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。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。我们正在与哥本哈根大学的Dimitrios Stamou合作，使用单分子方法来做这件事。为此，我们开发了一种强大的氯偶联质子运输的批量分析，然后将单个脂质体置于类似的条件下。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，并观察到复杂的pH变化，这表明目前的模型并不能完全解释CLC蛋白的功能。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。这种突变导致的疾病不同于其他ClC缺陷引起的疾病。我们已经通过在爪蟾卵母细胞或培养的哺乳动物细胞中表达WT和突变蛋白（加上将蛋白重新靶向到细胞表面的额外突变）来研究突变的功能影响，并分析由此产生的电流的特性。这些结果深入了解了患者缺陷的机制及其潜在的蛋白质原因。

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. 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 in collaboration with Dimitrios Stamou at the University of Copenhagen. Toward this end we have developed a robust bulk assay of chloride-coupled proton transport, and then subjected individual liposomes to similar conditions. We have now reproducibly observed ClC-mediated fluxes in individual liposomes and have obeserved complex pH variation, suggesting that current models do not fully explain the function of the CLC protein. 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 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.