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; 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.

从酵母到人类的每个真核生物细胞中都存在高尔基体复合物，并且在翻译后蛋白质修饰和这些分子的分类中起作用。这两个过程都需要酸性的液体pH，并将基材和反应物和反应物转移到高尔基腔中。预计内源离子通道对于调节高尔基管内的离子环境很重要。该建议是隔离和表征高尔基复合物的这些内源离子通道，并且是两个实验室之间的合作，一个实验室研究了离子通道，一个研究高尔基体功能。单个离子通道研究现在是可行的，因为我们最近改善了大鼠肝脏的高尔基分数分离。我们消除了转移高尔基体的蛋白质，并获得了400-700倍的内源性高尔基蛋白。富集分数中的离子通道已纳入平面脂质双层。我们命名了最普遍的离子通道Golac1（Golgi阴离子通道1）。该通道具有新的特性，并由pH调节在腔表面上。提出了两种假设，即Golac 1的功能：首先，它提供了通过电基质H+ATPase酸化高尔基管的必要的反面，其次是从高尔基流明中去除磷酸盐（由糖基化和硫酸化）。有三个特定的目标。 （1）电生理学表征高尔基体的阴离子和阳离子通道。 （2）通过富集洗涤剂 - 溶解的高尔基蛋白的分流来获得肽和cDNA序列。与通道活性并行富集的蛋白质将使用2D凝胶电泳，质谱和肽测序鉴定。鉴定出的cDNA将通过免疫荧光表达，纯化并确认为双层研究中的Golac和高尔基蛋白。 （3）研究通过候选分子和细胞因子对高尔基通道的调节。从临床角度来看，越来越多的疾病分类为离子通道病。某些人类疾病可能是由于内源性高尔基通道的突变引起的。