ACETYLCHOLINE TRANSPORTER OF SYNAPTIC VESICLES

描述：（改编自申请人的摘要）该项目是 研究突触囊泡的乙酰胆碱转运体 的 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ACh， 胆碱能神经末梢 生物化学和分子生物学 技术将被用来探讨结构-功能关系， 囊泡乙酰胆碱转运体（VAChT）。 长期目标是 了解VAChT的活性是如何调节的锄头， 可以开发出一种特定的药理学， 胆碱能功能不全疾病的干预。 最近克隆了许多物种的VAChT。 的可用性 这些克隆将被用来调查的关系， 在这个传送器中发挥作用。 申请人希望 揭示了囊泡内部结合质子的残基 并使它们穿过细胞膜， 并使其穿过膜和残留物， 结合维索霉素。 第一个目标是产生抗几种 由克隆大鼠的cDNA序列指定的合成多肽 VAChT。 它们将表征抗体的特异性， 它们结合在囊泡膜的一侧。 第二个目标是 完整的cDNA序列，指定的鱼雷加州VAChT， 我们将知道VAChT的完整氨基酸序列， 迄今为止，大多数生物化学工作已经完成。 第三个目的是净化 并对加利福尼亚电鳐的光亲和标记肽进行测序 形成ACh和vesamicol结合位点的一部分的VAChT。 第四 目的是开发一种表达大鼠VAChT的系统， 允许定量动力学参数和配体结合特性。 在哺乳动物细胞系和分泌途径突变体中的表达 酵母将被测试。 表达的VAChT将定量 其特征在于亚细胞靶向、表达水平、ACh 主动转运和配体结合。 第五个目标是确定 VAChT中的重要残基。 经典的蛋白质化学 应用于来自不同物种的表达的VAChT，大鼠VAChT将被 在选定的位点进行诱变。 变种人的特性 使用在AIM 4中开发的表达系统定量。 的 所得数据将允许氨基酸的实质性规格 对VAChT中的特定功能至关重要。

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.