Transport and Inhibition in a Biogenic Amine Transporter

生物胺转运蛋白的转运和抑制

• 批准号: 8066133
• 负责人: SATINDER Kaur SINGH
• 金额: $ 24.9万
• 依托单位: YALE 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 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8066133
• 关键词: Adverse effects; Affect; Antibodies; Antidepressive Agents; Area; Bacteria; Bacterial Proteins; Binding; Binding Sites; Biogenic Amines; Biological Assay; Caenorhabditis elegans; 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 Models; Molecular Sieve Chromatography; Movement; Neurons; Neurotransmitters; Norepinephrine; Organism; Phase; Play; Proteins; Psychotropic Drugs; Pump; Resistance; Resolution; Role; Screening procedure; Serotonin; Shapes; Signal Transduction; Site-Directed Mutagenesis; Sodium; Solutions; Structure; Synapses; Synaptic Transmission; System; Techniques; Testing; Therapeutic; Time; Vestibule; Work; X-Ray Crystallography; base; crosslink; design; extracellular; falls; in vivo; inhibitor/antagonist; insight; member; molecular modeling; monomer; neuropsychiatry; presynaptic; 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）：钠依赖性神经递质转运蛋白或神经递质钠同向转运蛋白（NSS）是完整的膜蛋白，在终止突触传递中发挥关键作用，从而在塑造突触信号传导的持续时间和强度中发挥关键作用。它们的工作原理是将预先存在的离子梯度与小分子神经递质从突触到神经元和神经胶质细胞质的热力学不利运动耦合起来。生物胺泵具有特别重要的意义，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，并且因为它们是抗抑郁药和可卡因等多种精神活性药物的目标。尽管具有如此的临床重要性，但底物易位和抑制机制的原子级细节仍然难以捉摸。原核 NSS 成员 LeuT 的结构/功能研究揭示了这两个主题的关键要素，但由于这种细菌蛋白与真核对应物之间的同一性如此之低，因此比较必然受到限制。本申请的目的是确定针对 LeuT 的转运和抑制的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的生物胺转运蛋白的单分散性及其结晶倾向来实现。然后，一个代表性成员将被结晶，其结构也将被解析。随后将结合定点诱变、半胱氨酸交联、稳态动力学、解离/缔合动力学和晶体学来测试基于结构的转运和抑制假设。虽然 LeuT 的很多内容似乎在真核 NSS 对应物中得到了反映，但其他领域还存在不足，因此这些实验的数据将填补一个关键的知识空白。此外，也许更重要的是，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，并可能研究耐药性的分子基础，从而为合理的药物设计工作开辟道路。当最终与超出本应用范围的体内工作相结合时，结构/功能研究也可能揭示致病多态性的分子基础。实现这些目标中的任何一个都可能对实验室和临床产生重大影响。