Psychostimulant Recognition by Serotonin Transporters

描述（由申请人提供）：本提案的广泛长期目标是确定5-羟色胺转运蛋白（SERT）识别精神兴奋剂（可卡因和安非他明）的分子决定因素，从而揭示涉及SERT功能和药物调节的基本机制。该项目与健康直接相关，因为SERT是大多数抗抑郁药物以及许多滥用药物的分子靶点。抗抑郁药如帕罗西汀、氟西汀和丙咪嗪与转运蛋白结合并抑制5-羟色胺摄取，从而延长神经递质的细胞外寿命。滥用的精神兴奋剂可卡因和安非他明也与转运蛋白结合，但具有不同的药理作用，导致滥用潜力和可能的神经毒性。尽管SERT在20世纪90年代早期被克隆，但围绕这种转运蛋白功能的分子和细胞神经生物学仍有待充分阐明。该项目采用多学科的方法，旨在显着推进有关SERT精神兴奋剂的分子药理学知识。这些研究将使用多种技术，包括哺乳动物细胞中重组转运蛋白的表达和表征、取代半胱氨酸可及性方法（SCAM）、实时荧光底物测定、电生理学和定点诱变，以鉴定与特定转运蛋白功能（包括拮抗剂和底物结合）相关的氨基酸。此外，计算机辅助可卡因和安非他明衍生物的结构活性研究将与诱变结合起来，试图确定直接的配体-转运蛋白相互作用。因此，本项目的具体目标是：1）确定形成底物渗透途径的核心结构域，2）阐明SERT结构域的方向信息，3）鉴定和阐明与精神兴奋剂识别相关的核心结构域中氨基酸的作用。拟议的战略将提供关键的新信息连接蛋白质结构的转运蛋白的功能特性，并提高对精神治疗和滥用药物的分子作用机制的理解。滥用可卡因和安非他明（包括甲基安非他明（“Meth”）和摇头丸或“迷魂药”）等兴奋剂引起社会极大关注。拟议中的研究将揭示关于这些滥用药物如何改变其大脑靶点的重要新信息（即，5-羟色胺转运体），并提供新的策略来对抗这些物质的成瘾特性。

DESCRIPTION (provided by applicant): The broad long-term objectives of this proposal are to identify the molecular determinants of psychostimulant recognition (cocaine and amphetamine) by serotonin transporters (SERTs), thereby revealing fundamental mechanisms involved SERT function and modulation by drugs. This project has direct health relatedness as SERTs are the molecular target for most antidepressant drugs as well as many drugs of abuse. Antidepressants such as paroxetine, fluoxetine, and imipramine bind to the transporter and inhibit serotonin uptake, thereby prolonging the extracellular lifespan of the neurotransmitter. The abused psychostimulants cocaine and amphetamine also bind to the transporter, but have distinct pharmacologic effects leading to abuse potential and possible neurotoxicity. Despite the cloning of SERT in the early 1990's, the molecular and cellular neurobiology surrounding the function of this transport protein remains to be fully elucidated. This project uses a multidisciplinary approach aimed at significantly advancing knowledge regarding the molecular pharmacology of psychostimulants at SERTs. The studies will use multiple techniques including expression and characterization of recombinant transporters in mammalian cells, the substituted cysteine accessibility method (SCAM), real-time fluorescent substrate assays, electrophysiology, and site-directed mutagenesis to identify amino acids involved with specific transporter functions including antagonist and substrate binding. In addition, computer-assisted structure-activity studies of cocaine and amphetamine derivatives will be coupled with mutagenesis in an attempt to identify direct ligand-transporter interactions. Therefore, the specific aims of this project are: 1) To determine the core domains forming the substrate permeation pathway, 2) To elucidate information about the orientation of SERT domains, and 3) To identify and elucidate the role of amino acids in the core domains involved with recognition of psychostimulants. The proposed strategies will provide critical new information linking protein structure to functional properties of the transporter and an enhanced understanding of the molecular mechanisms of action for psychotherapeutic and abused drugs. Abused stimulants such as cocaine and amphetamine (including methamphetamine ("Meth") and MDMA or 'ecstasy') are of great societal concern. The proposed studies will reveal important new information on how these abused drugs alter their target in the brain (i.e., the serotonin transporter) and offer new strategies to combat the addictive properties of these substances.