Fukuda M, Moreira J E, Lewis F M, Sugimori M, Niinobe M, Mikoshiba K, Llinás R
Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Ibaraki, Japan.
Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10708-12. doi: 10.1073/pnas.92.23.10708.
Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.
突触结合蛋白（Syt）是一种能与肌醇高聚磷酸系列（肌醇 1,3,4,5 - 四磷酸（IP4）、肌醇 1,3,4,5,6 - 五磷酸和肌醇 1,2,3,4,5,6 - 六磷酸）结合的突触小泡蛋白。制备了一种针对 C2B 结构域（一种肌醇高聚磷酸结合位点）的多克隆抗体（抗 Syt - C2B）。通过比较其抑制 IP4 与 C2B 结构域结合的能力和抑制 Ca2 + /磷脂与 C2A 结构域结合的能力，来确定该抗体对 C2B 结构域的特异性。将抗 Syt - C2B IgG 注入枪乌贼巨大突触前膜并未阻断突触释放。共注射 IP4 和抗 Syt - C2B IgG 未能阻断递质释放，而 IP4 本身是一种强大的突触释放阻断剂。对注射了抗 Syt - C2B IgG 的突触前纤维进行重复刺激，显示突触后反应幅度迅速下降，这可能是由于其阻断了突触小泡的循环利用。对抗 Syt - C2B 注射突触前膜的电子显微镜观察显示突触小泡数量减少了 90%。综合这些结果表明，Syt 分子在 Ca2 + 介导的突触小泡融合及其受肌醇高聚磷酸调控过程中，以及在突触小泡的循环利用中起着核心作用。
