MOLECULAR MECHANISMS OF TRANSMITTER RELEASE

本研究项目与法国研究组合作，旨在阐明神经递质释放的分子机制。我们的研究主要集中在1)囊泡招募和启动机制，2)响应Ca^<2+>信号的囊泡融合机制，我们得到了以下结果。囊泡募集和启动的机制。1)我们已经提出肌醇多磷酸结合到synaptotagmin的C2结构域作为胞吐的抑制钳。我们澄清了在染色质细胞中，结合的肌醇多磷酸在Ca^<2+>的作用下与synaptotagmin分离。微注射磷酸己肌醇对诱导胞吐和自发胞吐均有抑制作用，而硫酸己肌醇无抑制作用。2)我们明确了质下肌动蛋白网络和肌动蛋白-肌球蛋白相互作用对囊泡募集和启动至关重要。我们还发现蛋白激酶C （PKC）的激活是通过激活的PKC α或PKC β及其受体分子RACK1和皮质f -actin 2之间的复合物介导的。小泡融合响应Ca^<2+>信号的机制。1)对PC12细胞亚克隆的研究表明，syntaxin、VAMP-2、SNAP-25和Munc-18的表达降低降低了胞外活性。2)突触传递研究阐明了SNARE蛋白的循环以及SNARE蛋白与n型Ca^<2+>-通道的结合。还发现Doc2 α与Munc13-1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.