Dopamine Transporters: Mechanisms of Ligand Interaction

多巴胺转运蛋白：配体相互作用的机制

• 批准号: 7286036
• 负责人: MAARTEN E REITH
• 金额: $ 28.49万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286036
• 关键词: Address; Amphetamine Dependence; Attention deficit hyperactivity disorder; Binding; Binding Sites; Biological Assay; Biotinylation; Catechols; Cell membrane; Cells; Chemicals; Cocaine; Condition; Cysteine; Dendrites; Development; Disease; Dopamine; Extracellular Space; Face; Family; Functional disorder; Gilles de la Tourette syndrome; Glycine; Goals; Human; Intracellular Transport; Ions; Lead; Ligands; Link; Mediating; Membrane; Membrane Proteins; Mental disorders; Modification; Molecular; Mutation; Nerve Degeneration; Neurons; Neurotransmitters; Parkinson Disease; Parkinsonian Disorders; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Play; Preparation; Proline; Proteins; Role; Schizophrenia; Serotonin; Side; Site-Directed Mutagenesis; Sodium; Structure; Structure-Activity Relationship; Substance Addiction; Sulfhydryl Reagents; Testing; Therapeutic Intervention; Toxin; analog; crosslink; dopamine transporter; dopaminergic neuron; established cell line; gamma-Aminobutyric Acid; member; monomer; mutant; neurotoxic; neurotransmission; noradrenaline transporter; poly(L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10)); protein structure; psychostimulant; symporter; tool; uptake; voltage

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.

描述（由申请人提供）：多巴胺（DA）转运蛋白（DAT）是一种膜蛋白，通过以Na+依赖性方式介导DA的摄取和反向转运（释放）来调节多巴胺能神经传递。我们希望阐明靶向DAT的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，例如精神刺激剂（可卡因和苯丙胺）依赖性，神经变性，神经变性，以及精神疾病。该提案中的具体目的是：1）表征DAT活动与细胞内Na+水平之间的关系。假设是在去极化条件下的细胞内Na+水平，因此Na+通道的作用调节DAT活动。 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制备，化学修饰和功能测定法，从底物的质膜作用的哪一侧开始，我们将结构性研究与位置指导的诱变相结合，以探索底物，以供底物和DAT，涉及外部和内部启动的结构性确定性。底物的易位，可卡因类似物的结合，构象变化以及底物诱导的DAT内在化。我们将使用共表达两种不同DAT蛋白的单元格来确定用于结合和传输的最小函数单元，以探索间之间的相互作用对DAT构象变化的贡献，并解决寡聚化与底物诱导的DAT内部化的关系。计划的研究将提供有关DAT在蛋白质结构以及底物型精神刺激剂或帕金森氏症诱导毒素的分子机制方面的功能的信息，并可能导致对可能是DAT研究的工具的不对称作用底物的鉴定。结果还可能有助于了解与精神疾病有关的密切相关的5-羟色胺和去甲肾上腺素转运蛋白。