Molecular mechanisms of bacterial homologs of neurotransmitter:sodium symporters

神经递质细菌同系物的分子机制：钠转运体

• 批准号: 8477155
• 负责人: Jonathan A Javitch
• 金额: $ 60.53万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8477155
• 关键词: Accounting; Address; Affinity; Amino Acid Transporter; Amino Acids; Amphetamines; Antidepressive Agents; Archaeal Genome; Award; Bacillus (bacterium); Bacterial Genome; Binding; Binding Sites; Biochemical; Biogenic Amine Neurotransmitters; Biogenic Amines; Biological; Biological Assay; Biological Models; Cell membrane; Chloride Ion; Chlorides; Cocaine; Collaborations; Computer Analysis; Computer Simulation; Coupled; Coupling; Crystallization; Crystallography; DNA Sequence Rearrangement; Data; Detergents; Development; Disease; Drug Addiction; Drug Interactions; Drug Targeting; Drug abuse; Drug effect disorder; Epilepsy; Family; Family member; Fluorescence Spectroscopy; Fusobacterium; GABA transporter; Glean; Glycine; Goals; Grant; Guidelines; Health; Homeostasis; Homologous Gene; Human; Indium; Investments; Ion Cotransport; Ions; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Molecular Target; Monitor; Movement; Mutation; Neurotransmitters; Norepinephrine; Nutrient; Orphan; Pathway interactions; Pharmaceutical Preparations; Property; Proteins; Publishing; Regulation; Research Personnel; Resolution; Rewards; Role; Schizophrenia; Serotonin; Side; Signal Transduction; Site; Sodium; Solid; Specificity; Structure; Substrate Interaction; Testing; Tricyclic Antidepressive Agents; Tryptophan; Tyrosine; Vestibule; Work; abstracting; base; design; dopamine transporter; drug of abuse; electron density; experimental analysis; extracellular; gamma-Aminobutyric Acid; inhibitor/antagonist; insight; interdisciplinary approach; member; molecular dynamics; mutant; noradrenaline transporter; novel; psychostimulant; reconstitution; research study; serotonin transporter; single molecule; sodium ion; stem; stoichiometry; structural biology; success; symporter; therapy design; therapy development

6. Project Summary/Abstract Neurotransmitter:sodium symporters (NSS) couple the accumulation of substrate to the movement of sodium ions down their concentration gradient across the plasma membrane, and as such constitute key elements in cellular signaling and homeostasis. NSS include the transporters for dopamine, serotonin and norepinephrine-targets for amphetamine, cocaine, and antidepressant drugs-as well as the transporters for GABA and glycine, which are targeted for treatment of epilepsy and schizophrenia. In 2005 the Gouaux group solved at 1.65 ¿ the structure of LeuT, a bacterial NSS homolog, crystallized with 1 Leu and 2 Na+ bound in an occluded binding pocket (referred to as primary substrate binding (S1) site). The structure provided no easy clues to the pathway of substrate to the S1 site from the extracellular or the intracellular side. An unexpected second substrate binding (S2) site located in the extracellular vestibule was identified during the previous project period; binding and flux experiments showed that the two binding sites can be occupied simultaneously. Substrate in the S2 site allosterically triggers intracellular release of Na+ and substrate from the S1 site, thereby functioning as a "symport effector." Because tricyclic antidepressants (TCA) bind differently to this S2 site, they do not promote substrate release from the S1 site and thus act as symport uncouplers to inhibit transport. Identifying the conformational changes associated with transport and the permeation pathways that are formed within the transporter are long term goals of this project critical to understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. To achieve this goal, an integrated approach has been developed based on active collaborations with investigators whose expertise in computational modeling (Harel Weinstein), membrane protein crystallography (Poul Nissen), and single-molecule fluorescence spectroscopy (Scott Blanchard) enables the combined multidisciplinary approach described in this application. The following specific aims are proposed: 1) To use our novel discoveries regarding the specificity and modulation of S2 binding, by detergents, mutations, and ionic substitution, to develop conditions that enable us to understand the regulation of LeuT properties by the S2 binding site and to solve a structure of LeuT with substrate bound to the S2 site. This will provide atomic resolution data to inform our mechanistic hypothesis as to the essential role in transport of substrate binding to this site. 2) To characterize the mechanism of substrate transport in terms of specific conformational changes in the transporter that propagate the allosteric signal triggered by substrate binding to the S2-site towards the intracellular gate of the transporter and allow inward release of substrate. 3) To establish the relevance of our structural and functional findings in bacterial transporters to understanding the function of SERT and DAT. We will: a) demonstrate the essential functional role of the S2 site in these human transporters, and b) use a Cl-- dependent LeuT mutant to determine the structure of the Cl- binding site and thus to explicate the functional role of Cl- in SERT and DAT.

6.项目总结/摘要 神经递质：钠转运体（NSS）将底物的积累与钠的运动耦合起来 离子沿着它们的浓度梯度穿过质膜，因此构成了 细胞信号和体内平衡。NSS包括多巴胺、5-羟色胺和 去甲肾上腺素是安非他明、可卡因和抗抑郁药物的靶点，也是 GABA和甘氨酸，用于治疗癫痫和精神分裂症。2005年，Gouaux集团 在1.65 ° C下解析了LeuT的结构，LeuT是一种细菌NSS同系物，与1个Leu和2个Na+结合在一起结晶， 封闭的结合口袋（称为初级底物结合（S1）位点）。该结构提供了不容易 底物从细胞外或细胞内到达S1位点的途径的线索。一个意想不到 第二个底物结合（S2）位点位于细胞外前庭，在先前的研究中被鉴定出来。 结合和通量实验表明，这两个结合位点可以同时被占据。 S2位点的底物变构触发Na+和底物从S1位点的细胞内释放， 从而起到“同端口效应器”的作用。“因为三环类抗抑郁药（TCA）与S2 在S1位点，它们不促进底物从S1位点释放，因此作为共输送解偶联剂抑制 运输识别与运输和渗透途径相关的构象变化， 是该项目的长期目标，对于理解运输机的功能至关重要。 人类神经递质转运体的机制以及药物如何作用于这些机制。到 为实现这一目标，在与下列机构积极合作的基础上，制定了一项综合办法： 研究人员的专业知识在计算建模（哈雷尔温斯坦），膜蛋白晶体学 (Poul Nissen）和单分子荧光光谱法（Scott Blanchard）使组合 本申请中描述的多学科方法。提出了以下具体目标：1）使用 我们关于特异性和调节S2结合的新发现，通过去污剂，突变， 离子取代，开发条件，使我们能够了解LeuT性质的调节， S2结合位点，并解决了LeuT与结合到S2位点的底物的结构。这将提供原子 分辨率数据通知我们的机制假设，在运输的底物结合到 本网站2)根据特定构象变化表征底物转运机制 在转运蛋白中，通过底物与S2-位点的结合，将变构信号传递到 转运蛋白的细胞内门控并允许底物的向内释放。3)为了确定我们的 细菌转运蛋白的结构和功能发现，以了解SERT和DAT的功能。我们 将：a）证明S2位点在这些人类转运蛋白中的基本功能作用，和B）使用Cl-- 依赖性LeuT突变体来确定Cl-结合位点的结构，从而解释其功能 Cl-在SERT和DAT中的作用。

项目成果

Structure-activity relationships for a novel series of citalopram (1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile) analogues at monoamine transporters.

• DOI: 10.1021/jm1005034
• 发表时间: 2010-08-26
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Zhang P;Cyriac G;Kopajtic T;Zhao Y;Javitch JA;Katz JL;Newman AH
• 通讯作者: Newman AH