Transport of Neurotransmitter Into Synaptic Vesicles

描述（由申请人提供）：通过胞吐作用调节神经递质释放取决于递质进入神经分泌囊泡的转运。我们之前发现，几种所谓的I型磷酸盐转运蛋白实际上将谷氨酸转运到突触囊泡中，为使用谷氨酸作为递质的神经元提供了第一个明确的标记。泡状谷氨酸转运蛋白（VGLUT1） 1和VGLUT2在成人大脑中表现出几乎互斥的表达模式，VGLUT1在皮层和海马中表达，而VGLUT2在丘脑和脑干中表达。这两种异构体在内在运输活性上没有明显差异，但对VGLUT1敲除小鼠的分析表明，这两种异构体对高频刺激的反应存在差异，可能是由于膜运输的差异。我们现在已经发现VGLUT1包含多个内化基序，其中只有一个与VGLUT2共享，并且我们已经开始确定与这些序列相互作用的蛋白质。此外，VGLUT1敲除表明，VGLUT2在皮层和海马神经元中具有短暂的发育作用，注定在成年期只产生VGLUT1。与VGLUT1和vglut2相反，VGLUT1是由一些传统上不被认为释放谷氨酸的细胞群表达的，包括纹状体的胆碱能中间神经元，皮层和海马的gaba能中间神经元，血清素和多巴胺神经元。该项目的长期目标是了解谷氨酸的释放如何影响神经发育、突触传递和行为。该策略是利用生物物理方法研究VGLUTs的功能和运输，并通过基因操作在小鼠体内确定其作用。在具体目标方面，我们将：1)表征VGLUT1的蛋白相互作用。为了了解蛋白质相互作用如何影响VGLUT1的运输，我们将在活神经元中使用光学成像。2)确定瞬时VGLUT2表达在发育过程中的作用。我们将使用条件VGLUT2敲除小鼠来确定VGLUT2早期表达对轴突引导、突触发育、功能和可塑性的影响。3)确定谷氨酸释放在表达VGLUTS细胞群中的作用。我们将使用VGLUTS敲除和BAG转基因小鼠，用GFP标记VGLUT3+神经元，以评估不同神经系统中VGLUTS表达的生理和行为作用。我们还将产生一个条件敲除，以区分不同神经元表达的VGLUTS的行为效应。

DESCRIPTION (provided by applicant): The regulated release of neurotransmitter by exocytosis depends on transport of the transmitter into neurosecretory vesicles. We previously found that several so-called type I phosphate transporters in fact transport glutamate into synaptic vesicles, providing the first unambiguous markers for neurons that use glutamate as a transmitter. Vesicular glutamate transporters (VGLUTs) 1 and 2 exhibit an almost mutually exclusive pattern of expression in the adult brain, with VGLUT1 in cortex and hippocampus, and VGLUT2 in the thalamus and brainstem. The isoforms do not apparently differ in intrinsic transport activity, but the analysis of VGLUT1 knock-out mice suggests differences in response of the two isoforms to high frequency stimulation, presumably due to differences in membrane trafficking. We have now found that VGLUT1 contains multiple internalization motifs, only one of which is shared with VGLUT2, and we have begun to identify the proteins that interact with these sequences. In addition, the VGLUT1 knock-out has suggested a transient, developmental role for VGLUT2 in cortical and hippocampal neurons destined to make only VGLUT1 in adulthood. In contrast to VGLUT1 and 2, VGLUTS is expressed by a number of cell populations not traditionally considered to release glutamate, including cholinergic interneurons of the striatum, GABAergic interneurons in the cortex and hippocampus, serotonin and perhaps dopamine neurons. The long-term objectives of this program are to understand how the release of glutamate influences neural development, synaptic transmission and behavior. The strategy is to study the function and trafficking of VGLUTs using biophysical methods, and to determine their role in vivo by genetic manipulation in mice. In terms of specific aims, we will: 1) Characterize the protein interactions of VGLUT1. To understand how protein interactions influence the trafficking of VGLUT1, we will use optical imaging in live neurons. 2) Determine the role of transient VGLUT2 expression during development. We will use conditional VGLUT2 knock-out mice to determine the effects of early VGLUT2 expression on axon guidance, synapse development, function and plasticity. 3) Identify the roles of glutamate release by cell populations expressing VGLUTS. We will use VGLUTS knock-out and BAG transgenic mice that label VGLUT3+ neurons with GFP to assess the physiological and behavioral roles of VGLUTS expression by diverse neural systems. We will also produce a conditional knock-out to distinguish among the behavioral effects of VGLUTS expressed by different neurons.