MOLECULAR MECHANISMS OF TRANSMITTER RELEASE

本研究项目的目的是与法国研究小组合作，阐明神经递质释放的分子机制。本研究主要集中在1）囊泡募集和启动的机制，2）囊泡对Ca^<2+>信号应答的融合机制等方面，取得了以下研究成果.囊泡募集和启动的机制：1）我们认为多磷酸肌醇与突触结合蛋白C2结构域的结合是胞吐的抑制性钳。我们阐明了在嗜铬细胞中，结合的肌醇多磷酸在对Ca^<2+>的反应中从突触结合蛋白上解离。显微注射肌醇六磷酸可抑制诱发的和自发的胞吐，而肌醇六硫酸盐则不能抑制胞吐。2）我们阐明了质膜下肌动蛋白网络和肌动蛋白-肌球蛋白相互作用对囊泡的募集和启动是必不可少的。我们还发现，激活的蛋白激酶C（PKC）α或PKC β及其受体分子RACK 1与皮层F-actin之间的复合物介导了蛋白激酶C（PKC）启动的易化作用。1）对PC 12细胞亚克隆的研究表明，突触融合蛋白、VAMP-2、SNAP-25和Munc-18的表达降低，胞吐活性降低。2）对突触传递的研究阐明了SNARE蛋白的再循环以及SNARE蛋白与N型Ca^2+通道的结合。还发现Doc 2 α与Munc 13 -1的结合先于胞吐作用。

The aim of this research project is to elucidate the molecular mechanism of neurotransmitter release in collaboration with French group. The studies focused on 1) the mechanism of vesicle recruitment and priming, 2) the mechanism of fusion of vesicles in response to Ca^<2+> signals, and we have obtained the following results.1. The mechanism of vesicle recruitment and priming.1) We have suggested that binding of inositol polyphosphates to C2 domain of synaptotagmin acts as an inhibitory clamp for the exocytosis. We clarified that the bound inositol polyphosphates dissociate from synaptotagmin in response to Ca^<2+> in chromaffin cells. Microinjected inositol hexakisphosphate inhibited both the evoked and spontaneous exocytosis, while inositol hexakissulfate failed to inhibit.2) We clarified that the subplasmalemmal actin network and actin-myosin interaction are essential for the vesicle recruitment and priming. We also found that the facilitation of the priming by protein kinase C (PKC) is mediated through the complex between the activated PKC α or PKC β, their receptor molecule RACK1 and cortical F-actin.2. The mechanism of fusion of vesicles in response to Ca^<2+> signals.1) Studies with subclones of PC12 cells demonstrated that the reduced expression of syntaxin, VAMP-2, SNAP-25 and Munc-18 decreased the exocytotic activity.2) Studies on synaptic transmission clarified the recycle of SNARE proteins and binding of SNARE proteins with N-type Ca^<2+>-channels. It was also found that binding of Doc2 α to Munc13-1 precedes exocytosis.