Biogenic Amine Transporters: Mechanisms of Ligand Interaction.

描述（由申请人提供）：多巴胺转运蛋白（DAT）清除细胞外多巴胺，从而限制其释放后的寿命。清除过程包括DAT将多巴胺从多巴胺能神经元的外部转运到内部。DAT是精神兴奋剂药物的靶点，精神兴奋剂药物作为DA是DAT（安非他明）吸收的底物，或者是抑制底物转运的阻断剂（可卡因）。其他生物胺转运蛋白是血清素转运蛋白（SERT）和去甲肾上腺素转运蛋白（NET），它们是许多不同抗抑郁药物的靶点。这些蛋白质的结构洞察最近来自细菌同系物，亮氨酸转运蛋白LeuT的结晶，显示底物结合位点（以下称为S1）的位置，DAT，SERT和NET之间具有高度相似性的区域。新的证据表明，在LeuT的第二个底物位点（S2）的存在下，在外前庭，发挥了重要作用，在底物摄取。在反向摄取（流出）中，可以假设内前庭中存在S3位点，与摄取中的S2对称。不知道DAT、SERT或NET中是否存在S2或S3位点。我们将在结合（解离）实验以及摄取和外排试验中研究这些位点。将评估通过突变关键残基破坏S2或S3位点的影响。LeuT在初步实验中显示出DAT样性质，将与携带两个DA“头”的二价配体共结晶，使它们能够桥接S1和S2位点。此外，LeuT将与二价亮氨酸共结晶，化合物也将被设计用于桥接S1和S3。结合二价配体，如果需要与额外的修饰LeuT，可能会促进一个面向内的状态leuT，其结构的阐明一直难以捉摸的。据我们所知，在S2中还没有发现具有底物的LeuT晶体。虽然大量的证据表明DAT和其他生物胺转运蛋白的寡聚体组装，但它们的功能作用尚不清楚。我们将测试的假设，在一个寡聚体DAT大会个别原聚体不独立运作，并建立积极或消极的合作相互作用。有证据表明，生物胺转运蛋白的寡聚化和糖基化是密切相关的，但有相反的证据，这是第一个成熟的转运蛋白，因为它从细胞核到内质网高尔基体表面。我们将研究寡聚化，糖基化和运输DAT之间的关系。工具将是Lec 4细胞，产生部分糖基化的转运蛋白，一种缺乏三个N-连接的糖基化位点的构建体，以及一种DAT突变体与人类编码变异的分类，我们已经证明与多巴胺转运蛋白缺乏综合征（DTDS），一种遗传性早发性婴儿帕金森病-肌张力障碍有关。该项目的长期目标是更好地了解生物胺转运蛋白的结构-功能，这对于靶向DAT，SERT和NET的化合物的作用很重要：滥用药物，注意力缺陷多动障碍药物，环境毒素和抗抑郁药。

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) clears extracellular dopamine thereby limiting its lifetime after release. The clearance process consists of DAT translocating dopamine from the outside to the inside of dopaminergic neurons. The DAT is a target for psychostimulant drugs which, as DA, are substrates taken up by DAT (amphetamine), or are blockers inhibiting substrate translocation (cocaine). Other biogenic amine transporters are the serotonin transporter (SERT) and the norepinephrine transporter (NET), targets for many different antidepressant drugs. Structural insight into these proteins has recently come from crystallization of a bacterial homolog, the leucine transporter LeuT, showing the location of the substrate binding site (termed S1 in the following), a region with high similarity among DAT, SERT, and NET. Novel evidence indicates the presence in LeuT of a secondary substrate site (S2) in the outer vestibule, playing a fundamental role in substrate uptake. In reversed uptake (efflux), one can postulate an S3 site in the inner vestibule, in symmetry to S2 in uptake. It is nt known whether S2 or S3 sites exist in DAT, SERT, or NET. We will study these sites in binding (dissociation) experiments, and in uptake and efflux assays. The impact will be assessed of disrupting S2 or S3 sites by mutating key residues. LeuT, which in preliminary experiments shows DAT-like properties, will be co-crystallized with bivalent ligands carrying two DA "heads" enabling them to bridge S1 and S2 sites. In addition, LeuT will be co-crystallized with bivalent leucine, and compounds will also be designed for bridging S1 and S3. Binding of bivalent ligands, if needed with additional modifications of LeuT, may promote an inward-facing state of leuT, the structural elucidation of which has been elusive so far. No LeuT crystals with substrate in S2 have yet been visualized to our knowledge. Although abundant evidence points to oligomeric assemblies of DAT and other biogenic amine transporters, their functional role is not clear. We will test the hypothesis that individual protomers in an oligomeric DAT assembly are not functioning independently, and establish positive or negative cooperative interactions. There is evidence that oligomerization and glycosylation of biogenic amine transporters are intimately linked, but there is contrasting evidence as to which comes first in maturation of the transporter as it moves from nucleus to endoplasmic reticulum to Golgi to surface. We will study the relationship between oligomerization, glycosylation, and trafficking of DAT. Tools will be Lec 4 cells that produce partially glycosylated transporter, a construct that lacks the three N-linked glycosylation sites, and an assortment of DAT mutants with coding variations in humans we have shown to be linked with Dopamine Transporter Deficiency Syndrome (DTDS), an inherited early-onset infantile parkinsonism-dystonia. The long-term objectives of the project are to better understand biogenic amine transporter structure-function, important for the action of compounds that target DAT, SERT, and NET: drugs of abuse, medications for attention-deficit hyperactivity disorder, environmental toxins, and antidepressants.