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，因此取决于H+电化学驱动力（？H+）。然而，不同的发射机在不同程度上取决于？H+，化学成分（？pH）和电气组件（？？），我们目前对调节基因表达的因素知之甚少？H+也是？pH值还是？？以前的研究几乎完全集中在氯的表达的作用？pH值，但负责的载体和促进的因素？仍然知之甚少。此外，囊泡谷氨酸转运体（VGLUT）显示变构调节氯，本身可能表现出氯电导，但这些属性之间的关系仍然未知。我们也了解很少的离子耦合的VGLUT的机制，尽管重要的影响突触前调节的量子大小和谷氨酸受体的激活。因此，我们将1）阐明驱动囊泡谷氨酸转运的因素，通过促进表达？H+为？？。囊泡谷氨酸转运主要依赖于？？，我们最近发现了一种阳离子/H+交换活性，pH值变为？？并促进囊泡充满谷氨酸。该活性具有与Na+/H+交换剂家族相关的性质，我们现在将确定负责的同种型，并表征它们在递质释放中的作用。2)表征与VGLUT 2相关的电流。为了开发一个更强大的VGLUT活性测定，我们表达了一个内吞缺陷的突变体VGLUT 2在非洲爪蟾卵母细胞的质膜。我们现在将使用相关的电流来理解VGLUT的离子耦合和归因于它们的不同性质之间的关系，包括Na+依赖性磷酸盐转运，Cl-电导和Cl-的变构调节。3)鉴定负责不同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转运，我们将使用VGLUT 2和纯化的细菌亲戚E. coli DgoT.通过纯化蛋白的功能重建，我们发现DgoT介导H+共转运，而不是？？-驱动运输特性的VGLUT，我们现在将确定这些差异的运输机制的结构基础。 公共卫生相关性：突触传递涉及充满递质的囊泡的受调节的胞吐作用，但我们仍然对调节神经递质转运到突触囊泡的基本机制知之甚少。该计划将确定促进膜电位驱动囊泡谷氨酸转运的因素。它还将通过相关电流和细菌相对的分析表征囊泡谷氨酸转运的特性。这些结果将对我们理解兴奋性神经传递、行为和神经精神疾病具有重要意义。

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.