Chloride fluxes in organellar membranes

这个项目是使用多种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，尽管在某些组织中它们被用于其他功能。溶酶体功能紊乱可导致多种疾病，包括神经功能障碍（溶酶体贮积病）和骨质疏松症（骨过度钙化）。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。我们最近使用分离的溶酶体来鉴定和表征溶酶体膜中的氯离子通透性，它具有这种反离子途径所需的特征，并证明氯离子是由ClC-7运输的，这是一种专门针对溶酶体膜的Cl-/H+反转运体。在过去的一年中，我们一直在开发准确测量活细胞中溶酶体pH值的方法，以确定ClC-7和其他转运体对溶酶体pH值的影响。这些方法使用与葡聚糖连接的双波长比例荧光团特异性靶向溶酶体。pH值是通过处理两个波长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敲低抑制溶酶体酸化，并正在改进我们用来进行这些测量的方法。关于ClC-7的作用的另一种假设是，它对确定溶酶体中Cl-的管腔浓度很重要，而Cl-浓度又对激活溶酶体降解酶很重要。我们正在用各种各样的技术来检验这个假设。我们也开始使用荧光法来测量分离溶酶体的膜电压。这对于分析不同渗透性对确定溶酶体ph值的相对贡献是有用的。为了将这些方法结合在一起，我们与匹兹堡大学的Michael Grabe博士合作，正在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。总之，这些方法提供了一个综合的方法来了解溶酶体的动力学，并最终细胞器，pH调节。

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.