Membrane Trafficking of Vesicular Neurotransmitter Transporters

描述(申请人提供)：突触传递涉及通过调节神经递质从突触小泡释放，将电信号转化为化学信号。高频率的突触传递依赖于释放后神经递质的循环回到神经末梢，在那里它被位于囊泡膜的囊泡神经递质转运蛋白重新包装成突触小泡。神经递质的重复释放还需要在神经末梢局部回收突触小泡膜和蛋白质的机制。突触囊泡循环动力学的变化可能影响神经递质输出的数量和模式，从而有助于信息处理和突触可塑性。囊泡在功能池中的分布由它们的位置和融合概率决定，也可以影响重复放电时神经递质的释放。对于突触囊泡成分在胞吐后的循环，已经提出了多种途径，但这些途径与不同突触囊泡池的关系尚不清楚。突触囊泡蛋白被认为是作为一个单位进行再循环的。然而，新的数据表明，与不同的内吞适配器蛋白结合的不同可能会影响单个突触小泡蛋白的运输，从而影响突触小泡的组成，从而影响它们的功能特征。这项研究的长期目标是了解单个囊泡蛋白的膜运输如何影响突触小泡的蛋白质组成、突触小泡池的维持以及特定回路对递质的释放。这项建议的策略是结合生物化学、活细胞成像、电生理学和电子显微镜来研究突触小泡蛋白的运输。囊泡神经递质转运体与pH敏感的荧光蛋白的融合为研究蛋白质相互作用和调控机制对囊泡转运体蛋白循环途径的影响提供了光学探针。这项建议的具体目的是研究囊泡谷氨酸转运体VGLUT1和VGLUT 2的运输调控，1)表征两种密切相关的异构体运输的差异，2)确定蛋白质决定因素，3)VGLUT循环的生化和细胞机制。神经递质释放的调节可能是治疗干预的重要途径，也是开发更好的治疗癫痫、精神分裂症和焦虑症等神经精神疾病治疗的新靶点。

DESCRIPTION (provided by applicant): Synaptic transmission involves the transformation of electrical into chemical signals by the regulated release of neurotransmitter from synaptic vesicles. High frequency synaptic transmission depends on the recycling of neurotransmitter back into the nerve terminal after release, where it is re-packaged into synaptic vesicles by vesicular neurotransmitter transporter proteins located in the vesicle membrane. Repetitive neurotransmitter release also requires mechanisms to recycle synaptic vesicle membrane and proteins locally at the nerve terminal. Variation in the kinetics of synaptic vesicle recycling may shape the amount and pattern of neurotransmitter output, and hence contribute to information processing and synaptic plasticity. The distribution of vesicles in functional pools, defined by their location and fusion probability, can also influence neurotransmitter release in response to repetitive firing. Multiple pathways have been proposed for the recycling of synaptic vesicle components after exocytosis, but the relationship of these pathways to the different synaptic vesicle pools has remained unclear. Synaptic vesicle proteins have been assumed to undergo recycling as a unit. However, emerging data indicates that differences in the association with distinct endocytic adaptor proteins may influence the trafficking of individual synaptic vesicle proteins, affecting the composition of synaptic vesicles and hence their functional characteristics. The long-term goal of the proposed research is to understand how membrane trafficking of individual vesicular proteins influences the protein composition of synaptic vesicles, the maintenance of synaptic vesicle pools, and the release of transmitter by specific circuits. The strategy of this proposal is to study the trafficking of synaptic vesicle proteins by a combination of biochemistry, live cell imaging, electrophysiology, and electron microscopy. Fusions of vesicular neurotransmitter transporters with pH-sensitive fluorescent proteins provide optical probes to study the effect of protein interactions and regulatory mechanisms on the recycling pathways of vesicular transporter proteins. The specific aims of this proposal are designed to study the regulation of trafficking of vesicular glutamate transporters VGLUT1 and 2 by 1) characterizing differences in the trafficking of two closely related isoforms, 2) identifying the protein determinants and 3) biochemical and cellular mechanisms underlying VGLUT recycling. Regulation of neurotransmitter release may be an important approach to therapeutic intervention and the molecular machinery new targets for the development of better treatments for neuropsychiatric disorders such as epilepsy, schizophrenia, and anxiety disorders.