Mode of actions and functions of the Rab3A and SNARE systems in neurotransmitter release

研究表明，Rab3A是一个小的GTP酶，SNARE系统参与了神经递质的释放。Rab3A系统由其靶蛋白和几个调节蛋白组成，后者与GTP结合的Rab3A相互作用，而不与GDP结合的Rab3A相互作用。我们已经确定RabPhilin-3是Rab3A的靶蛋白，并揭示了RabPhilin-3在突触前中作为钙感受器发挥作用的可能性。另一方面，现有证据表明，SNARE系统在Rab3A系统下游发挥作用，并与神经递质释放有关。我们已经确定Tomosyn是SNARE系统的一种调节蛋白。Tomosyn可能是连接Rab3A和SNARE系统的中心参与者。然而，Rab3A系统调节SNARE系统神经递质释放的机制尚不清楚。因此，在本研究中，我们试图阐明其机制。我们揭示了Tomosyns至少存在三种亚型，即b-、m-和S-Tomosynns，并且所有的Tomosyn亚型都通过其C端的VAMP样区与Synaxin-1相互作用，而不与其他Synaxin-2、-3和-4亚型相互作用。此外，我们还鉴定了一个新的Synaxin-1结合蛋白。根据其生化特性，推测该蛋白可能是一种细胞骨架相关蛋白。1摩尔的合成素-1结合蛋白与约0.8摩尔的合成素-1结合，使合成素-1与合成素-1结合一半的剂量约为90 nm。Munc18以剂量依赖的方式抑制合成素-1结合蛋白与合成素-1的相互作用。在未来，我们试图纯化合成素-1结合蛋白，确定其一级结构，并揭示其在神经递质释放中的功能。

It has been revealed that the Rab3A, a small GTPase, and SNARE systems are involved in neurotransmitter release. The Rab3A system consists of its target protein, which interacts with the GTP-bound form of Rab3A but not with the GDP-bound form of Rab3A, and its several regulatory proteins. We have identified Rabphilin-3 as a target protein for Rab3A and revealed the possibility that Rabphilin-3 functions as a Ca^<2+>-sensor in the presynapse. On the other hand, available evidence suggests that the SNARE system functions at the downstream of the Rab3A system and is implicated in neurotransmitter release. We have identified Tomosyn as a regulatory protein of the SNARE system. It is possible that Tomosyn is a central player which links between the Rab3A and SNARE systems. However, the mechanism by which the Rab3A system regulates the SNARE system in neurotransmitter release remains to be clarified. Therefore, in this study we attempted to clarify the mechanism. We revealed that there were at least three isoforms of Tomosyn, b-, m-, and s-Tomosyns and that all of the tomosyn isoforms interacted through their C-terminal VAMP-like regions with syntaxin-1 but not with other syntaxin isoforms such as syntaxin-2, -3, and -4. Moreover, we identified a novel syntaxin-1-binding protein. On the basis of its biochemical properties, it was possible that the syntaxin-1-binding protein was a cytoskeleton-related protein. One mole of the syntaxin-1-binding protein bound to about 0.8 mole of syntaxin-1 and the dose giving the half maximal binding of syntaxin-1 to the syntaxin-1-binding protein was about 90 nM.The interaction of the syntaxin-1-binding protein with syntaxin-1 was inhibited by Munc18 in a dose-dependent manner. In future we attempt to purify the syntaxin-1-binding protein, determine its primary structure, and reveal its function in neurotransmitter release.