Chloride fluxes in organellar membranes

本计画是利用一系列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。溶酶体功能障碍导致多种疾病，包括神经功能障碍（溶酶体贮积病）和骨硬化症（骨过度钙化）。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。这种泵只有在伴随额外的离子转运以耗散由ATP酶建立的跨膜电压时才有效。遗传学证据表明，这一额外的离子途径是ClC型阴离子转运蛋白，但功能实验尚未证明这样的途径。我们确定了溶酶体中的离子转运途径，表征了它们的性质，并确定了负责的蛋白质。今年，我们开始检查内体，以确定Cl转运对其酸化过程的影响，并开始构建ClC-7敲除小鼠，以进一步了解这种转运蛋白的作用。我们还在开发测量活培养细胞中溶酶体pH值的方法，以检查ClC-7对这些细胞器的影响。

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. Genetic evidence suggests that this additional ion pathway a ClC-type anion transporter, but functional experiments have not yet demonstrated such a pathway. We identified the ion transport pathways in lysosomes, characterized their properties, and to identified the responsible proteins. This year we began examining endosomes to determine the effects of Cl transport on their acidification processes and began constructing a ClC-7 knockout mouse to further understand the effects of this transporter. We are also developing methods to measure pH in lysosomes in living cultured cells to examine the influence of ClC-7 on these organelles.