Molecular Mechanisms of Synaptic Vesicle Fusion

突触小泡融合的分子机制

• 批准号: 15300132
• 负责人: KIDOKORO Yoshiaki
• 金额: $ 9.79万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15300132/
• 关键词: Synaptic transmission; Drosophila; neuromuscular junction; synaptotagmin I; Ca^<2+> sensor; Ca^<2+> cooperativity; シナプス伝達; カルシウム・センサー; 自発性シナプス放出

Function of Synaptotagmin I(Syt I) ; Identification of Ca^<2+> binding sitesSyt I is considered to be a major Ca^<2+> sensor for fast synaptic transmission. Syt I has two Ca^<2+> binding domains, C2A and C2B. It is still debated which of these domains is sensing Ca^<2+>. In this study, we attempted to identify the Ca^<2+> binding domain for fast synaptic transmission using Drosophila syt I mutants.1)Syt I is a major Ca^<2+> sensor for fast synaptic transmission at the Drosophila neuromuscular junction We used three syt I mutants : syt I^<AD4> ; a null mutant, syt I^<AD1> ; lacks C2B while C2A remains intact, syt I^<AD3> ; has one amino-acid substitution in C2B. All of these mutants have severe impairment in synaptic transmission and embryonic lethal. In syt I^<AD4> synaptic transmission was severely impaired but not abolished. Remaining synaptic currents were Ca^<2+>-dependent. The apparent cooperativity, N, was 0.95. In syt I^<AD1> synaptic transmission was also severely impaired but … More slightly better than syt I^<AD4>. N was also 1.06, suggesting that the remaining C2A does not work as a Ca^<2+> sensor by itself. In syt I^<AD3> synaptic transmission was better than other two mutants but the mean amplitude was about 1/20 of the control, and N was 1.54 which was significantly smaller than 3.01 in the control. Since the AD3 mutation is known to block Ca^<2+>-dependent oligomerization of Syt I, this defect in syt I^<AD3> could be due to lack of oligomerization (Okamoto et al., 2005).2)Ca^<2+> binding sites, Ca1 and Ca2, in the Syt I C2B domain are sensing Ca^<2+> for fast synaptic transmission For this study we used two transformants in which two aspartates at Ca1 or Ca2 was changed to arginines to block Ca^<2+> binding. In the Ca1 mutant no fast synaptic transmission was detected, while in the Ca2 mutant synaptic transmission was strongly reduced but remained. Remaining synaptic currents were Ca^<2+>-dependent with smaller N, 1.86, suggesting that at least two Ca^<2+> molecules bind to induce fast vesicle fusion. Since in this mutant there is only one Ca^<2+> binding site remained, we had to look for another Ca^<2+> binding site. At this moment, we Ca^<2+>-dependent oligomerization is providing another Ca^<2+> binding site in this mutant.3)Ca^<2+>-dependent oligomerization is essential for fast synaptic transmission It has been reported that Sr^<2+> does not induce Ca^<2+>-dependent oligomerization of Syt I (Chapman et al.,1996). Taking advantage of this property of Sr^<2+> we tested whether Ca^<2+>-dependent oligomerization is required for synaptic transmission. Synaptic transmission in Sr^<2+> was strongly reduced and the cooperativity, N, was 2.01, which is significantly smaller than that, 3.01, in Ca^<2+>. Thus we conclude that Ca^<2+>-dependent oligomerization is essential for fast synaptic transmission. Less

