EDNOGENOUS ION CHANELS OF THE GOLGI COMPLEX

高尔基复合体的内源性离子通道

• 批准号: 6125330
• 负责人: JOHN H CALDWELL
• 金额: $ 29.19万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6125330
• 关键词: Golgi apparatus; acidity /alkalinity; adenosinetriphosphatase; carbohydrate receptor; complementary DNA; electrophysiology; endoplasmic reticulum; gel electrophoresis; immunofluorescence technique; inositol phosphates; intracellular membranes; ion channel blocker; laboratory rat; lipid bilayer membrane; liposomes; mass spectrometry; membrane channels; membrane reconstitution /synthesis; membrane transport proteins; organelles; protein sequence; protein structure function; solubility; tissue /cell culture

The Golgi complex is present in every eukaryotic cell, from yeast to humans, and functions in posttranslational protein modifications and sorting of these molecules to post-Golgi destinations. Both processes require an acidic lumenal pH and transport of substrate and reactants into and out of the Golgi lumen. Endogenous ion channels are expected to be important for regulating the ionic environment within the Golgi lumen. This proposal is to isolate and characterize these endogenous ion channels of the Golgi complex and is a collaboration between two laboratories, one that studies ion channels and one that studies Golgi function. Single ion channel studies are now feasible because we have recently improved the isolation of a Golgi fraction from rat liver. We eliminated proteins transiting the Golgi and achieved a 400-700 fold enrichment of endogenous Golgi proteins. Ion channels in the enriched fraction have been incorporated into planar lipid bilayers. We named the most prevalent ion channel GOLAC1 (Golgi Anion Channel 1). This channel has novel properties and is modulated by pH on the lumenal surface. Two hypotheses are proposed for the function of the GOLAC 1: first, it provides counterions necessary for acidification of the Golgi lumen by an electrogenic H+ATPase and second, it removes phosphate (generated by glycosylation and sulfation) from the Golgi lumen. There are three specific aims. (1) Electrophysiologically characterize anion and cation channels of the Golgi. (2) Obtain peptide and cDNA sequence by enriching for channel activity with subfractionation of detergent- solubilized Golgi proteins. Proteins that enrich in parallel with channel activity will be identified using 2D-gel electrophoresis, mass spectrometry, and peptide sequencing. The cDNA identified will be expressed, purified, and confirmed to be a GOLAC in bilayer studies and a Golgi protein by immunofluorescence. (3) Study modulation of Golgi channels by candidate molecules and cell factors. From a clinical viewpoint, there are an increasing number of diseases classified as ion channelopathies. It is likely that some human diseases will be due to mutations of endogenous Golgi channels.

高尔基复合体存在于从酵母到人类的每个真核细胞中，并在翻译后蛋白质修饰和将这些分子分类到高尔基后目的地中发挥作用。这两个过程都需要酸性的腔内 pH 值以及底物和反应物进出高尔基腔的运输。预计内源离子通道对于调节高尔基体腔内的离子环境非常重要。该提案旨在分离和表征高尔基复合体的这些内源离子通道，是两个实验室之间的合作，一个研究离子通道，另一个研究高尔基体功能。单离子通道研究现在是可行的，因为我们最近改进了从大鼠肝脏中分离高尔基体部分。我们消除了通过高尔基体的蛋白质，并实现了内源高尔基体蛋白质 400-700 倍的富集。富集部分中的离子通道已被纳入平面脂质双层中。我们将最常见的离子通道命名为 GOLAC1（高尔基阴离子通道 1）。该通道具有新颖的特性，并受腔表面 pH 值调节。对于 GOLAC 1 的功能，提出了两种假设：首先，它通过生电 H+ATP 酶提供高尔基体腔酸化所需的抗衡离子，其次，它从高尔基体腔中去除磷酸盐（由糖基化和硫酸化产生）。具体目标有三个。 (1) 电生理学表征高尔基体的阴离子和阳离子通道。 (2) 通过对去污剂溶解的高尔基体蛋白进行亚分级来富集通道活性，从而获得肽和 cDNA 序列。与通道活性同时富集的蛋白质将使用 2D 凝胶电泳、质谱和肽测序进行鉴定。鉴定出的 cDNA 将被表达、纯化，并通过免疫荧光证实为双层研究中的 GOLAC 和高尔基体蛋白。 (3) 研究候选分子和细胞因子对高尔基体通道的调节。从临床角度来看，越来越多的疾病被归类为离子通道病。一些人类疾病很可能是由于内源性高尔基体通道的突变造成的。