Membrane Trafficking of Vesicular Neurotransmitter Transporters

描述（由申请人提供）：突触传递涉及通过从突触囊泡中调节释放神经递质将电信号转化为化学信号。高频突触传递依赖于神经递质释放后再循环回到神经末梢，在那里它被位于囊泡膜上的囊泡神经递质转运蛋白重新包装到突触囊泡中。重复的神经递质释放还需要在神经末梢局部回收突触囊泡膜和蛋白质的机制。突触囊泡循环动力学的变化可以塑造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定义，也可以影响神经递质的释放，以响应重复放电。已经提出了多个途径的突触囊泡成分胞吐后的再循环，但这些途径的关系，不同的突触囊泡池仍然不清楚。突触囊泡蛋白被认为是作为一个单位进行再循环的。然而，新出现的数据表明，在协会与不同的内吞衔接蛋白的差异可能会影响个别突触囊泡蛋白的贩运，影响突触囊泡的组成，因此其功能特性。拟议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囊泡的蛋白质组成，突触囊泡池的维持以及特定回路的递质释放。该建议的策略是通过生物化学，活细胞成像，电生理学和电子显微镜的组合来研究突触囊泡蛋白的运输。囊泡神经递质转运蛋白与pH敏感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蛋白再循环途径的影响提供了光学探针。该提案的具体目的是通过以下方式研究囊泡谷氨酸转运蛋白VGLUT 1和2的转运调控：1）表征两种密切相关亚型的转运差异，2）鉴定蛋白质决定簇和3）VGLUT再循环的生化和细胞机制。调节神经递质的释放可能是治疗干预的重要途径，分子机制可能是开发更好的神经精神疾病（如癫痫、精神分裂症和焦虑症）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.