Chloride fluxes in organellar membranes

该项目使用多种方法来分析溶酶体膜的离子转运特性，特别关注它们在产生细胞器的酸性管腔pH值中的作用。溶酶体是细胞内的细胞器，在大多数细胞中充当消化细胞器，尽管在某些组织中它们用于其他功能。溶酶体利用ATP驱动的质子泵来维持酸性的管腔pH值并促进其消化功能。这样的泵只有在伴随额外的离子转运以耗散由ATP酶（一种反向途径）建立的跨膜电压时才能有效。在过去，我们使用分离的溶酶体来鉴定和表征溶酶体膜中的氯离子渗透性，其具有这种反转运途径所需的特征，并证明氯离子由ClC-7转运，ClC-7是一种特异性靶向溶酶体膜的Cl-/H+反向转运蛋白。我们最近优化了使用双波长比率荧光团连接到葡聚糖特异性靶向溶酶体的方法。通过处理在两个波长下拍摄的细胞图像来测量pH。我们已经制定了方法来准确地校准系统，以将图像测量与实际pH值相关联。我们现在已经分析了C1 C-7基因敲低对siRNA对照不影响溶酶体pH的细胞的影响，并使用这些系统来证明C1 C-7敲低在至少一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。我们还几乎完成了对分离的溶酶体中酸化的离子需求的分析，该分析将使用该系统来证明ClC-7敲除严重损害了从敲除小鼠分离的肝溶酶体中的酸化。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。 除了我们在溶酶体上的工作，我们已经开始探索其他细胞器中的酸化过程。我们已经完成了今年发表的关于脑中网格蛋白包被囊泡中氯依赖性酸化的分析工作。 最后，我们一直在与基因组研究所的Gahl实验室合作，分析他们在未诊断疾病项目中发现的一种ClC-7突变形式，该患者的疾病模式完全不同于其他ClC-7突变。在过去的一年里，我们在理解这种突变的影响方面取得了实质性进展，仔细分析了突变对ClC-7功能的影响，这些变化对溶酶体pH值的影响，以及治疗这种疾病的可能方法。我们目前正在准备出版这部作品。我们证明了突变导致患者溶酶体变得过酸性，细胞器pH值的变化导致细胞代谢的破坏。我们还表明，我们可以通过用已知的碱化溶酶体的试剂处理细胞来纠正细胞缺陷。这些方法提示了治疗该病的可能性。这项工作已于今年发表，我们目前正在跟进这一主题的进一步实验。

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.