Doc2-munc13蛋白复合体在胞吐作用中强化囊泡启动的机理研究

胞吐作用中，脂双层膜囊泡必需经过启动（priming）才能与质膜融合。神经系统中突触囊泡的启动直接影响突触传递与可塑性。然而启动的机制却不完全清楚。申请人近期发现Doc2-munc13蛋白复合体能使囊泡进入强于正常启动的超级启动（superpriming）状态，但分子机制未知。基于初步研究结果，申请人对这种强化型启动提出如下假说：Doc2-munc13在Ca2+调控下，通过与RIM、munc18等蛋白相互作用，加强分子启动，即增加以SNARE为核心的膜融合分子机械的成熟度，实现对启动的强化，从而在兴奋性与抑制性突触中支持短时程可塑性的形成。本项目计划利用分子生物学、电生理等手段验证该假说，鉴定复合蛋白中的钙感受器结构域，确认强化启动的方式与关键性蛋白间相互作用，并深入了解其对突触可塑性的影响，从而阐明超级启动的分子机制，推进对胞吐中启动机理的认识，并为了解神经突触运作机制提供新的切入点。

During exocytosis, vesicles with lipid bilayer membranes must undergo ‘priming’ to get prepared for the fusion of vesicle membrane versus plasma membrane. In the nervous system, the priming of synaptic vesicles is directly involved in synaptic transmission and plasticity. However, the molecular mechanisms underlying vesicle priming is not fully understood. The applicant recently found that Doc2-munc13 protein complex confers vesicles a state of ‘superpriming’, which is more strengthened than normal priming, via an unknown mechanism. Based on preliminary results, the applicant proposed a hypothesis for this enhanced version of priming: Doc2-munc13 induced superpriming is mediated by Ca2+ signals, and achieved by reinforcing molecular priming (increasing the maturation level of the fusion machinery with SNARE complex as the core), depending on its interactions with RIM, munc18 and etc; this type of priming supports the formation of short-term plasticity in both excitatory and inhibitory synapses. This project plans to test this hypothesis using molecular biological and electrophysiological approaches. The Ca2+ sensing motif within the protein complex will be identify. The priming mode (molecular priming or positional priming) of the superpriming and the key protein interaction mediating superpriming will be determined. And its roles in synaptic plasticity will be further clarified. This study will enhance the understand of vesicle priming, a crucial step during exocytosis, and provide new insights for the mechanisms of the activity of neuronal synapses.