Involvement of Inositol Polyphosphates in Neurotermuinal Mechanism and Analysis of the Molecular Mechanism

突触聚集素的C2a和C2b结构域是参与突触小泡内吞和外吞的功能结构域，我们的研究表明，肌醇多磷酸(IP)与C2b结构域的结合抑制了突触小泡的胞吐作用。在本研究中，我们利用哺乳动物细胞，如大鼠颈上神经节(SCG)神经元或牛肾上腺嗜铬细胞，研究了这两个结构域的功能细节，并研究了与C2B结构域的结合蛋白。作为另一种方法，我们研究了淀粉样前体蛋白(APP)的胞内结构域结合蛋白，结果表明：(1)ip对大鼠脊髓背角神经节神经元的Ca~(2+)-Lt；2+-gt；依赖的融合和自发释放，但这种抑制作用经抗C2B抗体处理后消失。(2)C2a结构域以Ca^-lt；2+&gt；这些结果表明，突触凝集素是通过C2a和C2B结构域来调节突触囊泡功能的关键分子。此外，在突触体中还发现了一种分子质量为115 kDa的新蛋白，它与突触小泡的胞内结构域结合。APP胞内结构域结合蛋白的发现为阿尔茨海默病的生化阐明提供了可能。目前对该结合蛋白的鉴定研究正在进行中。

C2A and C2B domains of synaptotagmin are functional domains which are involved in exo- and endocytosis of synaptic vesicles, and we have shown that exocytosis is suppressed with the binding of inositol polyphosphates (IP) to C2B domain.In this studies, we investigated about the details of function of both domains using mammalian cells, such as rat supeior cervical ganglion (SCG) neurons or bovine adrenal chromaffin cells, and also investigated about the binding protein to C2B domain.As an another approach, we investigated about the binding protein to the intracellular domain of amyloid precursor protein (APP), because neurotransmission was suppressed by injection of C-terminal peptide of APP into rat SCG neurons.The results revealed that (1) IP inhibited Ca^<2+> -dependent fusion and spontaneous release, but the inhibition disappeared by the treatment with anti-C2B antibody, (2) C2A domain was involved in fusion step of synaptic vesicles as a Ca^<2+> sensor, while C2B domain was involved in both steps of exo- and endocytosis of synaptic vesicles, (3) clathrin assembly protein which is involved in endocytosis bound to C2B domain with high affinity, and this binding was inhibited by IP, (4) novel protein with a molecular mass of 115kDa which binds to the intracellular domain of APP was found in synaptosomes.These results clearly demonstrated that synaptotagmin was a central key molecule which regulates the functions of synaptic vesicles via C2A and C2B domains.Further, it is possible that discover of the novel protein which binds to the intracellular domain of APP contributes to biochemical elucidation for Alzheimer's desease.The studies are now in progress about identification of the binding protein.