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: 7773012
• 负责人: Jonathan A Javitch
• 金额: $ 67.64万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7773012
• 关键词: Accounting; Address; Affinity; Amino Acid Transporter; Amino Acids; Amphetamines; Antidepressive Agents; Archaeal Genome; Award; Bacillus (bacterium); 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; Coupling; Crystallization; Crystallography; DNA Sequence Rearrangement; Data; Detergents; Development; Disease; Drug Addiction; Drug Delivery Systems; Drug Interactions; Drug effect disorder; Epilepsy; Family; Family member; Fluorescence Spectroscopy; Fusobacteria; 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; base; design; dopamine transporter; drug of abuse; electron density; extracellular; gamma-Aminobutyric Acid; inhibitor/antagonist; insight; interdisciplinary approach; member; molecular dynamics; mutant; noradrenaline transporter; novel; psychostimulant; public health relevance; reconstitution; research study; single molecule; sodium ion; stem; stoichiometry; structural biology; success; symporter; therapy design; therapy development

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Neurotransmitter transporters are the target of psychostimulant drugs such as cocaine and amphetamine and are targets for antidepressants as well as for new drugs in development for the treatment of epilepsy and schizophrenia. Identifying the conformational dynamics of transport, the permeation pathways within the transporter, and the role of substrate and inhibitor binding are critical for understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. The powerful approaches we have established will allow us to reach this understanding and address drug action and guidelines for therapy design anchored in solid structural and functional information.

描述（由申请人提供）：神经递质：钠协同转运体（NSS）将底物的积累与钠离子沿其浓度梯度穿过质膜的运动偶联，因此构成细胞信号传导和体内平衡的关键要素。NSS包括多巴胺、5-羟色胺和去甲肾上腺素的转运蛋白-苯丙胺、可卡因和抗抑郁药物的靶点-以及GABA和甘氨酸的转运蛋白，这些转运蛋白用于癫痫和精神分裂症的治疗。在2005年，Gouaux小组在1.65 º解析了LeuT的结构，LeuT是一种细菌NSS同源物，在封闭的结合口袋（称为初级底物结合（S1）位点）中与1个Leu和2个Na+结合。该结构没有提供简单的线索，底物的途径，从细胞外或细胞内的S1位点。一个意想不到的第二个底物结合（S2）位于细胞外前庭的网站被确定在以前的项目期间，结合和通量实验表明，这两个结合位点可以同时被占用。 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“共转运效应物”的作用。“由于三环类抗抑郁药（TCA）与S2位点的结合方式不同，它们不会促进底物从S1位点释放，因此可以作为同向解偶联剂抑制转运。确定与运输相关的构象变化和在转运蛋白内形成的渗透途径是本项目的长期目标，这对于理解人类神经递质转运蛋白的功能机制以及药物如何作用于这些机制至关重要。为了实现这一目标，基于与研究人员的积极合作，开发了一种综合方法，这些研究人员在计算建模（Harel Weinstein），膜蛋白晶体学（Poul Nissen）和单分子荧光光谱学（Scott Blanchard）方面的专业知识使本申请中描述的组合多学科方法成为可能。提出了以下具体目标：1）利用我们关于S2结合的特异性和调节的新发现，通过去污剂，突变和离子取代，开发使我们能够理解S2结合位点对LeuT性质的调节的条件，并解决LeuT与S2位点结合的底物的结构。这将提供原子分辨率的数据，告知我们的机械假说，在运输的底物结合到这个网站的重要作用。2)根据转运蛋白中的特定构象变化表征底物转运机制，该构象变化将底物结合至S2位点触发的变构信号传播至转运蛋白的细胞内门，并允许底物向内释放。3)为了确定我们在细菌转运蛋白中的结构和功能发现与理解SERT和DAT功能的相关性，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，从而阐明Cl-在SERT和DAT中的功能作用。神经递质转运蛋白是可卡因和安非他明等精神兴奋剂药物的靶点，也是抗抑郁药以及正在开发的治疗癫痫和精神分裂症的新药的靶点。确定运输的构象动力学，运输内的渗透途径，以及底物和抑制剂结合的作用对于理解人类神经递质转运蛋白的功能机制以及药物如何作用于这些机制至关重要。我们已经建立的强大的方法将使我们能够达到这种理解，并解决药物作用和治疗设计的指导方针锚定在坚实的结构和功能信息。