Mechanisms of fusion and transmitter release in neurosecretion

描述(申请人提供)：该项目的主要长期目标是对内分泌细胞和神经末梢的胞吐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吐机制释放各种神经递质和神经肽。这一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，因此是调节大脑功能、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，对胞吐事件的详细了解是特别有意义的。它还将促进我们对许多其他细胞功能在健康和疾病中的理解，因为胞吐融合事件在对抗细菌或寄生虫入侵宿主的免疫反应中发挥核心作用，因为许多其他发生在细胞内的融合事件或病毒进入期间的融合事件似乎使用了密切相关的机制。在刺激下，分泌囊泡的内容物通过连接囊泡腔和细胞外空间的融合孔释放。因此，形成融合孔的机制和形成融合孔的分子组成是理解胞吐作用机制的中心。SNARE(可溶性NSF附着受体)复合体由突触素、合成素和SNAP-25蛋白组成，形成一个平行的四股螺旋卷曲，在胞吐过程中起关键作用，可能与囊泡和质膜之间的紧密结合和融合有关。这一建议是基于这样的假设，即融合孔是由SNARE蛋白打开和扩大的，但额外的蛋白质参与了融合孔的形成和扩大。我们研究了嗜铬细胞中单个囊泡的胞吐作用，结合了融合孔电导的电生理测量和通过融合孔的儿茶酚胺释放的安培测量。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的性质调节递质从单个囊泡释放的机制。对SNARE蛋白SNAP-25和VAMP2进行分子操作，结合融合孔开度、融合孔电导、寿命、波动、选择性和递质释放的测量，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递质的机制。与公共卫生相关：神经递质和激素以及各种其他化合物高度储存在细胞内被称为分泌囊泡的膜结合细胞器中。在刺激时，形成一个融合孔，将泡状腔连接到细胞外空间，从而释放存储的分子。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.