Mechanism of Presynaptic Function as Studied Using Giant Synaptosomes

1.本研究有两个主要目的。一是突触体膜和突触囊泡膜离子通道的检测和表征。为此，我们采用了将生物膜重组为平面脂双层或巨型蛋白脂质体的方法。第二个目标是开发一种新的实验系统，它允许我们应用到目前为止还不适用于重建系统的各种技术。为此，我们尝试在不使用外源磷脂的情况下，通过融合生物膜囊泡来制备巨大的生物膜囊泡。我们开发了一种简单的方法，从大鼠脑中获得突触小体和突触小泡的制备，它们是受其他膜组分污染最小的。建立了一种快速、简便、无需洗涤剂制备巨型蛋白脂质体的方法。这项技术比涉及增溶/透析过程的传统方法有一些优势。使用这个新开发的…更多的技术以及平面膜技术，我们已经检测到了几种阳离子和阴离子通道。突触体膜上含有电压非依赖性钾通道，这可能有助于静息电位的测定。此外，还观察到钙依赖性钾通道。突触小泡膜上存在两种单价阳离子通道和一种阴离子通道。这些通道可能在突触前终末神经递质的胞吐过程中发挥重要作用。采用聚乙二醇类融合化合物处理，电融合，部分酶消化和/或冷冻解冻技术，我们能够在不添加外源性脂质的情况下制备直径10-20微米的巨型突触体。人们试图将电生理学和细胞操作的各种程序应用于这些巨大的突触体。到目前为止，它们中的一些是不可能通过重组蛋白脂质体实现的。成功地进行了微电极的插入和注射。这些结果表明，巨型突触体应该是一个非常有用的系统，以研究突触前功能及其机制。较少

1. This study has two main objectives. One is the detection and characterization of ion channels of synaptosomal membranes and synaptic vesicle membranes. For this purpose, we adopted reconstitution of biomembranes into planar lipid bilayer or into giant proteoliposomes. The second objective is the development of a new experimental system, which allow us to apply various techniques not applicable, so far, to the reconstitution system. For this purpose, we attempted to prepare giant biomembrane vesicles, by fusion of biomembrane vesicles without use of exo-genous phospholipids.2. We developed a simple method to obtain preparations of synaptosomes and synaptic vesicles from rat brain, which are minimal of contamina-tions by other membrane fractions.3. A rapid and simple method to prepare giant proteoliposomes without use of detergent has been established. This technique has some advantages over conventional methods involving the solubilization/dialysis procedure. Using this newly develop … More ed technique as well as the planar membrane technique, we have detected several kinds of cation and anion channels. Synaptosomal membrane contains voltage-independent potassium channels, which may contribute to the determination of resting potential. A calcium-dependent potassium channel was also observed. Two kinds of mono-valent cation channels and an anion channel were found to occur in the membrane of synaptic vesicles. These channels may play an important role in the exocytosis of neurotransmitters from presynaptic terminals.4. Using the treatment with a fusogenic compound such as polyethyleneglycol, electrofusion, partial digestion with proteases and/or freez-ing-thawing technique, we were able to prepare giant synaptosomes, 10-20 micrometers in diameter, without addition of exogenous lipid. Attempts to apply various procedures of electrophysiology and cell manipulation to these giant synaptosomes were made. Some of them had been so far impossible with reconstitute proteoliposomes. Insertion of a microelectrode and injection were carried out successfully. These results suggest that giant synaptosome should be a very useful system for the study of presynaptic functions and their mechanisms. Less