| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1988: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
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
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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
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