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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.