Chloride fluxes in organellar membranes

细胞器膜中的氯离子通量

• 批准号: 8342238
• 负责人: Joseph A Mindell
• 金额: $ 65.94万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8342238
• 关键词: ATP phosphohydrolase; Albers-Schonberg disease; Anions; Behavior; Biological Assay; CLC Gene; Carrier Proteins; Cells; Chloride Ion; Chlorides; Collaborations; Dextrans; Disease; Enzymes; Fluorescence; Genes; Goals; Ion Transport; Ions; Knock-out; Lead; Life; Link; Lysosomal Storage Diseases; Lysosomes; Measurement; Measures; Membrane; Methods; Molecular; Neurologic Dysfunctions; Organelles; Pathway interactions; Permeability; Play; Process Measure; Property; Proton Pump; Pump; Regulation; Relative (related person); Role; Shunt Device; Simulate; Small Interfering RNA; Techniques; Testing; Tissues; Universities; antiporter; base; bone; cellular imaging; dextran; fluorophore; image processing; ratiometric; shunt pathway; voltage

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.

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。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。在过去的一年中，我们一直在开发方法，以准确地meaure在活细胞中的溶酶体的pH值，以确定的影响，CLC-7和其他转运蛋白的溶酶体pH值。这些方法使用双波长的比例荧光团连接到葡聚糖，专门针对溶酶体。通过处理在两个波长下拍摄的细胞图像来测量pH。今年，我们已经初步证实，基于siRNA的ClC-7敲低抑制了溶酶体酸化，并正在改进我们用于进行这些测量的方法。关于ClC-7的作用的另一种假设是，它对于确定溶酶体中Cl-的内腔浓度是重要的，这反过来对于激活溶酶体降解酶是重要的。我们正在使用各种技术来验证这一假设。我们也开始使用基于荧光的测定来测量分离的溶酶体中的膜电压。这是一个有用的，以分析不同的渗透性的相对贡献，以确定溶酶体的pH值。为了将这些方法联系在一起，我们，在合作与博士迈克尔Grabe在匹兹堡大学，计算模拟已知的特征的溶酶体酸化mecahnism，以确定这些是否可以解释观察到的酸化行为。这些方法一起提供了一个综合的方法来了解溶酶体的动力学，和ultimatly细胞器，pH调节。