The role of V-type ATPase in synaptic vesicle fusion

V型ATP酶在突触小泡融合中的作用

• 批准号: 6835954
• 负责人: GLEN G ERNSTROM
• 金额: $ 4.3万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6835954
• 关键词: Caenorhabditis elegans; acidity /alkalinity; adenosinetriphosphatase; alternatives to animals in research; calcium flux; electrophysiology; exocytosis; fluorescent dye /probe; gene mutation; intermolecular interaction; membrane fusion; motor neurons; neurotransmitter transport; postdoctoral investigator; protein structure function; synaptic vesicles

DESCRIPTION (provided by applicant): Synaptic vesicle exocytosis is a special case of membrane fusion. Despite extensive research in neurons and other cells, controversies remain concerning the basic mechanism of vesicle fusion. Two models have been proposed to describe the process of how vesicles fuse with a target membrane. In one model, cognate SNARE proteins on apposed membranes form a trans complex that is necessary and sufficient to catalyze the fusion of the two membranes. In a second model, fusion is initiated by protein-protein interactions between integral membrane V0 sectors of the vacuolar-type ATPase in apposed membranes. The goal of this proposal is to test the hypothesis that V0 proteins are involved in synaptic vesicle fusion in the nematode C. elegans. To test this we will characterize the impact vacuolar ATPase mutations have on the fusion of synaptic vesicles. We will use with electrophysiological imaging techniques to assay vesicle fusion at the neuromuscular junction. Since the V0 sector is involved with acidifying synaptic vesicles when associated with the V1 ATPase sector, we will also examine whether the genetic mutants in the V0 solely affect vesicle acidification. This approach will resolve a major controversy in synaptic vesicle exocytosis; specifically whether the V-ATPase participates solely function in neurotransmitter loading or whether it has in additional role in priming and/or fusion. First, using a vesicle-targeted pH-sensitive GFP, we will measure the effects V-ATPase mutations have on vesicle acidification. Second, we will measure spontaneous postsynaptic miniature currents to determine whether V0 mutations decrease the frequency of vesicle fusion. Third, fusion rates in V-ATPase mutants will be measured more directly by measuring the exocytosis of fluorescent dye tracer FM4-64 from the membranes of stained vesicles.

描述（由申请人提供）：突触囊泡胞吐作用是膜融合的一种特殊情况。尽管对神经元和其他细胞进行了广泛的研究，但关于囊泡融合的基本机制，仍有争议。已经提出了两个模型来描述囊泡如何与靶膜融合的过程。在一个模型中，融合膜上的同源snare蛋白形成了一种反式复合物，这是必要且足以催化两个膜的融合的复合物。在第二个模型中，融合是由粘液型ATPase的整体膜V0扇区之间的蛋白质 - 蛋白质相互作用引发的。该提案的目的是检验以下假设：V0蛋白参与线虫C.秀隐杆线虫中的突触囊泡融合。为了测试这一点，我们将表征液泡ATPase突变对突触囊泡融合的影响。我们将与电生理成像技术一起在神经肌肉连接处测定囊泡融合。由于V0扇区与V1 ATPase扇区相关时与酸化突触囊泡有关，因此我们还将检查V0中的遗传突变体是否仅影响囊泡酸化。这种方法将解决突触囊泡胞吐作用的重大争议。具体而言，V-ATPase是否仅参与神经递质加载中的功能，还是它在启动和/或融合中具有其他作用。首先，使用囊泡靶向的pH敏感GFP，我们将测量V-ATPase突变对囊泡酸化的影响。其次，我们将测量自发的突触后微型电流，以确定V0突变是否会降低囊泡融合的频率。第三，通过测量从染色囊泡的膜上测量荧光染料示踪剂FM4-64的胞吐作用，可以更直接地测量V-ATPase突变体中的融合速率。