Molecular Mechanisms of Synaptic Vesicle Fusion

Synaptotagmin I（Syt I）的功能Ca^<2+>结合位点的鉴定syt I被认为是快速突触传递的主要Ca^<2+>传感器。Syt I有两个Ca^<2+>结合域，C2A和C2B。这些结构域中哪一个感知Ca^<2+>仍然存在争议。在这项研究中，我们试图在果蝇syt I突变体中鉴定Ca^<2+>结合域用于快速突触传递。1)Syt I是果蝇神经肌肉连接处快速突触传递的主要Ca^<2+>传感器。我们使用了三个Syt I突变体：Syt I^<AD4>；一个零突变体，syt I^<AD1>；缺乏C2B，而C2A保持完整，syt I^<AD3>；在C2B中有一个氨基酸取代。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。syt I^<AD4>突触传递严重受损，但未完全消除。其余突触电流依赖于Ca^<2+>。表观协同度N为0.95。syt I^<AD1>的突触传递也严重受损，但…略好于syt I^<AD4>。N也为1.06，这表明剩余的C2A本身不能作为Ca^<2+>传感器工作。在syt I^<AD3>中，突触传递优于其他两个突变体，但平均振幅约为对照的1/20，N为1.54，显著小于对照的3.01。由于已知AD3突变会阻断Syt I的Ca^<2+>依赖性寡聚化，因此Syt I^<AD3>的这种缺陷可能是由于缺乏寡聚化（Okamoto et al., 2005）。2) Syt I C2B结构域的Ca^<2+>结合位点Ca1和Ca2感知Ca^<2+>以实现快速突触传递。在本研究中，我们使用了两个转化子，其中Ca1或Ca2上的两个天冬氨酸被改变为精氨酸以阻断Ca^<2+>结合。在Ca1突变体中没有检测到快速突触传递，而在Ca2突变体中突触传递强烈减少但仍然存在。剩余的突触电流依赖于Ca^<2+>， N较小，为1.86，这表明至少有两个Ca^<2+>分子结合诱导快速囊泡融合。由于在这个突变体中只剩下一个Ca^<2+>结合位点，我们必须寻找另一个Ca^<2+>结合位点。此时，Ca^<2+>依赖性寡聚化在该突变体中提供了另一个Ca^<2+>结合位点。3)Ca^<2+>依赖性寡聚化是快速突触传递的必要条件。据报道，Sr^<2+>不会诱导Syt I的Ca^<2+>依赖性寡聚化（Chapman et al.,1996）。利用Sr^<2+>的这一特性，我们测试了突触传递是否需要Ca^<2+>依赖的寡聚化。Sr^<2+>的突触传递明显减弱，协同度N为2.01，显著小于Ca^<2+>的3.01。因此我们得出结论，Ca^<2+>依赖性寡聚化是快速突触传递所必需的。少

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