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

Rab3A，一个小的GTPase，和SNARE系统参与神经递质释放。Rab3A系统由其靶蛋白（与gtp结合形式的Rab3A相互作用，而不与gdp结合形式的Rab3A相互作用）及其几个调节蛋白组成。我们已经确定Rabphilin-3是Rab3A的靶蛋白，并揭示了Rabphilin-3在突触前作为Ca^<2+>-传感器的可能性。另一方面，现有证据表明SNARE系统在Rab3A系统的下游起作用，并与神经递质释放有关。我们已经确定Tomosyn是SNARE系统的调节蛋白。有可能Tomosyn是连接Rab3A和SNARE系统的核心参与者。然而，Rab3A系统调控SNARE系统在神经递质释放中的作用机制尚不清楚。因此，在本研究中，我们试图阐明其机制。我们发现Tomosyn至少有三种异构体，b-， m-和s-Tomosyn，并且所有Tomosyn异构体都通过其c端vamp样区域与syntaxin-1相互作用，但不与syntaxin-2， -3和-4等其他syntaxin异构体相互作用。此外，我们还发现了一种新的syntaxin-1结合蛋白。根据其生化特性，syntaxin-1结合蛋白可能是一种细胞骨架相关蛋白。一摩尔的syntaxin-1结合蛋白与约0.8摩尔的syntaxin-1结合，使syntaxin-1与syntaxin-1结合蛋白的一半最大结合剂量约为90 nM。Munc18以剂量依赖的方式抑制syntaxin-1结合蛋白与syntaxin-1的相互作用。未来我们将尝试纯化syntaxin-1结合蛋白，确定其初级结构，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.