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首页> 外文期刊>The European Journal of Neuroscience >Calcium-dependent acetylcholine release from Xenopus oocytes: simultaneous ionic currents and acetylcholine release recordings.
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Calcium-dependent acetylcholine release from Xenopus oocytes: simultaneous ionic currents and acetylcholine release recordings.

机译：非洲爪蟾卵母细胞钙依赖性乙酰胆碱释放：同时离子电流和乙酰胆碱释放记录。

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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.
机译：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synaptobrevin（SNARE复合物）被认为参与了膜融合机核心的形成，但对SNARE相互作用的分子基础尚不完全了解。因此，设计实验以测试新模型中的这些关系将很有趣。非洲爪蟾卵母细胞是研究离子通道和神经递质受体的分子结构和功能的有价值的工具。在这里，我们显示SNARE蛋白存在于天然的非洲爪蟾卵母细胞中，并且那些注入了乙酰胆碱和突触前质膜的卵母细胞从水Tor鱼的电器官中提取，承担了胆碱能神经末梢的某些功能。在单个去极化的卵母细胞中同时记录并分析神经递质的释放和宏观电流：使用化学发光法检测乙酰胆碱的释放，并利用两电极电压钳利用卵母细胞的内源性Ca2 +激活的氯化物电流来测量钙的进入系统。神经递质的释放是钙和电压依赖性的，在几种钙通道阻滞剂的存在下部分减少。梭菌神经毒素，既是全毒素又是注射的轻链形式，也抑制了乙酰胆碱的释放。我们还通过使用针对SNAP-25，syntaxin或VAMP / synaptobrevin的单克隆抗体，研究了SNARE复合物在突触传递和膜电流中的作用。与梭菌毒素一样，使用针对VAMP /突触臂蛋白，SNAP-25和语法蛋白的抗体可抑制乙酰胆碱的释放。然而，宏观电流仅通过语法蛋白抗体或肉毒杆菌-C1神经毒素被修饰。该模型构成了一种了解囊泡胞吐过程的新方法。

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