Ohara-Imaizumi M, Fukuda M, Niinobe M, Misonou H, Ikeda K, Murakami T, Kawasaki M, Mikoshiba K, Kumakura K
Life Science Institute, Sophia University, Chiyoda-ku, Tokyo, Japan.
Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):287-91. doi: 10.1073/pnas.94.1.287.
Synaptotagmin that contains two repeats of C2 regulatory domains is considered to be involved in neurotransmitter release. To reveal the roles of synaptotagmin in the regulation of exocytosis, we examined the effects of antibodies against C2A and C2B domains on Ca2+-evoked catecholamine (CA) release from digitonin-permeabilized adrenal chromaffin cells, resolving the Ca2+-evoked release into ATP-dependent priming and ATP-independent Ca2+-triggered steps. Anti-C2A antibody clearly reduced the ATP-independent release, suggesting that the C2A domain directly facilitate or promote Ca2+-triggered step, vesicular fusion. In contrast, anti-C2B antibody did not affect Ca2+-evoked release by itself, but significantly increased the spontaneous Ca2+-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C2B domain inhibited both the ATP-independent Ca2+-evoked release and the spontaneous release in a dose-dependent manner. The inhibition by IHPS was totally reversed by anti-C2B antibody and significantly reversed by high concentration of Ca2+. These results suggest that IHPS binding to C2B domain arrests membrane fusion by presumably preventing interaction of synaptotagmin with phospholipids or with proteins of plasma membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of intracellular Ca2+. Binding of the increased intracellular Ca2+ to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.
含有两个C2调节结构域重复序列的突触结合蛋白被认为参与神经递质释放。为了揭示突触结合蛋白在胞吐作用调节中的作用，我们检测了针对C2A和C2B结构域的抗体对洋地黄皂苷通透的肾上腺嗜铬细胞中Ca2+诱发的儿茶酚胺（CA）释放的影响，将Ca2+诱发的释放解析为ATP依赖的引发步骤和ATP非依赖的Ca2+触发步骤。抗C2A抗体明显降低了ATP非依赖的释放，表明C2A结构域直接促进或推动Ca2+触发步骤，即囊泡融合。相反，抗C2B抗体本身不影响Ca2+诱发的释放，但显著增加了自发的Ca2+非依赖释放。此外，结合C2B结构域的肌醇高聚磷酸系列（IHPS）以剂量依赖的方式抑制了ATP非依赖的Ca2+诱发释放和自发释放。IHPS的抑制作用被抗C2B抗体完全逆转，高浓度的Ca2+使其显著逆转。这些结果表明，IHPS与C2B结构域的结合可能通过阻止突触结合蛋白与磷脂或质膜蛋白的相互作用来阻止膜融合。因此，IHPS与C2B结构域的结合可能使停靠或引发的囊泡在细胞内Ca2+的静息水平下远离自发融合。细胞内Ca2+增加与C2A结构域的结合可能通过解除IHPS的这种抑制作用来促进或触发囊泡融合。
