Molecular Genetic Dissection of Synaptic Transmission

突触传递的分子遗传学剖析

• 批准号: 09480237
• 负责人: KIDOKORO Yoshiaki
• 金额: $ 1.79万
• 依托单位: GUNMA UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480237/
• 关键词: mutants; SNARE hypothesis; Drosophila; neuronal-synaptobrevin; miniature synaptic currents; cyclic AMP; synaptic vesicle fusion; csp; シナプス; シナプトブレビン; 誘発性シナプス電流; 自発性微小シナプス電流; shibire; シナプス小胞; リサイクリング

For synaptic transmission vesicles in the presynaptic terminal containing neurotransmitter have to fuse to the membrane and release the content to the synaptic cleft. Various proteins are known to be involved in this fusion process. A hypothesis, SNARE hypothesis, is proposed to account for the arrangement of those proteins. We have investigated the molecular mechanism of this process by using Drosophila mutant larvae which lack key molecules.Neuronal synaptobrevin (n-syb) is vesicle membrane protein and a target of tetanus toxin. In mutant larvae which lack n-syb no nerve-evoked synaptic currents were observed. However, miniature synaptic currents (mSC) remained in lower frequencies. The frequency of mSG's was dependent on external Ca^<2+> concentrations in high potassium saline. Furthermore, the mSG frequency increased with black widow spider venom extract and Ca^<2+> ionophore, A23187.These results indicate that the remaining mSC's in n-syb mutants are Ca^<2+> dependent. In wild typ … More e larvae an elevation of cAMP in the presynaptic nerve terminal either by an activator of adenylate cyclase, forskolin, or by a membrane permeable analog of cAMP, db-cAMP, resulted in an increase in msc's. However, in n-syb mutants neither of these drugs increased the mSG frequency. Thus we conclude that the n-syb independent pathway for vesicle fusion is insensitive to cAMP (Yoshihara et at., 1999).Cysteine-string protein (csp) is also vesicle protein and supposed to interact with voltage-gated Ca^<2+> channels in the terminal membrane. In a temperature sensitive mutant of csp no nerve-evoked synaptic currents were observed in non-permissive temperature (32 ﾟC). This could be due to lack of function of voltage-gated Ca^<2+> channels at high temperature. To test this possibility we visualized terminal Ca^<2+> by a Ca^<2+> sensitive dye. At room temperature the Ca^<2+> concentration increase upon repetitive stimulation of nerve. However, at non-permissive temperature the Ca^<2+> signal decreased significantly, thus suggesting a failure of Ca^<2+> channel function.Since csp is on the synaptic vesicle membrane it is possible that Ca^<2+> channels have to be associated with synaptic vesicles to operate properly. To test this.possibility we used another mutant call shibire in which endocytosis is blocked at non-permissive temperature. We also found in shibire that Ca^<2+> signals were lower in non-permissive temperature. In conclusion, for voltage-gated Ca^<2+> channels in the presynaptic terminal to work properly vesicles might have to be docked to release sites (Umbach et al., 1998). Less

对于突触传递，突触前末梢中含有神经递质的囊泡必须融合到膜上并将内容物释放到突触间隙。已知多种蛋白质参与该融合过程。一个假说，陷阱假说，提出了这些蛋白质的安排。我们利用果蝇突变体幼虫研究了这一过程的分子机制。神经小突触蛋白（neuronalsynaptobrevin，n-syb）是一种囊泡膜蛋白，是破伤风毒素的作用靶点。在缺乏n-syb的突变幼虫中，没有观察到神经诱发的突触电流。然而，微型突触电流（mSC）保持在较低的频率。在高钾生理盐水中，mSG的频率依赖于外部Ca^<2+>浓度。此外，用黑寡妇蜘蛛毒提取物和Ca^<2+>离子载体A23187，mSG频率增加。这些结果表明，在n-syb突变体中剩余的mSC是Ca^<2+>依赖性的。在野生类型中 ...更多信息 用腺苷酸环化酶的激活剂毛喉素或cAMP的膜渗透性类似物db-cAMP升高突触前神经末梢的cAMP，可导致突触后神经元的增加。然而，在n-syb突变体中，这些药物都没有增加mSG频率。因此，我们得出结论，囊泡融合的n-syb非依赖性途径对cAMP不敏感（Yoshihara等人，半胱氨酸串蛋白（csp）也是囊泡蛋白，并且被认为与终末膜中的电压门控Ca^<2+>通道相互作用。在csp的温度敏感突变体中，在非允许温度（32 ℃）下没有观察到神经诱发的突触电流。这可能是由于电压门控Ca^<2+>通道在高温下功能丧失所致。为了验证这种可能性，我们用一种Ca^2+敏感染料观察了末端Ca^2+。在室温下，反复刺激神经后，Ca^<2+>浓度增加。然而，在非允许温度下，Ca^<2+>信号显著降低，这表明Ca^<2+>通道功能失效，因为csp位于突触囊泡膜上，所以Ca^<2+>通道可能必须与突触囊泡结合才能正常工作。为了测试这种可能性，我们使用了另一种称为shibire的突变体，其中内吞作用在非允许温度下被阻断。我们还发现在非允许温度下，Shibire的Ca^<2+>信号较低。总之，为了使突触前末梢中的电压门控Ca^<2+>通道正常工作，囊泡可能必须停靠在释放位点（Umbach等人，1998年）。少

项目成果

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黑美，H.，吉原，M.

Deitcher,D.L.,Ueda,A.,...,Kidokoro,Y.et al.: "Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin" J.Neurosci.18. 2028-2039 (1998)

Deitcher,D.L.,Ueda,A.,...,Kidokoro,Y.et al.：“果蝇基因神经突触短蛋白的突变揭示了神经递质诱发和自发释放的不同要求”J.Neurosci.18。

Saitoe,M., Koshimoto,H., Kidokoro,Y.et al.: "Distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes revealed with focal release of L-glutamate from caged compound by laser." Journal of Neuroscience Methods. (in press). (1

Saitoe,M.、Koshimoto,H.、Kidokoro,Y.等人：“通过激光从笼状化合物中集中释放 L-谷氨酸，揭示了培养的胚胎果蝇肌管中功能性谷氨酸受体的分布。”

Rheuben, M. B., Yoshihara, M.& Kidokoro, Y.: "Ultrastructural orrelates of neuromuscular junction development.in "Neuromuscular Junction in Drosophila"eds.Vivian Budnik et al." Landes Bioscience.(in press),

鲁本，M.B.，吉原，M.

Umbach, U.A., Saitoe, M., Kidokoro, Y., et al.: "Attenuated influx of calcium ions at nerve endings of csp and shibire mutant Drosophila" J.Neurosci.18. 3233-3240 (1998)

Umbach, U.A.、Saitoe, M.、Kidokoro, Y. 等人：“csp 和 shibire 突变果蝇神经末梢钙离子流入减弱”J.Neurosci.18。