformation between high and low pH forms of the proteins in the lipid bilayer membrane. We are currently focusing our mechanistic efforts on the lysosomal CLC, ClC-7. Recent reports demonstrate that another mammalian ClC, ClC-7, though usually targeted to lysosomes, can be retargeted to the plasma membrane by mutating a dileucine sorting motif. We have used this method to retarget ClC-7 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.