Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells.-猫眼课题宝

突触结合蛋白 C2A 和 C2B 结构域在肾上腺嗜铬细胞胞吐作用调节中的不同作用。

DOI：

发表时间：

1997

期刊：

Proceedings of the National Academy of Sciences of the United States of America

影响因子：

11.1

通讯作者：

中科院分区：

综合性期刊1区

文献类型：

作者：

M. Ohara;M. Fukuda;M. Niinobe;H. Misonou;K. Ikeda;Takeshi Murakami;M. Kawasaki;K. Mikoshiba;K. Kumakura

含有两个重复的C2调节结构域的突触结合蛋白被认为参与神经递质的释放。为了揭示synaptotagmin在胞吐调节中的作用，我们研究了C2 A和C2B结构域的抗体对毛地黄皂苷透化的肾上腺嗜铬细胞Ca 2+诱发的儿茶酚胺（CA）释放的影响，将Ca 2+诱发的释放分解为ATP依赖性启动和ATP非依赖性Ca 2+触发步骤。抗C2 A抗体明显减少ATP非依赖性释放，表明C2 A结构域直接促进或促进Ca 2+触发的步骤，囊泡融合。相反，抗C2B抗体本身不影响Ca 2+诱发的释放，但显著增加自发的Ca 2+非依赖性释放。此外，肌醇高聚磷酸盐系列（IHPS），结合C2B结构域抑制ATP非依赖性Ca 2+诱发的释放和自发释放的剂量依赖性方式。IHPS的抑制作用可被抗C2B抗体完全逆转，并可被高浓度Ca ~（2+）显著逆转。这些结果表明，IHPS结合到C2B结构域逮捕膜融合，大概是通过防止与磷脂或质膜蛋白质的突触结合蛋白的相互作用。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Ca 2+的静息水平下远离自发融合。增加的细胞内Ca 2+与C2 A结构域的结合可以通过释放IHPS的这种抑制来促进或触发囊泡融合。

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.