Conformational changes in CIC chloride transporters

该项目继续研究ClC型氯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，并参与各种细胞内隔室的pH动态平衡。尽管它们具有重要的生理意义，但人们对它们的功能机制知之甚少。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。我们目前在这一领域的重点是观察单个蛋白脂质体中单个CLC转运体的通量。我们正在使用单分子方法来探索单个CLC转运蛋白的转运活性。我们现在已经在单个脂质体中重复性地观察到了ClC介导的通量，并观察到了质子通过ClC氯-质子逆向转运体的传输。 利用突变的CLCs和改变的运输过程，我们正在使用这一测试来探索运输机制的基本方面。 此外，我们一直在探索另一种CLC的患者突变，CLC-7，这在溶酶体酸化中很重要。这种突变会导致一种不同于其他ClC-7C缺陷引起的疾病。我们一直在通过在非洲爪哇卵母细胞或培养的哺乳动物细胞中表达WT和突变蛋白(以及将蛋白重定向到细胞表面的额外突变)并分析产生的电流的特性来研究突变的功能影响。这些结果为深入了解患者缺陷的机制及其潜在的蛋白质原因提供了线索。 最后，在一个有些无关的项目中，我们参与了一个合作项目，探索哺乳动物柠檬酸转运蛋白的结构和功能之间的关系，该转运蛋白是实验室长期感兴趣的一个家族的成员。与纽约大学王大能的实验室合作，我们确定了这种蛋白质的结构，并证明了一种药物抑制剂的工作原理是将转运域固定在一个地方，使其不能以运输所需的方式移动。这篇文章发表在去年的《自然》杂志上。

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.