Transport of Neurotransmitter Into Synaptic Vesicles

描述（由申请人提供）：所有经典神经递质进入突触囊泡的运输涉及H+交换机制，因此依赖于H+电化学驱动力(？由液泡型H+- atp酶产生的¿H+)。然而，不同的发射机在不同程度上依赖于？H+，化学成分(？pH)和电气成分（??），目前我们对调节？谁也不喜欢？pH值还是？？以前的研究几乎完全集中在氯在？pH，但负责的载体和促进的因素？？仍然知之甚少。此外，泡状谷氨酸转运蛋白（VGLUTs）表现出氯离子的变构调节，并可能表现出氯离子电导，但这些性质之间的关系尚不清楚。尽管对量子大小的突触前调节和谷氨酸受体的激活具有重要意义，但我们对VGLUTs离子偶联的机制知之甚少。因此，我们将1)阐明通过促进？H+ as ?？水疱谷氨酸运输主要依靠？？，我们最近发现了一种阳离子/H+交换活性，转化为？pH值？？促进谷氨酸囊泡充盈。该活性具有与Na+/H+交换器家族相关的特性，我们现在将确定负责的同工异构体，以及表征它们在传递体释放中的作用。2)表征与VGLUT2相关的电流。为了开发一种更可靠的VGLUT活性检测方法，我们在爪蟾卵母细胞的质膜上表达了VGLUT2的内吞缺陷突变体。我们现在将使用相关电流来了解vglut的离子偶联以及归因于它们的不同性质之间的关系，包括Na+依赖的磷酸盐运输，Cl-电导和Cl-的变构调节。3)确定不同VGLUT家族成员离子偶联差异的残基。为了了解密切相关的蛋白质如何通过不同的离子偶联机制介导运输，我们将使用VGLUT2和纯化的细菌亲戚E. coli DgoT的定点诱变。通过对纯化蛋白的功能重构，我们发现DgoT介导的是H+共转运，而不是H+共转运。驱动的输运特性，我们现在将确定这些输运机制差异的结构基础。

DESCRIPTION (provided by applicant): The transport of all classical neurotransmitters into synaptic vesicles involves a mechanism of H+ exchange and hence depends on a H+ electrochemical driving force (?¿H+) generated by the vacuolar-type H+-ATPase. However, different transmitters depend to varying extents on the two components of ?¿H+, the chemical component (?pH) and the electrical component (??), and we currently understand little about the factors that regulate expression of ?¿H+ as either ?pH or ??. Previous studies have focused almost exclusively on the role of chloride in expression of ?pH, but the carriers responsible and the factors that promote ?? remain poorly understood. In addition, the vesicular glutamate transporters (VGLUTs) show allosteric regulation by chloride and may themselves exhibit a chloride conductance, but the relationship between these properties remains unknown. We also understand little about the mechanism of ionic coupling by the VGLUTs, despite important implications for the presynaptic regulation of quantal size and the activation of glutamate receptors. We will thus 1) Elucidate the factors that drive vesicular glutamate transport by promoting expression of ?¿H+ as ??. Vesicular glutamate transport depends primarily on ??, and we have recently identified a cation/H+ exchange activity that converts ?pH into ?? and promotes vesicle filling with glutamate. The activity has properties associated with the family of Na+/H+ exchangers and we will now identify the isoform(s) responsible, as well as characterize their role in transmitter release. 2) Characterize the currents associated with VGLUT2. To develop a more robust assay for VGLUT activity, we have expressed an endocytosis-defective mutant of VGLUT2 at the plasma membrane of Xenopus oocytes. We will now use the associated currents to understand the ionic coupling of the VGLUTs and the relationship between different properties ascribed to them, including Na+-dependent phosphate transport, a Cl- conductance and allosteric regulation by Cl-. 3) Identify residues responsible for the differences in ionic coupling by different VGLUT family members. To understand how closely related proteins can mediate transport with different mechanisms of ionic coupling, we will use site-directed mutagenesis of VGLUT2 and a purified bacterial relative, E. coli DgoT. By functional reconstitution of the purified protein, we have found that DgoT mediates H+ cotransport rather than the ??-driven transport characteristic of the VGLUTs, and we will now determine the structural basis for these differences in transport mechanism. PUBLIC HEALTH RELEVANCE: Synaptic transmission involves the regulated exocytosis of vesicles filled with transmitter, but we still understand little about the basic mechanisms that regulate neurotransmitter transport into synaptic vesicles. This program will identify factors that promote the membrane potential driving vesicular glutamate transport. It will also characterize the properties of vesicular glutamate transport through the analysis of associated currents and a bacterial relative. The results will have important implications for our understanding of excitatory neurotransmission, behavior and neuropsychiatric disease.