Dopamine Transporters: Mechanisms of Ligand Interaction

描述（由申请人提供）：多巴胺（DA）转运蛋白（DAT）是一种膜蛋白，通过Na+依赖的方式介导DA的摄取和反向转运（释放）来调节多巴胺能神经传递。我们希望阐明控制靶向DAT的物质结合和易位的分子细节，作为开发涉及DAT的疾病的治疗干预措施的第一步，如精神兴奋剂（可卡因和安非他明）依赖、神经变性和精神疾病。本文的具体目的是：1)表征DAT活性与细胞内Na+水平之间的关系。假设是在去极化条件下细胞内Na+水平和Na+通道的作用调节DAT活性。我们将把表达DAT和电压门控Na+通道的细胞内的Na"水平与DAT结合和运输DA的能力联系起来；我们将探讨细胞内Na+引起的构象变化。2)描述各种底物在DAT中的作用。假设是具有修饰的儿茶酚部分的底物与DA在从DAT的细胞内侧进入结合位点的能力上不同。我们将使用不同的DAT制剂，化学修饰和功能分析来研究底物的质膜作用是从哪一边开始的；我们将结合结构-活性研究和定点诱变来探索底物和DAT的外部和内部通路的结构决定因素。3)评价DAT寡聚化的功能作用。该假说认为，DAT的寡聚结构在底物的不对称结合和易位、可卡因类似物的结合、构象变化和底物诱导的DAT内化中发挥作用。我们将使用共表达两种不同DAT蛋白的细胞来确定结合和运输的最小功能单元，探索单体间相互作用对DAT构象变化的贡献，并解决寡聚化与底物诱导的DAT内化的关系。计划中的研究将提供有关DAT与蛋白质结构的功能以及底物型精神兴奋剂或帕金森诱发毒素的分子机制的信息，并可能导致识别不对称作用底物，这可能是DAT研究的工具。研究结果也有助于了解与精神疾病密切相关的血清素和去甲肾上腺素转运体。

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) is a membrane protein that regulates dopaminergic neurotransmission by mediating uptake and reverse transport (release) of DA in a Na+-dependent manner. We wish to elucidate the molecular details governing binding and translocation of substances targeting DAT as a first step in the development of therapeutic interventions for diseases involving DAT, such as psychostimulant (cocaine and amphetamine) dependence, neurodegeneration, and psychiatric disorders. The specific aims in this proposal are: 1) Characterizing the relationship between DAT activity and the intracellular Na+ level. The hypothesis is that the intracellular Na+ level under depolarizing conditions and thus the action of Na+ channels regulates DAT activity. We will correlate the Na" level inside cells expressing both DAT and voltage-gated Na+ channel with the ability of DAT to bind and transport DA; and we will probe conformational changes induced by intracellular Na+. 2) Delineating sidedness for action of various substrates at DAT. The hypothesis is that substrates with a modified catechol moiety differ from DA in their ability to access the binding site from the intracellular side of DAT. We will use different DAT preparations, chemical modification, and functional assays to investigate which side of the plasma membrane action of substrates is initiated from; and we will combine structure-activity studies with site-directed mutagenesis to explore, for both substrate and DAT, the structural determinants involved in external and internal access. 3) Assessing functional role of DAT oligomerization. The hypothesis is that oligomeric structures of DAT play a role in asymmetric binding and translocation of substrates, binding of cocaine analogs, conformational changes, and substrate-induced DAT internalization. We will use cells co-expressing two different DAT proteins to determine the minimal function unit for binding and transport, to explore the contribution of inter-monomer interactions to conformational changes of DAT, and to address the relation of oligomerization to the substrate-induced DAT internalization. The planned studies will provide information on the function of DAT in relation to protein structure as well as on the molecular mechanisms of substrate-type psychostimulants or parkinsonism-inducing toxins, and may lead to identification of asymmetrically-acting substrates that could be tools for DAT studies. Results may also help understanding the closely related serotonin and norepinephrine transporters that are linked to mental illness.