PSYCHOSTIMULANT RECOGNITION BY SEROTONIN TRANSPORTERS

血清素转运体（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体结合，抑制血清素的摄取，从而延长神经递质在细胞外的寿命。滥用的精神兴奋剂可卡因和安非他明也与转运体结合，但具有不同的药理作用，导致滥用和可能的神经毒性。尽管在20世纪90年代早期克隆了SERT，但关于这种转运蛋白功能的分子和细胞神经生物学信息很少。该项目采用多学科方法，旨在显著推进SERTs精神兴奋剂分子药理学方面的知识。这些研究将使用多种技术，包括在哺乳动物细胞中表达和表征重组转运蛋白、电生理学、免疫印迹、嵌合蛋白的形成和位点定向诱变，以识别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安非他明衍生物的结构-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，该项目的具体目标是：1)识别参与识别SERT拮抗剂（如可卡因）的转运体结构域和残基；2)确定特定SERT结构域对底物特性（如易位、外排和通道样活性）的贡献。所提出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，并增强对精神治疗和滥用药物的分子作用机制的理解。

The serotonin transporter (SERT) is 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, very little information is available regarding the molecular and cellular neurobiology surrounding the function of this transport protein. 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, electrophysiology, immunoblotting, the formation of chimeric proteins, 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 identify transporter domains and residues involved in recognition of SERT antagonists such as cocaine and 2) to determine the contributions of specific SERT domains to substrate properties such as translocation, efflux, and channel- like activity. 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.