Transport of Neurotransmitter Into Synaptic Vesicles

摘要 所有经典的神经递质进入突触囊泡的运输都涉及一个 H+交换机制，因此取决于H+电化学驱动力（<$H+） 由液泡型H+-ATP酶产生。然而，不同的发射器取决于不同的 对<$$> H+的两个组分，化学组分（<$pH）和电 成分（），我们目前对调节表达的因子知之甚少。 H+为pH或pH。以前的研究几乎完全集中在 氯化物在表达的pH值，但负责的载体和促进的因素， 仍然知之甚少。此外，囊泡谷氨酸转运蛋白（VGLUT）显示， 氯离子的变构调节，本身可能表现出氯离子电导，但 这些属性之间的关系仍然未知。我们也不太了解 VGLUT的离子耦合机制，尽管对突触前 量子大小的调节和谷氨酸受体的激活。因此我们将 1)阐明通过促进谷氨酸受体的表达来驱动囊泡谷氨酸转运的因素。 H+也是。囊泡谷氨酸转运主要依赖于谷氨酸，我们最近 确定了阳离子/H+交换活性，将pH值转化为pH值，并促进囊泡填充 谷氨酸盐该活性具有与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，以及表征它们在传递中的作用。 release. 2)表征与VGLUT 2相关的电流。为了开发一种更耐用的检测方法， VGLUT活性，我们已经表达了一个内吞缺陷的VGLUT 2突变体在血浆中， 非洲爪蟾卵母细胞膜。我们现在将使用相关的电流来理解 离子耦合的VGLUT和不同属性之间的关系归因于 它们包括Na+依赖的磷酸盐转运、Cl-传导和别构调节 的Cl-。 3)通过不同的VGLUT家族鉴定负责离子偶联差异的残基 成员为了了解密切相关的蛋白质如何介导不同蛋白质的转运， 离子偶联机制，我们将使用VGLUT 2的定点突变和纯化的 细菌近缘种E. coli DgoT.通过纯化蛋白的功能重建，我们得到 发现DgoT介导H+协同运输，而不是由H2驱动的运输特征。 VGLUT，我们现在将确定这些运输差异的结构基础 机制

Abstract 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.