THE ACETYLCHOLINE TRANSPORTER OF SYNAPTIC VESICLES

描述：（改编自申请人的摘要）：囊泡乙酰胆碱 乙酰胆碱转运蛋白（VAChT）储存乙酰胆碱（ACh），用于从神经释放 终端VAChT的结构-功能分析将通过定点PCR进行。 突变，表达和转运蛋白变化的表征， 抑制剂结合，pH依赖性和ACh洗脱特性的突变体。一 VAChT运输循环的微观动力学模型将是重要的， 解释。相关囊泡单胺转运蛋白（VMAT）的序列 和细菌多药耐药性（MDR）转运蛋白将指导策略。的 目标是将特定的氨基酸分配给特定的功能。目标1： 确定两个质子易位位点中的第二个。的可能性 核心质子转运机制在VAChT和VMAT之间并不保守 将在运输救援实验中进行测试目标2旨在确定 必须去质子化以结合ACh的pKa 7.1残基和pKa > 9必须质子化运输结合ACh。目标3寻求本地化 乙酰胆碱结合位点。此外，可能的静电指导乙酰胆碱结合 将进行调查。目标4将测试六个保守的 首次在细菌MDR转运蛋白控制中发现的构象基序 VAChT运输循环中的构象变化。保守残基 也将测试基序侧翼的构象作用。项目 将确定VAChT介导或控制运输的特征，包括 那些可能限制乙酰胆碱储存的。这可能会揭示一个合适的目标 用于开发通过调节VAChT增加ACh储存的药物 功能长期目标是有效治疗胆碱能 机能减退

DESCRIPTION: (Adapted from applicant's abstract): The vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) for release from the nerve terminal. A structure-function analysis of VAChT will be done by site-directed mutagenesis, expression and characterization of changes in transporter, inhibitor binding, pH dependence and ACh washout properties of the mutants. A microscopic kinetics model of the VAChT transport cycle will be important to interpretation. The sequences of related vesicular monoamine transporter (VMAT) and bacterial multidrug resistance (mdr) transporters will guide strategy. The goal is to assign specific amino acids to specific functions. Aim 1 seeks to identify the second of the two proton-translocation sites. The possibility that the core proton-translocation mechanism is not conserved between VAChT and VMAT will be tested in transport-rescue experiments. Aim 2 seeks to identify the residue of pKa 7.1 that must be deprotonated to bind ACh and the residue of pKa > 9 that must be protonated to transport bound ACh. Aim 3 seeks to localize the ACh binding site. Also, possible electrostatic guidance for ACh binding will be investigated. Aim 4 will test whether any of six conserved conformational motifs first identified in bacterial mdr transporters control conformational changes in the VAChT transport cycle. Conserved residues flanking the motifs also will be tested for conformational roles. The project will identify features of VAChT mediating or controlling transport, including those potentially limiting to ACh storage. This might reveal a suitable target for development of a drug that increases ACh storage by modulating VAChT function. The long term goal is effective treatment of cholinergic hypofunction.