Transport and Inhibition in a Biogenic Amine Transporter

生物胺转运蛋白的转运和抑制

• 批准号: 7451429
• 负责人: SATINDER Kaur SINGH
• 金额: $ 8.92万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7451429
• 关键词: Adverse effects; Affect; Antibodies; Antidepressive Agents; Area; Bacteria; Bacterial Proteins; Binding; Binding Sites; Biogenic Amines; Biological Assay; Caenorhabditis elegans; Chromosome Pairing; Class; Clinic; Clinical; Cocaine; Complex; Coupled; Coupling; Crystallization; Crystallography; Cysteine; Cytoplasm; DNA Sequence Rearrangement; Data; Disease; Dissociation; Dopamine; Drug Design; Drug Interactions; Drug resistance; Elements; Exhibits; Family member; Fluorescence; Functional disorder; Genetic Polymorphism; Goals; Homologous Gene; Human; Incubated; Insecta; Integral Membrane Protein; Investigation; Ions; Kinetics; Laboratories; Leucine; Light; Location; Mammalian Cell; Modeling; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Movement; Neurons; Neurotransmitters; Norepinephrine; Numbers; Organism; Phase; Play; Proteins; Psychotropic Drugs; Pump; Range; Rate; Research Personnel; Resistance; Resolution; Role; Screening procedure; Serotonin; Shapes; Signal Transduction; Site-Directed Mutagenesis; Sodium; Solutions; Structure; Synapses; Synaptic Transmission; System; Techniques; Testing; Therapeutic; Time; Urination; Vestibule; Work; X-Ray Crystallography; base; crosslink; design; extracellular; falls; in vivo; inhibitor/antagonist; insight; member; molecular modeling; monomer; neuropsychiatry; presynaptic; programs; protein expression; radioligand; research study; small molecule; symporter

DESCRIPTION (provided by applicant): Na?dependent neurotransmitter transporters or neurotransmitter sodium symporters (NSS) are integral membrane proteins that play a critical role in terminating synaptic transmission and, thus, in shaping the duration and magnitude of synaptic signaling. They work by coupling preexisting ion gradients to the thermodynamically unfavorable movement of small molecule neurotransmitters from the synapse to neuronal and glial cytoplasms. Pumps for the biogenic amines are of particular significance because their dysfunction has been implicated in a number of debilitating neuropsychiatric diseases and because they are the target of a broad array of psychoactive agents such as antidepressants and cocaine. Despite such clinical importance, atomic-level detail into the mechanism of substrate translocation and inhibition has remained elusive. Structure/function studies of LeuT, a prokaryotic NSS member, has revealed critical elements on both topics, but because the identity between this bacterial protein and the eukaryotic counterparts is so low, the comparisons will necessarily be limited. The goal of this application is to determine if the molecular models of transport and inhibition advanced for LeuT also apply to the eukaryotic NSS members. This will be accomplished by screening known biogenic amine transporters in heterologous expression systems for monodispersity and their propensity for crystallization. A representative member will then be crystallized, and its structure solved. Structure-based hypotheses of transport and inhibition will subsequently be tested with a combination of site-directed mutagenesis, cysteine crosslinking, steady-state kinetics, dissociation/association kinetics, and crystallography. While there is certainly much about LeuT that seems to be reflected in eukaryotic NSS counterparts, other areas fall short, so data from these experiments will fill a critical intellectual void. In addition, and perhaps more importantly, structural studies of a eukaryotic biogenic amine transporter will permit further investigation into specific antagonist binding sites and perhaps into the molecular basis for drug resistance, thereby opening the way to rational drug design efforts. When eventually coupled with in vivo work beyond the scope of this application, structure/function studies may also shed light on the molecular underpinnings of disease-causing polymorphisms. Achieving any one of these objectives would likely have significant impact in both the laboratory and in the clinic.

描述（由申请人提供）：NA？依赖性神经递质转运蛋白或神经递质钠钠象征（NSS）是积分的膜蛋白，在终止突触传播方面起着至关重要的作用，因此，在塑造突触信号的持续时间和幅度中。它们是通过将先前存在的离子梯度耦合到小分子神经递质从突触到神经元和神经胶质细胞肿瘤的热力学不利运动的工作起作用。生物胺的泵具有特殊意义，因为它们的功能障碍与许多使人衰弱的神经精神疾病有关，并且因为它们是众多精神活性剂（例如抗抑郁药和可卡因）的靶标。尽管如此临床重要性，但原子水平的细节涉及底物易位和抑制作用的机制。原核生物NSS成员Leut的结构/功能研究已经揭示了这两个主题的关键要素，但是因为这种细菌蛋白与真核对应物之间的身份如此之低，所以比较必须受到限制。该应用的目的是确定LEUT的转运和抑制分子模型是否也适用于真核NSS成员。这将通过在异源表达系统中筛选已知生物胺转运蛋白的单分散性及其结晶倾向来实现。然后，代表成员将结晶，其结构已解决。基于结构的转运和抑制假设将通过位置定向的诱变，半胱氨酸交联，稳态动力学，分离/关联动力学和晶体学结合进行测试。虽然关于Leut的很多肯定会反映在真核NSS对应物中，但其他领域却短缺，因此来自这些实验的数据将填补关键的知识分子空白。此外，也许更重要的是，对真核生物胺转运蛋白的结构研究将允许进一​​步研究特定的拮抗剂结合位点，并可能成为耐药性的分子基础，从而为有理药物设计工作开辟了道路。当最终与该应用范围之外的体内工作结合在一起时，结构/功能研究也可能阐明了引起疾病的多态性的分子基础。实现这些目标中的任何一个都可能对实验室和诊所产生重大影响。