Chloride fluxes in organellar membranes

本项目使用多种方法相结合的方法来分析溶酶体膜的离子传输特性。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提供服务，尽管在某些组织中它们也用于其他功能。溶酶体功能障碍会导致多种疾病，包括神经功能障碍(溶酶体储存性疾病)和骨化症(骨骼过度钙化)。溶酶体利用三磷酸腺苷驱动的质子泵来维持酸性的管腔pH，并促进其消化功能。这样的泵只有伴随着额外的离子传输才能有效，以消除由ATPase建立的跨膜电压，ATPase是一种反离子途径。我们最近使用分离的溶酶体来鉴定和表征溶酶体膜上的氯离子通透性，它具有这种反离子途径所需的特征，并证明了氯离子是由ClC-7运输的，ClC-7是一种针对溶酶体膜的氯-/H+逆向转运蛋白。在过去的一年里，我们一直在开发准确测量活细胞溶酶体pH的方法，以确定ClC-7和其他转运蛋白对溶酶体pH的影响。这些方法使用与葡聚糖连接的双波长比率荧光团来特异性地靶向溶酶体。PH值是通过处理在两个波长上拍摄的细胞图像来测量的。今年，我们已经初步证实，基于siRNA的ClC-7基因敲除可以抑制溶酶体的酸化，并正在改进我们用来进行这些测量的方法。关于ClC-7作用的另一种假说是，ClC-7对确定溶酶体中氯离子的腔内浓度很重要，这反过来又对激活溶酶体降解酶很重要。我们正在使用各种技术来检验这一假设。我们还开始使用基于荧光的方法来测量分离的溶酶体中的膜电压。这有助于分析不同通透性对测定溶酶体pH的相对贡献。为了将这些方法联系在一起，我们与匹兹堡大学的Michael Grabe博士合作，通过计算模拟溶酶体酸化机制的已知特征，以确定这些特征是否可以解释观察到的酸化行为。这些方法共同提供了一种综合的方法来理解溶酶体和最终细胞器的pH调节的动力学。

This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). 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. We recently 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. In the past year we have been developing methods to accurately meaure the pH in lysosomes in living cells in order to determine the influence of ClC-7 and other transporters on the lysosomal pH. These methods 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. This year, we have obtained preliminary confirmation that siRNA-based knockdown of ClC-7 inhibits lysosomal acidifciation and are refining the methods we are using to make these measurements. An alternate hypothesis regarding the role of ClC-7 is that it is important for determining the lumenal concentration of Cl- in lysosomes, which in turn is important for activating lysosomal degradative enzymes. We are testing this hypothesis using a variety of techniques. We have also begun to use fluorescence-based assays to measure the membrane voltage in isolated lysosomes. This is a useful to to analyze the relative contributions of different permeabilities to determining the lysosomal pH. To tie these approaches together, we, in collaboration with Dr. Michael Grabe at the University of Pittsburgh, are computationally simulating the known features of the lysosomal acidfication mecahnism to determine whether these can explain observed acdification behaviors. Together these methods provide an integrated approach to understanding the dynamics of lysosomal, and ultimatly organellar, pH regulation.