Conformational changes in CIC chloride transporters

该项目继续研究ClC型氯离子通道蛋白的构象变化。ClC家族的氯离子传导离子通道参与了一系列的生物过程，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，并参与多种细胞内隔室的pH动态平衡。尽管它们具有重要的生理意义，但人们对这些通道的作用机制知之甚少。我们试图通过研究几个家族成员来了解这些蛋白质的功能特性，包括真核和原核同源物。在这个项目中，我们正在使用荧光方法来证明细菌CLC逆向转运蛋白中存在与运输相关的构象变化。目前，我们正在与瑞士的Henning Stahlberg合作，在一系列条件下形成这种蛋白质的2D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，随着我们发现晶体形态在高pH值和低pH值下都稳定，该项目取得了进展。来自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。我们目前正在集中我们的机械努力在溶酶体CLC，CLC-7。最近的报道表明，另一种哺乳动物ClC，ClC-7，虽然通常以溶酶体为靶点，但也可以通过突变二亮氨酸分类基序重定向到质膜上。我们已经用这种方法重定了ClC-7，并正在用电生理学来研究它的传输特性。我们最初的描述，即将出版，检查了ClC-7转运体的外部pH依赖性，并探索了在CLC中观察到的一种新的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. 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.