Structural Basis of Vesicular Neurotransmitter Transport

囊泡神经递质运输的结构基础

• 批准号: 8964141
• 负责人: ROBERT H EDWARDS
• 金额: $ 65.13万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964141
• 关键词: Allosteric Regulation; Anions; Artificial Membranes; Bacteria; Bacterial Proteins; Behavior; Binding; Biological Assay; Carrier Proteins; Cells; Chemicals; Chimeric Proteins; Chloride Ion; Chlorides; Coupling; Crystallization; Crystallography; Dependence; Detergents; Diffusion; Disease; Environment; Escherichia coli; Exhibits; Exocytosis; Family; Fluorescence; Glutamate Transporter; Glutamates; Homologous Gene; In Vitro; Inorganic Phosphate Transporter; Insecta; Ion Cotransport; Lipids; Liposomes; Lysosomes; Measurement; Mediating; Membrane; Membrane Potentials; Methods; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Monitor; Movement; Mutagenesis; Mutation; Neurotransmitters; Phase; Physiological; Post-Translational Protein Processing; Production; Property; Protein Family; Proteins; Recombinant Proteins; Recycling; Regulation; Reporting; Resolution; Role; Sialic Acids; Structure; Synaptic Transmission; Synaptic Vesicles; System; Testing; Time; Vesicle; Vesicle Transport Pathway; Work; base; driving force; genetic manipulation; improved; in vivo; member; mutant; neuropsychiatry; neurotransmitter transport; novel; pH gradient; programs; protein structure; public health relevance; radiotracer; reconstitution; screening; sialic acid permease; thermophilic organism; tool; vacuolar H+-ATPase; vapor

 DESCRIPTION (provided by applicant): The transport of all classical transmitters into synaptic vesicles depends on an outwardly directed H+ electrochemical driving force (µH+) produced by the vacuolar H+-ATPase. However, vesicular glutamate transport differs from the vesicular transport of other classical transmitters, and relies almost entirely on the electrical component o this gradient () rather than the chemical gradient (pH). Indeed, it remains unclear whethe the vesicular glutamate transporters (VGLUTs) mediate H+ exchange at all. They may simply catalyze facilitated diffusion, or even function as anion channels. In contrast, the closely relate transporter sialin catalyzes the electroneutral cotransport of H+ with sialic acid, and it remains unknown how two members of the SLC17 family can mediate such apparently different activities. However, sialin has also been reported to mediate vesicular glutamate transport, suggesting that the two different activities reflect a common underlying mechanism. The long-term objective of this program is to understand how the SLC17 family confers both -driven diffusion and H+ cotransport. The strategy is to determine the structure of proteins in this family and use this information to guide studies of mechanism. Screening a number of bacterial proteins related to the VGLUTs, we have identified one that can be crystallized under a number of different conditions, and that diffracts to 3.7 Å in the lipidic cubic phase. We have also reconstituted the recombinant protein into artificial membranes and shown that it catalyzes the cotransport of an organic anion with H+, similar to sialin. We now propose to 1) refine the structure of DgoT at atomic resolution; 2) determine the structure of DgoT in different functional states, including substrate-bound; 3) test the role of specific residues implicated by the structur in substrate recognition and H+ movement; and 4) determine the structure of a metazoan VGLUT. The results will help us to understand how one class of transport proteins and perhaps even one protein can couple in apparently different ways to the H+ electrochemical driving force. At the same time, structural analysis should illuminate the mechanism for allosteric regulation of the VGLUTs by chloride, which remains poorly understood, and by H+, which we have recently discovered. The identification of mutants with altered properties also provides us with tools to test the physiological role of these properties by genetic manipulation in vitro and n vivo.

 描述（由申请人提供）：所有经典递质向突触小泡的运输取决于液泡 H+-ATP 酶产生的向外定向的 H+ 电化学驱动力 (μH+)。然而，囊泡谷氨酸转运不同于其他经典递质的囊泡转运，并且几乎完全依赖于该梯度的电分量()而不是化学梯度(pH)。事实上，目前尚不清楚囊泡谷氨酸转运蛋白 (VGLUT) 是否介导 H+ 交换。它们可能只是催化促进扩散，甚至充当阴离子通道。相比之下，密切相关的转运蛋白唾液酸蛋白催化 H+ 与唾液酸的电中性共转运，目前尚不清楚 SLC17 家族的两个成员如何介导这种明显不同的活动。然而，唾液酸蛋白也被报道可以介导囊泡谷氨酸转运，这表明这两种不同的活性反映了一个共同的潜在机制。该计划的长期目标是了解 SLC17 系列如何实现  驱动的扩散和 H+ 共转运。该策略是确定该家族中蛋白质的结构，并利用该信息来指导机制研究。通过筛选多种与 VGLUT 相关的细菌蛋白，我们发现了一种可以在多种不同条件下结晶的蛋白，并且在脂质立方相中衍射至 3.7 Å。我们还将重组蛋白重构到人工膜中，并表明它可以催化有机阴离子与 H+ 的共转运，类似于唾液酸蛋白。我们现在建议1）在原子分辨率上精炼DgoT的结构； 2) 确定DgoT在不同功能状态下的结构，包括底物结合状态； 3) 测试结构所涉及的特定残基在底物识别和H+移动中的作用； 4)确定后生动物VGLUT的结构。这些结果将帮助我们了解一类转运蛋白甚至一种蛋白质如何以明显不同的方式与 H+ 电化学驱动力耦合。同时，结构分析应该阐明 VGLUT 的变构调节机制，即氯化物（目前仍知之甚少）和 H+（我们最近发现的）。具有改变特性的突变体的鉴定还为我们提供了通过体外和体内遗传操作来测试这些特性的生理作用的工具。