Conformational changes in CIC chloride transporters

本项目继续研究clc型氯离子转运蛋白的构象变化。ClC家族的氯离子传导离子通道和转运体参与了许多生物过程；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。尽管这些蛋白具有重要的生理意义，但其功能机制却鲜为人知。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。我们目前在这一领域的重点是观察单个蛋白质脂质体中单个ClC转运体的通量。我们正在使用单分子方法来探测单个ClC转运蛋白的转运活性。我们现在已经在单个脂质体中可重复地观察到ClC介导的通量，并观察到质子通过Cl-相反运动驱动的ClC氯离子-质子反转运体进行转运。

This project continued studying conformational changes in ClC-type chloride transport proteins. The ClC family of chloride-conducting ion channels and transporters 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 proteins 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. Our current focus in this area is to observe the flux due to a single ClC transporter in a single proteoliposome. We are doing this using single molecule methods to probe the transport activity of individual ClC transport proteins. We have now reproducibly observed ClC-mediated fluxes in individual liposomes and have observed proton transport through a ClC chlroide-proton antiporter driven by opposing movement of Cl-. Using mutant ClCs with altered transport processes, we are using this assay to probe fundamental aspects of the transport mechanism. In addition, we have been exploring a patient mutation in another CLC, ClC-7, which is important in lysosomal acidification. This mutation leads to a disease different from those caused by other ClC-7C defects. We have been investigating the functional effects of the mutation by expressing WT and mutant proteins (with additional mutations that retarget the protein to the cell surface) in Xenopus oocytes or cultured mammalian cells and analyzing the properties of the resulting currents. These results give insight into the mechanism of the defect in patients and its underlying protein cause. Finally, in a somewhat unrelated project, we have participated in a collaboration to explore the relationships between structure and function in a mammalian citrate transporter, a member of a family long of interest to the lab. In collaboration with Da-Neng Wang's lab at NYU we determined the structure of this protein and demonstrated that a pharmacologic inhibitor works by pinning the transport domain in one place so it cannot move in the ways required by transport. This was published last year in Nature.