Mechanisms of fusion and transmitter release in neurosecretion

描述（由申请人提供）：本项目的主要长期目标是对内分泌细胞和神经末梢的胞外囊泡融合和递质释放提供详细的生物物理和分子理解。在电刺激下，神经末梢和内分泌细胞通过胞外机制释放多种神经递质和神经肽。这种神经分泌过程在人体广泛的生理功能中具有突出的重要性。它允许突触传递以及激素和神经调节剂的释放，因此是介导脑功能，情绪反应，行为和各种其他生理过程的重要事件。对于神经分泌受损的疾病，详细了解胞吐事件具有特殊的意义。它还将促进我们对健康和疾病中许多其他细胞功能的理解，因为胞外融合事件在抵抗细菌或寄生虫入侵宿主生物的免疫反应中起着核心作用，因为发生在细胞内的许多其他融合事件或病毒进入过程中的融合事件似乎使用密切相关的机制。刺激后，分泌囊泡的内容物通过连接囊泡腔和细胞外间隙的融合孔释放。因此，融合孔形成的机制和形成融合孔的分子成分是理解胞吐作用机制的核心。SNARE（可溶性NSF附着受体）复合物由synaptobrevin、syntaxin和SNAP-25蛋白组成，形成一个螺旋状的平行四螺旋束，在胞外分泌中起关键作用，可能负责囊泡和质膜之间的紧密结合和融合。这一建议是基于一个假设，即融合孔是由SNARE蛋白打开和扩大的，但其他蛋白参与融合孔的形成和扩大。我们结合融合孔电导的电生理测量和通过融合孔释放儿茶酚胺的电流测量，研究了嗜铬细胞中单个囊泡的胞吐作用。我们对融合孔的选择性和融合孔动力学进行了详细的表征，以清楚地了解融合孔特性调节单个囊泡释放递质的机制。对SNARE蛋白SNAP-25和VAMP2进行分子操作，并测量融合孔的开度、融合孔的电导、寿命、波动、选择性和传递物释放，以确定融合孔形成的分子基础、融合孔结构、融合孔动力学和通过融合孔释放传递物的机制。公共卫生相关性：神经递质和激素以及各种其他化合物以高浓度储存在细胞内称为分泌囊泡的膜结合细胞器中。刺激后形成融合孔，将囊泡腔连接到细胞外空间，从而释放储存的分子。本项目旨在阐明融合孔的分子结构和动力学，以及通过融合孔释放递质的机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.