神经元离子通道-动作电位-量子化分泌关系研究

We have made a series of achievements in previous program with the same name: 1）In the aspect of secretion physiology/translational medicine, we found that the regulation of glutamate secretion in the synapse of calyx mainly depends on the number of action potentials（AP）instead of action potential time coding (APTC) in brain slice. In contrast, dopamine release in striatum of anaesthetic rats in vivo, similar to soma secretion, is strongly regulated by APTC; 2) In the aspect of the mechanism of cell secretion, we made important progresses: In 2002, we found a new and international concerned form of secretion mechanism which is dependent on voltage instead of calcium, and we called it “calcium independent but voltage dependent secretion (CIVDS)”. Spontaneous membrane depolarization with low frequencies (<4Hz) trigger CIVDS, while high frequency stimulations, which can cause pain, trigger CDS (calcium dependent secretion). The two vesicle pools for CIVDS and CDS exist independently in soma of DRG neurons. In order to keep our leading role in the field of “the regulation mechanism of cell secretion (mainly on monoamine neurotransmitters) under physiological condition”, we aim at studying the cellular and molecular regulation mechanisms of cell secretion in more physiological conditions (healthy and sick conditions) in this application: 1) to find out the molecular mechanism of CIVDS and studying its mechanism of function; 2) under physiological conditions (in vivo and brain slice), using major Parkinson’s disease anmal models to real time record APTC induced dopamine release in striatum to study the mechanism of Parkinson’s disease; 3) the regulation mechanism of G protein coupled receptor(GPCR) and calcium on quantal secretion，

我们上期同名项目取得系列成果：1）生理态分泌调控研究，发现脑片谷氨酸突触上动作电位（AP）调控分泌机制只是AP数目而不是AP时间编码（APTC）；但在体（in-vivo）大脑纹状体的多巴胺分泌却受APTC强烈调控。 2)培养细胞的分泌机制研究，在我室2002发现的电压(而不是钙离子)依赖性分泌CIVDS基础上，新发现静息DRG神经元自发的低频AP（<4Hz）特异触发CIVDS分泌，而疼痛高频刺激主要触发CDS（钙依赖性分泌）；发现同一细胞CIVDS和CDS两种囊泡库相互独立。为扩大我们“生理态单胺分泌”的优势，本申请将集中研究（健康和疾病态）分泌机制：1）在过去12年工作基础上，研究CIVDS分子机制；2）生理（in-vivo和脑片）条件下，研究主要帕金森病动物模型的多巴胺分泌及其相关易感基因致病机理；3) G蛋白受体和钙离子调控量子化分泌的机理。

1995-2013期间，在基金委杰青、重点等基金的连续支持下，我们在培养细胞胞体和脑组织薄片上研究了离子通道对动作电位编码（APC）和神经递质分泌的调控机理，并首次发现了不依赖钙离子、由动作电位直接诱发的囊泡分泌形式（CiVDS）。本期基金集中研究健康和疾病态胞体和突触分泌受离子通道和APC调控机理，主要进展包括：1）揭示了CiVDS的分子机制以及分泌物:电压门控N型钙离子通道（Cav2.2）作为CiVDS的电压感受器，与囊泡分泌所必须的SNARE蛋白偶联在一起，当发生动作电位时Cav2.2发生构象变化直接通过SNARE蛋白拉动囊泡发生融合，从而释放出ATP；2）结合帕金森病动物模型，应用电化学微电极记录多巴胺释放，发现parkin的底物Syt11是一种新型的PD致病基因。另外，还将多巴胺检测技术应用于干细胞治疗帕金森病研究。“离子通道-动作电位-神经分泌”课题揭示了这一神经系统基本问题在单细胞、脑薄片、和活体动物大脑的规律和机制，为以后的神经生理/病理机制研究提供了坚实的基础。5年发表相关论文30余篇，其中本项目资助发表SCI论文20篇。