突触素I的功能；钙结合位点的鉴定Syt I被认为是突触快速传递的主要感受器。Syt I有两个钙结合结构域，C2a和C2b。这些结构域中的哪一个是感知钙离子的，目前仍存在争议。在这项研究中，我们试图利用果蝇syt I突变体来鉴定快速突触传递的钙结合结构域。1)syt I是果蝇神经肌肉接头快速突触传递的主要感受器。我们使用了三个syt I突变体：syt I^&lt；ad4&gt；；一个零突变体，syt i^&lt；ad1&gt；缺失C2B，而C2a保持完整，syt I^&lt；ad3&gt；；在C2B中有一个氨基酸替换。所有这些突变体在突触传递和胚胎致死方面都有严重的损害。在系统中，突触传递严重受损，但并未被取消。剩余的突触电流是钙依赖的。表观协同系数N为0.95。在系统中，突触传递也受到严重损害，但…比syt i^&lt；ad4&gt；略好一些。N也是1.06，这表明剩余的C2a本身不能作为钙离子传感器。突触传递优于其他两个突变体，但平均波幅约为对照的1/20，N为1.54，显著小于对照的3.01。由于已知AD3突变可以阻止依赖于Ca^&lt；2+和gt；的Syt I齐聚，因此Syt I&lt；AD3&gt；中的这一缺陷可能是由于缺乏齐聚作用(Okamoto等人，2005)。2)在SYT I C2B结构域中，Ca^&lt；2+&gt；结合部位CA1和Ca 2+正在感应Ca^&lt；2+&gt；为了快速突触传递，我们使用了两个转化子，其中CA1或Ca 2+的两个天冬氨酸被改变为精氨酸来阻断Ca&lt；2+&gt；结合。在CA1突变体中没有检测到快速突触传递，而在突触中突触传递明显减少，但仍存在。剩余的突触电流是钙依赖的，N较小，为1.86，提示至少有两个钙离子结合的分子可以诱导快速的囊泡融合。由于在这个突变体中只有一个Ca^&lt；2+&gt；结合位点，我们不得不寻找另一个Ca^&lt；2+&gt；结合位点。3)钙依赖的寡聚是突触快速传递所必需的。据报道，锶不能诱导Syt I的钙依赖的寡聚(Chapman et al.，1996)。利用sr^&lt；2+&gt；的这一特性，我们测试了突触传递是否需要钙依赖的寡聚作用。Sr^&lt；2+&gt；突触传递明显减少，协同系数N为2.01，显著小于Ca^&lt；2+&gt；的3.01。因此，我们得出结论，钙离子依赖的寡聚作用是突触快速传递所必需的。较少

项目成果

The role of integrins in the modulation of neuromuscular release from motor nerve terminals by stretch and hypertonicity.

整合素在通过拉伸和高渗调节运动神经末梢神经肌肉释放中的作用。

• 发表时间: 2004
• 期刊: Journal of Neurocytology 32
• 作者: Grinnell;A.D.;Chen;B.-M.;Kashani;A.;Lin;J.;Suzuki;K.;Kidokoro;Y.
• 通讯作者: Y.

External Ca2+ dependency of synaptic transmission in drosophila synaptotagmin I mutants.

• DOI: 10.1152/jn.00205.2005
• 发表时间: 2005-08
• 期刊: Journal of neurophysiology
• 影响因子: 2.5
• 作者: T. Okamoto;T. Tamura;Kazuhiro Suzuki;Y. Kidokoro

Exocytosis and endocytosis of synaptic vesicles and functional roles of vesicle pools : Lessons from the Drosophila neuromuscular function.

突触小泡的胞吐作用和内吞作用以及囊泡池的功能作用：果蝇神经肌肉功能的教训。

• 发表时间: 2005
• 期刊: Neuroscientist 11
• 作者: Kuromi;H.;Kidokoro;Y.
• 通讯作者: Y.

Hou, D., Suzuki, K., Wolfgang, W.J., Clay, C., Forte, M., Kidokoro, Y.: "Presynaptic impairement of synaptic transmission in Drosophila embryos lacking Gsα."Journal of Neuroscience. 23. 5897-5905 (2003)

Hou, D.、Suzuki, K.、Wolfgang, W.J.、Clay, C.、Forte, M.、Kidokoro, Y.：“缺乏 Gsα 的果蝇胚胎中突触传递的突触前损伤。”神经科学杂志 23. 5897-。 5905 (2003)

Sakai, T., Kidokoro, Y.: "Overexpression of a CREB repressor isoform enhances the female receptivity in Drosophila."Behavior Genetics. 32. 413-422 (2003)

Sakai, T., Kidokoro, Y.：“CREB ​​阻遏物亚型的过度表达增强了果蝇的雌性接受性。”行为遗传学。