STRUCTURE/FUNCTION OF THE HUMAN DOPAMINE TRANSPORTER

释放后，多巴胺在突触内和周围的浓度通过再摄取机制迅速降低。多巴胺转运蛋白（DAT）是在质膜上进行这种再摄取过程的蛋白质，是可卡因和相关精神兴奋剂的奖励特性和滥用潜力的主要分子靶点。DT需要细胞外Na+和Cl-，并将多巴胺的易位与这些离子的运动结合起来。尽管最近克隆了DAT和相关的神经递质转运体，但这些转运体的分子结构和转运机制尚不清楚。该项目的长期目标是确定DAT对底物易位的结构基础以及可卡因等药物对底物易位的抑制作用。我们最近的工作表明，细胞外和细胞内环残基在DAT的结构和功能中起着关键作用。我们已经在DAT中鉴定了四种内源性半胱氨酸，它们与极性巯基试剂的反应影响可卡因类似物的结合。其中两个残基位于假定的细胞质环中，这表明这些环在决定结合位点的结构方面起着关键作用。值得注意的是，可卡因或多巴胺的存在显著延缓了极性巯基试剂与这些半胱氨酸的反应，即使在试剂可以直接接触细胞内残基的膜制备中也是如此。因此，底物或抑制剂的结合改变了构象，从而改变了细胞质环的可及性。相比之下，在可卡因的存在下，位于第一跨膜段胞外末端的内源性半胱氨酸Cys90变得更容易接近，这表明该区域也发生了构象变化。因此，我们提出以下具体目标：1)通过确定每个假定的环路是细胞外还是细胞内来确定DAT的拓扑结构。2)确定环残基对可卡因和多巴胺结合以及多巴胺转运的功能作用。3)确定细胞外环之间的近似接近度。这些研究将使我们能够推断有关膜跨越段的包装，环的排列以及伴随结合和运输的这些环的构象变化的关键信息。这些信息将通过提供一个组织实验结果的结构框架来补充我们正在进行的研究，以及计划项目资助的其他组成部分。它还将提供额外的实验数据，用于测试和完善DAT和相关转运体（如血清素转运体和去甲肾上腺素转运体）的分子模型，这些转运体是各种抗抑郁药物的靶点。

After its release, the concentration of dopamine in and around the synapse is rapidly reduced by reuptake mechanisms. The dopamine transporter (DAT), the protein which carries out this reuptake process at the plasma membrane, is the major molecular target responsible for the rewarding properties and abuse potential of cocaine and related psychostimulants. DT requires extracellular Na+ and Cl- and couples the translocation of dopamine to the movement of these ions. Despite the recent cloning of DAT and related neurotransmitter transporters, the molecular structure and mechanism of transport by these transporters are poorly understood. The long-term goals of this project are to determine the structural bases of substrate translocation by DAT and of its inhibition by drugs such as cocaine. Our recent work indicates that extracellular and intracellular loop residues play a critical role in the structure and function of DAT. We have identified four endogenous cysteines in DAT, the reaction of which with polar sulfhydryl regents affects the binding of cocaine analogues. Two of these residues are in putative cytoplasmic loops, suggesting that these loops play a critical role in determining the structure of the binding site. Remarkably, the presence of cocaine or dopamine dramatically retards the reaction of polar sulfhydryl reagents with these cysteines, even in a membrane preparation in which the regents have direct access to intracellular residues. Therefore, binding of substrate or inhibitor alters the conformation and hence the accessibility of the cytoplasmic loops. In contrast, Cys90, the endogenous cysteine at the putative extracellular end of the first membrane-spanning segment becomes more accessible in the presence of cocaine, suggesting that a conformational change occurs in this region as well. Thus, we propose the following specific aims: 1) To determine the topology of DAT by ascertaining whether each putative loop is extracellular or intracellular. 2) To determine the functional role of the loop residues for cocaine and dopamine binding and for dopamine transport. 3) To determine approximately proximity between extracellular loops. These studies will allow us to infer critical information about the packaging of the membrane spanning segments, the arrangement of the loops and conformation changes in these loops which accompany binding and transport. This information will complement our ongoing studies, as well as the other components of the Program Project Grant, by providing a structural framework within which to organize the experimental results. It will also provide additional experimental data with which to test and refine molecular models of DAT and related transporters such as the serotonin transporter and norepinephrine transporters, which are targets for various antidepressant drugs.