Mechanisms of fusion and transmitter release in neurosecretion

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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.