ACETYLCHOLINE TRANSPORTER OF SYNAPTIC VESICLES

突触小泡的乙酰胆碱转运蛋白

• 批准号: 2416262
• 负责人: STANLEY MONROE PARSONS
• 金额: $ 16.39万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-03-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2416262
• 关键词: Torpedo; acetylcholine; chemical binding; cholinergic receptors; complementary DNA; laboratory rat; membrane transport proteins; molecular cloning; molecular site; protein sequence; synaptic vesicles; synthetic peptide; tissue /cell culture

DESCRIPTION: (adapted from the applicant's abstract) This project is studying the acetylcholine transporter of synaptic vesicles. The transporter exchanges protons pumped into the vesicles with cytoplasmic acetylcholine (ACh) in order to store ACh for evoked release from cholinergic nerve terminals. Biochemical and molecular biological techniques will be used to probe the structure- function relationship of the vesicular ACh transporter (VAChT). The long range aim is to understand how the activity of the VAChT is regulated in the hoe that a specific pharmacology can be developed which will allow beneficial intervention in diseases of cholinergic insufficiency. The VAChT from a number of species recently was cloned. The availability of these clones will be exploited to investigate the relationship of structure to function in this transporter. The applicants wish to descover the residues that bind protons on the inside of the vesicles and move them through the membrane, the residues that bind ACh outside of the vesicles and move it through the membrane and the residues that bind vesamicol. The first aim is to raise antisera against several synthetic polypeptides specified by the cDNA sequence of the cloned rat VAChT. They will characterize the specificity of the antibodies and the side of the vesicular membrane that they bind to. The second aim is to complete sequencing of cDNA specifying the Torpedo californica VAChT so that we will know the full amino acid sequence of the VAChT, on which most biochemical work to date has been done. The third aim is to purify and sequence photo-affinity labeled peptides of the Torpedo californica VAChT that form part of the ACh and vesamicol binding sites. The fourth aim is to develop a system expressing rat VAChT under circumstances that allow quantitation of kinetic parameters and ligand-binding properties. Expression in both a mammalian cell line and in secretory pathway mutants of yeast will be tested. Expressed VAChT will be quantitatively characterized as to subcellular targeting, level of expression, ACh active transport and ligand binding. The fifth aim is to identify important residues in VAChT. Classical protein chemistry will be applied to expressed VAChT from different species and rat VAChT will be mutagenized at selected sites. The properties of the mutants will be quantitated using the expression system developed in aim 4. The resulting data will allow substantial specification of amino acids critical to specific functions in the VAChT.

描述：（改编自申请人的摘要）该项目是 研究突触小泡的乙酰胆碱转运蛋白。 这 转运蛋白与细胞质交换泵入囊泡的质子 乙酰胆碱（ACh），以储存乙酰胆碱以诱发释放 胆碱能神经末梢。 生物化学和分子生物学 技术将用于探究结构-功能关系 囊泡乙酰胆碱转运蛋白 (VAChT)。 长期目标是 了解 VAChT 的活性如何在锄头中受到调节 可以开发一种特定的药理学，这将有助于 干预胆碱能不足疾病。 最近从许多物种中克隆了 VAChT。 可用性 这些克隆将被用来研究 在此转运蛋白中发挥作用的结构。 申请人希望 发现囊泡内部结合质子的残基 并将它们移过膜，与膜外结合乙酰胆碱的残基 囊泡并使其穿过膜和残留物 结合维沙考。 第一个目标是提高针对多种病毒的抗血清 由克隆大鼠的 cDNA 序列指定的合成多肽 VAChT。 他们将表征抗体的特异性和 它们结合的囊泡膜的一侧。 第二个目标是 对加州鱼雷 VAChT 的 cDNA 进行完整测序 我们将知道 VAChT 的完整氨基酸序列，其中 迄今为止大部分生化工作已经完成。 第三个目的是净化 以及加州鱼雷光亲和标记肽的序列 VAChT 构成 ACh 和 vesamicol 结合位点的一部分。 第四个 目的是开发一种在以下情况下表达大鼠 VAChT 的系统： 允许对动力学参数和配体结合特性进行定量。 在哺乳动物细胞系和分泌途径突变体中表达 将测试酵母。 表达的 VAChT 将被定量 表征为亚细胞靶向、表达水平、ACh 主动运输和配体结合。 第五个目标是确定 VAChT 中的重要残留物。 经典的蛋白质化学将是 应用于来自不同物种和大鼠 VAChT 的表达 VAChT 在选定的位点进行诱变。 突变体的特性将是 使用目标 4 中开发的表达系统进行定量。 所得数据将允许对氨基酸进行实质性说明 对 VAChT 中的特定功能至关重要。