Seagar M, Lévêque C, Charvin N, Marquèze B, Martin-Moutot N, Boudier J A, Boudier J L, Shoji-Kasai Y, Sato K, Takahashi M
INSERM Unité 464, Institut Jean Roche, Faculté de Médecine Secteur Nord, Marseille, France.
Philos Trans R Soc Lond B Biol Sci. 1999 Feb 28;354(1381):289-97. doi: 10.1098/rstb.1999.0380.
Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.
神经递质从突触小泡的释放是由电压门控钙通过P/Q型或N型钙通道内流触发的。从大鼠脑突触体中纯化N型通道最初提示了钙通道与两种参与胞吐作用的关键蛋白之间的分子相互作用：突触结合蛋白I和 syntaxin 1。免疫共沉淀实验与以下假说一致，即N型和P/Q型钙通道而非L型钙通道与包含syntaxin 1、SNAP - 25、VAMP和突触结合蛋白I或II的7S复合物相关联。在青蛙神经肌肉接头处进行的免疫荧光共聚焦显微镜检查证实，钙通道、syntaxin 1和SNAP - 25共定位于突触前质膜发生递质释放的活性区。利用重组蛋白进行实验以绘制在形成P/Q型钙通道孔的α1A亚基上的突触蛋白相互作用位点。体外翻译的35S - 突触结合蛋白I结合到位于连接α1A亚基同源结构域II和III的胞质环上的一个位点。这种直接联系将突触结合蛋白（一种推测的胞吐作用钙传感器）靶向到靠近通道口的钙内流微区。半胱氨酸串珠蛋白（CSPs）含有与Hsp70协同作用的分子伴侣特有的J结构域。它们位于突触小泡上，并被认为参与调节突触前钙通道的活性。发现CSPs与钙通道的结合位点与突触结合蛋白相同，并且还与VAMP相关联。CSPs可能作为与Hsp70相关的分子伴侣，指导钙通道处多蛋白复合物的组装或解离。
