Transport of Neurotransmitter Into Synaptic Vesicles

摘要 所有经典神经递质进入突触小泡的运输涉及一种 H+交换的机制，因此依赖于H+电化学驱动力(？H+) 由液泡型H+-ATPase产生。然而，不同的发射器依赖于不同的 H+的两个组分--化学组分(PH)和电性 成分，我们目前对调节其表达的因素知之甚少。 H+表示为pH或。以往的研究几乎完全集中在脑血管紧张素转换酶的作用 氯化物在pH的表达中，但载体负责和促进因素 人们对此仍然知之甚少。此外，囊泡型谷氨酸转运体(VGLUT)显示 氯化物的变构调节，本身可能表现出氯化物的电导，但 这些属性之间的关系尚不清楚。我们也不太了解 VGLUT的离子耦合机制，尽管对突触前有重要的意义 量子大小的调节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是通过促进 H+As。囊泡谷氨酸的运输主要依赖于谷氨酸，最近我们发现 确定了一种阳离子/H+交换活性，它能将pH转化为pH，并促进囊泡充盈 含有谷氨酸。该活性具有与Na+/H+交换器家族相关的性质 我们现在将确定负责的异构体(S)，以及他们在递质中的作用 放手。 2)表征与VGLUT2相关的电流。为了开发一种更可靠的检测方法 VGLUT活性，我们在血浆中表达了VGLUT2的内吞缺陷突变体 非洲爪哇卵母细胞的膜。我们现在将使用相关的电流来理解 VGLUT的离子耦合及其不同性质之间的关系 它们包括钠离子依赖的磷酸盐转运、氯离子电导和变构调节。 被氯离子污染。 3)确定导致不同VGLUT家族离子偶联差异的残基 会员。为了了解密切相关的蛋白质如何通过不同的 离子偶联的机制，我们将使用VGLUT2的定点突变和一个纯化的 细菌近缘，E.coliDgoT。通过对纯化蛋白进行功能重组，我们获得了 发现DgoT介导H+共转运，而不是驱动的转运特性 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.