Structural Basis of Vesicular Neurotransmitter Transport

囊泡神经递质运输的结构基础

基本信息

批准号：
9258506
负责人：
ROBERT H EDWARDS
金额：
$ 61.06万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/9258506
关键词：
Allosteric Regulation Anions Artificial Membranes Bacteria Bacterial Proteins Behavior Binding Biological Assay Carrier Proteins Cells Chemicals Chimeric Proteins Chlorides Coupling Crystallization Crystallography Dependence Detergents Diffusion Electrophysiology (science)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 driving force genetic manipulation improved in vivo member mutant neuropsychiatric disorder neurotransmitter transport novel pH gradient programs protein reconstitution protein structure protein transport public health relevance radiotracer reconstitution screening sialic acid permease thermophilic organism tool vacuolar H+-ATPase vapor

项目摘要

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（由适用提供）：将所有经典发射器转运到突触蔬菜中，取决于真空h+-ATPase产生的外向定向的H+电化学驱动力（µH+）。然而，囊泡谷氨酸的转运与其他经典发射器的囊泡转运不同，几乎完全依赖于该梯度（）而不是化学梯度（PH）的电动成分。实际上，尚不清楚囊泡谷氨酸转运蛋白（VGLUTS）介质H+交换是否是否。它们可以简单地催化制备的扩散，甚至充当阴离子通道。相比之下，与唾液酸相关的小型转运蛋白sialin催化了H+的电up-utral共转移，而SLC17家族的两个成员如何介导这种明显不同的活动仍然未知。然而，据报道，唾液素可以介导囊泡谷氨酸的转运，这表明这两种不同的活性反映了一种共同的潜在机制。该计划的长期目标是了解SLC17家族如何赋予驱动的差异和H+共同运动。该策略是确定该家族中蛋白质的结构，并使用此信息来指导机制研究。筛选许多与vgluts相关的细菌蛋白，我们已经确定了一种可以在多种不同条件下结晶的细菌蛋白，并且在脂质立方相中衍射为3.7Å。我们还将重组蛋白重构为人造膜，并表明它催化了H+的有机阴离子的共转运，类似于Sialin。现在，我们建议1）在原子分辨率下完善DGOT的结构； 2）确定不同功能状态（包括底物结合）的DGOT的结构； 3）测试特定残差的作用是由结构在底物识别和H+运动中实现的； 4）确定后生VGLUT的结构。结果将有助于我们了解一种类别的运输蛋白，甚至一种蛋白质如何以明显不同的方式与H+电化学驱动力融为一体。同时，结构分析应阐明氯化物对vgluts的变构调节的机制，氯化物的变型（氯化物）保持不足，而H+是我们最近发现的。鉴定具有改变性质的突变体还为我们提供了通过基因操纵在体外和N vivo中测试这些性质的物理作用的工具。