Chloride fluxes in organellar membranes

该项目使用多种方法来分析溶酶体膜的离子运输特性，特别关注它们在产生细胞器酸性腔内pH值中的作用。溶酶体是细胞内的细胞器，在大多数细胞中作为消化细胞器，尽管在某些组织中它们被用于其他功能。溶酶体利用atp驱动的质子泵来维持酸性的腔内pH值并促进其消化功能。这种泵只有伴随着额外的离子传输才能有效，以消散由atp酶（一种反离子途径）建立的跨膜电压。过去，我们使用分离的溶酶体来鉴定和表征溶酶体膜中氯离子的通透性，它具有这种反离子途径所需的特征，并证明氯离子是由Cl- 7运输的，这是一种专门针对溶酶体膜的Cl-/H+反转运体。我们最近优化了使用双波长比例荧光团连接到葡聚糖特异性靶向溶酶体的方法。pH值是通过处理两个波长的细胞图像来测量的。我们已经制定了精确校准系统的方法，使图像测量与实际pH值相关联。我们现在分析了ClC-7基因敲低对siRNA控制不影响溶酶体pH的细胞的影响，并使用这些系统证明ClC-7敲低至少在某些细胞类型中损害酸化，我们目前正在完成完成这项工作所需的控制。我们也几乎完成了对分离溶酶体酸化的离子需求的分析，并将使用该系统证明ClC-7敲除严重损害从敲除小鼠分离的肝溶酶体的酸化。我们也开始探索其他代谢过程对溶酶体pH值的影响，因为溶酶体最近被证明是细胞代谢应激的重要监测站。

This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes with particular focus on their role in generating the acid luminal pH of the organelle. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase, a counterion pathway. In the past, we used isolated lysosomes to identify and characterize a Chloride permeability in the lysosomal membrane which has the features required of such a counterion pathway and demonstrated that the chloride is transported by ClC-7, a Cl-/H+ antiporter specifically targeted to the lysosomal membrane. We have recently optimized the methods to use dual-wavelength ratiometric fluorophores linked to dextran to specifically target lysosomes. pH is measured by processing images of the cells taken at the two wavelengths. We have worked out methods to accurately calibrate the system to correlate image measurements with actual pH values. We have now analyzed the effects of ClC-7 gene knockdown on cells where siRNA controls do not affect lysosome pH and used those systems to demonstrate that ClC-7 knockdown compromises acidification in at least some cell types, we are currently completeing controls required to finalize this work. We have also nearly completed an analysis of the ion requirements of acidification in isolated lysosomes which will be and used that system to demonstrate that ClC-7 knockout severely impairs acidification in liver lysosomes isolated from knockout mice. We are also beginning to explore the influences of other metabolic processes on the pH of lysosomes, since lysosomes have recently been shown to serve as important monitoring stations for cellular metabolic stress. In addition to our work on lysosomes, we have begun to explore the acidification process in other organelles. We have completed work on analysis of chloride dependent acidification in clathrin coated vesicles in brain which was published this year. Finally, we have been collaborating with the Gahl lab in the Genome institute to analyze a mutant form of ClC-7 that they have found in a patient in the Undiagnosed Diseases Program who has a disease pattern completely unlike those found with other ClC-7 mutations. We have made substantial progress in understanding the effects of this mutation over the past year, with careful analysis of the effects of the mutation on ClC-7 function, the effects of these changes on pH in lysosomes, and possible approaches to treating this disease. We are currently preparing this work for publication. We demonstrated that the mutaion causes patient lysosomes to become hyperacidic, and that change in organellar pH leads to a host of disruptions of cellular metabolism. We also showed that we could correct the cellular defects by treating cells with agents known to alkalinize lysosomes. These approches suggest possibilities for therapy of the disease. This work was published this year and we are currently following up with further experiments on this topic.