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。在这种情况下，我们观察到单一脂质体的强劲酸化。我们目前正在优化这些检测方法，并试图提高它们的稳健性。

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. In this circumstances, we observe robust acidification in single liposomes. We are currently optimizing these assays and trying to improve their robustness.