Aleu Jordi, Blasi Joan, Solsona Carles, Marsal Jordi
Laboratory of Cellular and Molecular Neurobiology, Department of Cell Biology and Pathology, Medical School, Hospital of Bellvitge, University of Barcelona, Barcelona, Spain.
Eur J Neurosci. 2002 Oct;16(8):1442-8. doi: 10.1046/j.1460-9568.2002.02208.x.
The fusion of synaptic vesicles with presynaptic membranes is controlled by a complex network of protein-protein and protein-lipid interactions. SNAP-25, syntaxin and synaptobrevin (SNARE complex) are thought to participate in the formation of the core of the membrane fusion machine but the molecular basis of SNARE interactions is not completely understood. Thus, it would be interesting to design experiments to test those relationships in a new model. Xenopus laevis oocytes are valuable tools for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Here we show that SNARE proteins are present in native Xenopus oocytes and that those oocytes injected with acetylcholine and presynaptic plasma membranes extracted from the electric organ of Torpedo marmorata assume some of the functions of a cholinergic nerve terminal. Neurotransmitter release and macroscopic currents were recorded and analysed simultaneously in a single oocyte electrically depolarized: acetylcholine release was detected using a chemiluminiscent method and calcium entry was measured by exploiting the endogenous Ca2+-activated chloride current of the oocyte with a two-electrode voltage-clamp system. Neurotransmitter release was calcium- and voltage-dependent and partially reduced in the presence of several calcium channel blockers. Clostridial neurotoxins, both holotoxin and injected light-chain forms, also inhibited acetylcholine release. We also studied the role of the SNARE complex in synaptic transmission and membrane currents by using monoclonal antibodies against SNAP-25, syntaxin or VAMP/synaptobrevin. The use of antibodies against VAMP/synaptobrevin, SNAP-25 and syntaxin inhibited acetylcholine release, as did clostridial toxins. However, macroscopic currents were only modified either by syntaxin antibody or by Botulinium-C1 neurotoxin. This model constitutes a new approach for understanding the vesicle exocytosis processes.
突触小泡与突触前膜的融合受蛋白质 - 蛋白质和蛋白质 - 脂质相互作用的复杂网络控制。SNAP - 25、 syntaxin和突触小泡蛋白（SNARE复合体）被认为参与了膜融合机器核心的形成，但SNARE相互作用的分子基础尚未完全明了。因此，设计实验在新模型中测试这些关系将会很有趣。非洲爪蟾卵母细胞是研究离子通道和神经递质受体分子结构与功能的宝贵工具。在这里我们表明，SNARE蛋白存在于天然非洲爪蟾卵母细胞中，并且那些注射了乙酰胆碱和从电鳐电器官提取的突触前质膜的卵母细胞承担了胆碱能神经末梢的一些功能。在单个电去极化的卵母细胞中同时记录和分析神经递质释放和宏观电流：使用化学发光法检测乙酰胆碱释放，并用双电极电压钳系统利用卵母细胞内源性Ca2 + 激活的氯离子电流测量钙内流。神经递质释放依赖于钙和电压，并且在几种钙通道阻滞剂存在的情况下部分减少。梭菌神经毒素，无论是全毒素还是注射的轻链形式，也抑制乙酰胆碱释放。我们还通过使用针对SNAP - 25、 syntaxin或VAMP / 突触小泡蛋白的单克隆抗体研究了SNARE复合体在突触传递和膜电流中的作用。使用针对VAMP / 突触小泡蛋白、SNAP - 25和syntaxin的抗体抑制了乙酰胆碱释放，梭菌毒素也是如此。然而，宏观电流仅被syntaxin抗体或肉毒杆菌C1神经毒素改变。该模型构成了一种理解囊泡胞吐过程的新方法。
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