Biogenic Amine Transporters: Mechanisms of Ligand Interaction

生物胺转运蛋白：配体相互作用的机制

基本信息

批准号：
8675545
负责人：
MAARTEN E REITH
金额：
$ 6.9万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8675545
关键词：
Acetylglucosamine Amphetamines Antidepressive Agents Asparagine Attention Deficit Disorder Attention deficit hyperactivity disorder Binding Binding Sites Biogenic Amines Biological Assay Carrier Proteins Cell Nucleus Cells Cocaine Code Crystallization Cytosol Data Dissociation Dopamine Drug Binding Site Drug abuse Dystonia Endoplasmic Reticulum Family Funding Golgi Apparatus Grant Head Homologous Gene Human Individual Inherited Leucine Ligands Link Location Mannose Mental Depression Modeling Modification Mutate N-Acetylglucosaminyltransferases Neurotransmitters Oligosaccharides Parkinsonian Disorders Pharmaceutical Preparations Play Positioning Attribute Process Property Protein Conformation Proteins Protomer Reagent Regulation Relative (related person)Role Site Sodium Structure Surface Syndrome Testing Toxic Environmental Substances Variant Vestibule Work crosslink design dimer dopamine transporter dopaminergic neuron drug of abuse early onset extracellular glycosylation infancy insight member methanethiosulfonate monomer mutant noradrenaline transporter novel psychostimulant research study serotonin transporter symporter tool trafficking uptake

项目摘要

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.

多巴胺转运蛋白（DAT）清除细胞外多巴胺，从而限制其释放后其寿命。清除过程包括DAT将多巴胺从外部转移到多巴胺能神经元的内部。 DAT是心理刺激药物的靶标，作为DA，是由DAT（苯丙胺）占用的底物，或者是抑制底物易位（可卡因）的阻滞剂。其他生物胺转运蛋白是5-羟色胺转运蛋白转运蛋白（SERT）和去甲肾上腺素转运蛋白（NET），是许多不同抗抑郁药的靶标。对这些蛋白质的结构洞察力最近来自细菌同源物，亮氨酸转运蛋白Leut的结晶，显示了底物结合位点的位置（称为S1中的S1），该区域在DAT，SERT和NET之间具有很高的相似性。新的证据表明，在外侧前庭中二级底物位点（S2）的存在，在底物摄取中起着基本作用。在反向摄取（外排）中，可以假定内侧前庭中的S3位点，以对称性为对称性，以摄取的S2。尚不知道S2或S3位点是否存在于DAT，SERT或NET中。我们将研究这些位点的结合（解离）实验，以及摄取和排出分析。将通过突变关键残基来评估破坏S2或S3位点的影响。 Leut在初步的实验中显示了类似DAT的特性，将与带有两个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，化合物也将用于桥接S1和S3。二价配体的结合，如果需要进行其他修改，可能会促进leut的内向状态，到目前为止，其结构阐明是难以捉摸的。据我们所知，尚未对S2中底物的Leut晶体具有可视化。尽管大量证据表明DAT和其他生物胺转运蛋白的寡聚组件，但其功能作用尚不清楚。我们将检验以下假设：低聚DAT组装中的个体毒素不独立运行，并建立正或负合作相互作用。有证据表明，生物胺转运蛋白的寡聚化和糖基是密切相关的，但是有鲜明的证据表明，当转运蛋白从核从核向内质网向高尔基体移动时，它首先是在转运蛋白成熟时出现的。我们将研究DAT的寡聚，糖基化和运输之间的关系。 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.该项目的长期目标是更好地了解生物胺转运蛋白转运蛋白结构功能，这对于靶向DAT，SERT和NET的化合物的作用很重要：滥用药物，注意力缺陷多活跃障碍，环境毒素和抗抑郁药的药物。