Mechanisms of fusion and transmitter release in neurosecretion

该项目的主要长期目标是提供详细的生物物理和分子 了解内分泌细胞和神经末梢的胞吐囊泡融合和递质释放。 在电刺激时，神经末梢和内分泌细胞释放各种神经递质和 神经肽通过胞吐机制。这一神经分泌过程在 人体具有广泛的生理功能。它允许突触传递以及 荷尔蒙和神经调节剂的释放，因此是调节大脑功能的重要事件，情绪 反应、行为和其他各种生理过程。对胞吐作用的详细理解 事件对于神经分泌受损的疾病特别有意义。它还将推进我们的 了解健康和疾病中的许多其他细胞功能，因为胞吐融合事件在 在对入侵宿主生物体的细菌或寄生虫的免疫反应中起中心作用，因为 在细胞内发生的许多其他融合事件或病毒进入期间的融合事件似乎密切地使用 相关机制。 在刺激下，分泌小泡的内容物通过连接的融合小孔释放 泡状腔通向细胞外间隙。熔融气孔的形成机制和熔融气孔的形成 因此，形成融合孔的分子成分是理解融合孔形成机制的中心 胞吐。SNARE(可溶性NSF附着受体)复合体，由这些蛋白质组成 Synaptobrevin、Synaxin和SNAP-25形成一个卷曲的线圈，一个平行的四螺旋束，在 胞吐作用，可能与囊泡和质膜之间的紧密结合和融合有关。这 该提议是基于这样的假设，即融合孔由SNARE蛋白打开并扩展，但 更多的蛋白质参与融合孔的形成和扩张。 我们结合电生理测量研究了嗜铬细胞中单个囊泡的胞吐作用。 用安培测量儿茶酚胺在融合过程中释放的电导 毛孔。我们对熔融孔的选择性和熔融孔动力学进行了详细的表征 对熔融孔隙性质调节释放的机制有一个清晰的认识 来自单个囊泡的传递器。SNARE蛋白SNAP-25和VAMP2的分子调控 结合熔融孔开度、熔融孔电导、寿命 波动、选择性和递质释放决定融合孔形成、融合的分子基础 孔结构、融合孔动力学和通过融合孔释放递质的机制。神经递质和激素以及各种其他化合物在高浓度下储存在 细胞内有膜结合的细胞器，称为分泌囊。在刺激下，融合孔是 形成连接泡状腔和细胞外空间的连接，从而释放存储的分子。这 该项目旨在通过以下方式阐明融合孔的分子结构和动力学及其机制 哪些传输器通过融合孔释放。

The principle long-term objective of this project is to provide a detailed biophysical and molecular understanding of exocytotic vesicle fusion and transmitter release in endocrine cells and nerve terminals. Upon electrical stimulation nerve terminals and endocrine cells release a variety of neurotransmitters and neuropeptides by an exocytotic mechanism. This process of neurosecretion is of outstanding importance in a broad range of physiological functions in the human body. It allows for synaptic transmission as well as release of hormones and neuromodulators and is thus an essential event mediating brain function, emotional response, behavior and various other physiological processes. A detailed understanding of the exocytotic event is of particular interest for diseases where neurosecretion is impaired. It will also advance our understanding of many other cellular functions in health and disease because exocytotic fusion events play a central role in the immune response against bacteria or parasites invading a host organism and because many other fusion events that occur inside the cell or the fusion events during viral entry appear to use closely related mechanisms. Upon stimulation the contents of the secretory vesicles are released through a fusion pore that connects the vesicular lumen to the extracellular space. The mechanism by which the fusion pore is formed and the molecular components forming the fusion pore are therefore central to understanding the mechanisms of exocytosis. The SNARE (Soluble NSF Attachment REceptor) complex, composed of the proteins synaptobrevin, syntaxin and SNAP-25 forming a coiled coil, a parallel four-helix bundle, plays a key role in exocytosis and may be responsible for tight binding and fusion between vesicle and plasma membrane. This proposal is based on the hypothesis that the fusion pore is opened and expanded by SNARE proteins but that additional proteins participate in fusion pore formation and expansion. We study exocytosis of single vesicles in chromaffin cells combining electrophysiological measurements of fusion pore conductance with amperometric measurements of catecholamine release through the fusion pore. We perform a detailed characterization of the fusion pore selectivity and fusion pore dynamics to obtain a clear understanding of the mechanisms by which the fusion pore properties regulate the release of transmitter from individual vesicles. Molecular manipulations of the SNARE proteins SNAP-25 and VAMP2 are performed in conjunction with measurements of fusion pore opening, fusion pore conductance, lifetime, fluctuations, selectivity and transmitter release to determine molecular basis of fusion pore formation, fusion pore structure, fusion pore dynamics and the mechanism of transmitter release through the fusion pore. Neurotransmitters and hormones as well as various other compounds are stored at high concentration in membrane-bound organelles, called secretory vesicles, inside the cell. Upon stimulation a fusion pore is formed connecting the vesicular lumen to the extracellular space, thereby releasing the stored molecules. This project is aimed at elucidating the molecular structure and dynamics of the fusion pore and mechanism by which transmitters are released through the fusion pore.