Dopamine Transporters: Mechanisms of Ligand Interaction

描述(申请人提供)：多巴胺(DA)转运体(DAT)是一种膜蛋白，通过Na+依赖的方式介导DA的摄取和反向运输(释放)来调节多巴胺能神经传递。我们希望阐明靶向DAT的物质的结合和移位的分子细节，作为开发涉及DAT的疾病治疗干预的第一步，例如精神刺激剂(可卡因和苯丙胺)依赖、神经变性和精神障碍。这一建议的具体目的是：1)表征DAT活性与细胞内Na+水平的关系。该假说认为去极化状态下的细胞内Na+水平以及Na+通道的作用调节DAT的活性。我们将把同时表达DAT和电压门控Na+通道的细胞内Na“水平与DAT结合和转运DA的能力联系起来，并探讨细胞内Na+诱导的构象变化。2)确定了不同底物在DAT中的作用侧向。假设具有修饰的儿茶酚部分的底物与DA的不同之处在于它们从DAT的细胞内侧访问结合位置的能力。我们将使用不同的DAT准备、化学修饰和功能分析来研究底物的质膜作用是从哪一侧开始的；我们将结合结构活性研究和定点突变来探索底物和DAT涉及外部和内部访问的结构决定因素。3)评价DAT齐聚的功能作用。假设DAT的寡聚体结构在底物的不对称结合和移位、可卡因类似物的结合、构象变化以及底物诱导的DAT内化中发挥作用。我们将利用共表达两种不同DAT蛋白的细胞来确定结合和运输的最小功能单位，探索单体间相互作用对DAT构象变化的贡献，并探讨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与蛋白质结构相关的功能以及底物类型精神刺激剂或帕金森诱发毒素的分子机制的信息，并可能导致识别可作为DAT研究工具的不对称作用底物。研究结果也可能有助于理解与精神疾病密切相关的5-羟色胺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.