ARCHAEAL & BACTERIAL HOMOLOGS OF DOPAMINE TRANSPORTER

古菌

• 批准号: 6447888
• 负责人: Jonathan A Javitch
• 金额: $ 16.35万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6447888
• 关键词: Archaea; Escherichia coli; SDS polyacrylamide gel electrophoresis; Xenopus oocyte; bacterial genetics; bacterial proteins; clone cells; dopamine transporter; gel filtration chromatography; ion transport; membrane transport proteins; molecular cloning; neurotransmitter transport; polymerase chain reaction; protein structure function; tissue /cell culture; transfection /expression vector; transport proteins; western blottings

DESCRIPTION: (provided by the applicant) Following the release of dopamine, its concentration in and around the synapse is rapidly reduced by the dopamine transporter (DAT), the major molecular target of cocaine and related psychostimulants. DAT requires extracellular sodium and chloride and couples the translocation of dopamine to the movement of these ions down their concentration gradients. Despite the cloning of DAT and related neurotransmitter transporters, its molecular structure and transport mechanisms are poorly understood. Progress in this area has been hampered by the lack of sufficient functional, purified protein and the inability to develop high-level expression systems for these proteins. Bacterial membrane proteins are generally more amenable to structural analysis and high-level expression than are their eukaryotic counterparts. We have recently identified an entire family of proteins in archaea and in bacteria that are homologous to DAT. Currently 39 proteins from 21 different organisms appear to fall into this family. None of these transporter proteins have been studied, and their substrates are unknown. In this CEBRA application, we propose to intensively explore the properties of a subset of these DAT archaeal and bacterial homologs in order to assess their suitability for use in further direct and indirect structural studies. Thus we propose the following specific aims: 1) To clone the 9 sodium- and chloride-dependent transporters from archaea and bacteria that are most similar to DAT. 2) To express these proteins heterologously in E. coli, and to assess and optimize levels of plasma membrane expression as weft as solubilization and purification conditions. 3) To express a candidate transporter(s), which based on studies in Aim 2 is suitable for further structural analysis, in Xenopus laevis oocytes to facilitate studies using electrophysiological techniques, focusing initially on sodium-dependent transient currents to determine whether the transporter(s) is inserted into the membrane and is functional. 4) To identify the substrates and/or non-substrate inhibitors of this transporter(s) by the measurement of substrate-induced currents. At the end of the period of support we will be in a position to choose a limited number of these archaeal and bacterial transporters for use in crystallization trials as a preliminary step towards obtaining a high-resolution structure. Moreover, we will also be poised to pursue biochemical and biophysical methods to acquire functional data and indirect structural information about these transporters. The resulting information will likely revolutionize our structural understanding of the function of DAT and related human neurotransmitter transporters, such as the serotonin and norepinephrine transporters that are targets for antidepressant drugs and cocaine, in a way that is only a distant prospect through continued work on the eukaryotic transporters alone.

描述：（申请人提供） 释放多巴胺后，其集中在突触中和周围 多巴胺转运蛋白（DAT）迅速降低，主要分子 可卡因和相关心理刺激剂的靶标。 DAT需要细胞外 钠和氯化物以及夫妻多巴胺向运动的易位 这些离子降低了它们的浓度梯度。尽管dat的克隆 和相关的神经递质转运蛋白，其分子结构和 运输机制知之甚少。该领域的进展一直是 由于缺乏足够的功能，纯化的蛋白质和 无法为这些蛋白质开发高级表达系统。 细菌膜蛋白通常更适合结构分析 与他们的真核对应物相比，高级表达。我们有 最近确定了古细菌和细菌中的整个蛋白质家族 与DAT同源。目前来自21种不同生物的39种蛋白质 似乎属于这个家庭。这些转运蛋白都没有 研究了，它们的底物是未知的。在此CEBRA应用中，我们 建议深入探索这些Dat Archaeal子集的属性 和细菌同源物，以评估其进一步使用的适用性 直接和间接的结构研究。因此，我们提出以下特定 目的：1）从中克隆9个钠和氯化物依赖性转运蛋白 与DAT最相似的古细菌和细菌。 2）表达这些蛋白质 在大肠杆菌中异源，并评估和优化质膜的水平 表达为纬线作为溶解和纯化条件。 3）表达 基于AIM 2的研究的候选转运蛋白适用于 进一步的结构分析，在拉维斯卵母细胞中促进研究 使用电生理技术，最初集中于钠依赖性 瞬态电流以确定转运蛋白是否插入 膜，功能性。 4）识别底物和/或非底物 通过测量底物诱导的抑制剂 电流。在支持期结束时，我们将有能力 选择有限数量的这些古细菌和细菌转运蛋白用于 结晶试验是获得获得的初步步骤 高分辨率结构。而且，我们还将有望追求 获取功能数据和间接的生化和生物物理方法 这些转运蛋白的结构信息。由此产生的信息将 可能彻底改变了我们对DAT功能和 相关的人类神经递质转运蛋白，例如5-羟色胺和 去甲肾上腺素转运蛋白是抗抑郁药的靶标 可卡因，以一种持续的工作只是遥远的前景 单独的真核转运蛋白。