Psychostimulant Recognition by Serotonin Transporters

描述(由申请人提供)：这项提案的广泛长期目标是确定5-羟色胺转运体(SERT)识别精神刺激剂(可卡因和苯丙胺)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。这个项目与健康有直接的关系，因为SERTS是大多数抗抑郁药物以及许多滥用药物的分子靶标。抗抑郁药如帕罗西汀、氟西汀和丙咪嗪与转运体结合并抑制5-羟色胺的摄取，从而延长神经递质的细胞外寿命。被滥用的精神刺激剂可卡因和苯丙胺也与转运体结合，但具有不同的药理作用，导致滥用潜力和可能的神经毒性。尽管在20世纪90年代初S克隆了SERT，但围绕该转运蛋白功能的分子和细胞神经生物学仍未完全阐明。该项目使用多学科方法，旨在显著提高关于SERTS精神刺激剂的分子药理学的知识。这些研究将使用多种技术，包括在哺乳动物细胞中表达和鉴定重组转运蛋白、取代半胱氨酸可及性方法(SCAM)、实时荧光底物分析、电生理学和定点突变，以鉴定与特定转运蛋白功能相关的氨基酸，包括拮抗剂和底物结合。此外，计算机辅助的可卡因和苯丙胺衍生物的结构-活性研究将与诱变相结合，试图确定直接的配体-转运体相互作用。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)鉴定和阐明氨基酸在参与识别精神刺激剂的核心结构域中的作用。拟议的策略将提供关键的新信息，将蛋白质结构与转运体的功能特性联系起来，并加强对精神治疗和滥用药物的分子作用机制的理解。滥用兴奋剂，如可卡因和安非他明(包括甲基苯丙胺(“Meth”)和摇头丸或“摇头丸”)，引起了社会的极大关注。拟议的研究将揭示有关这些滥用药物如何改变其大脑靶标(即5-羟色胺转运体)的重要新信息，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.