Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.
Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.
复制标题
突触结合蛋白 C2A 结构域在递质释放中的作用，通过将特异性抗体注射到鱿鱼巨突触前末端来确定。
DOI：
10.1073/pnas.92.23.10703
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发表时间：
1995
影响因子：
11.1
通讯作者：
Llinás,R
中科院分区：
文献类型：
--
作者：
Mikoshiba,K;Fukuda,M;Moreira,JE;Lewis,FM;Sugimori,M;Niinobe,M;Llinás,R
克隆了鱿鱼突触结合蛋白（Syt）的cDNA及其开放阅读框架，用谷胱甘肽S-转移酶（GST）-Syt-C2 A免疫家兔获得了多克隆抗体。结合试验表明，抗Syt-C2 A抗体可识别鱿鱼Syt，并抑制Ca（2+）依赖性磷脂与C2 A结构域的结合。这种抗体，当注射到前终端在鱿鱼巨大的突触，阻止发射器释放的方式类似于先前报道的突触前注射的肌醇高聚磷酸盐系列的成员。阻滞不伴随突触前动作电位或突触前Ca 2+电流的振幅或电压依赖性的任何变化。突触后电位对重复突触前刺激不敏感，表明抗体对递质释放系统有直接作用。发射器释放块后，共聚焦显微镜分析注射罗丹明共轭抗Syt-C2 A的前终末连接表现出荧光点在突触前质膜的内表面旁边的活动区。相同制剂的结构分析表明，突触囊泡的积累在大小和分布上对应于共焦显示的荧光点。连同发现这种抗体阻止Ca 2+结合C2 A结构域中的特异性受体，这些结果表明Ca 2+通过激活Syt的C2 A结构域触发递质释放。我们的结论是，C2 A结构域是直接相关的突触囊泡的融合，结果在发射器的释放。
Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.