ACETYLCHOLINE TRANSPORTER OF SYNAPTIC VESICLES

描述：(改编自申请者的摘要)这个项目是 研究突触小泡的乙酰胆碱转运体。这个 转运蛋白将泵入小泡的质子与细胞质交换 乙酰胆碱(ACh)以储存ACh以从 胆碱能神经末梢。生化与分子生物学 将使用技术来探索结构与功能的关系 泡状ACh转运体(Vacht)。远景目标是 了解Vacht的活动是如何在锄头中进行调节的 可以开发一种特定的药理学，它将使有益的 胆碱能缺乏症的干预。 最近，一些物种的游艇被克隆出来。可得性 这些克隆中的一个将被用来研究 结构以在此传送器中发挥作用。申请者希望 发现囊泡内侧结合质子的残留物 并将它们通过膜，结合在外面的残基 并使其通过膜和残留物 捆绑维沙米考。第一个目标是提高对几个 克隆大鼠cDNA序列所特有的合成多肽 瓦希特。它们将表征抗体的特异性和 它们与之结合的囊泡膜的一侧。第二个目标是 鱼雷Calfornica Vacht So基因的全序列测定 我们将知道游艇的完整氨基酸序列，在它上面 到目前为止，大多数生化工作已经完成。第三个目标是净化 和序列光亲和标记肽的鱼雷 Vacht形成ACh和维沙米考结合位点的一部分。第四次 目的是开发一种在以下情况下表达大鼠游艇的系统 允许量化动力学参数和配体结合性质。 哺乳动物细胞系和分泌途径突变体中的表达 将进行酵母菌测试。快递的Vacht将是定量的 具有亚细胞靶向性、表达水平、ACh的特征 主动转运和配体结合。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 适用于表达不同物种的Vacht和大鼠Vacht 在选定的地点进行了诱变。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。 由此产生的数据将使氨基酸的实质性规范 对游艇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.