C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.
C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.
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DOI：
10.1523/jneurosci.3360-11.2012
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发表时间：
2012-02-22
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影响因子：
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通讯作者：
Südhof TC
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作者：
Kaeser-Woo YJ;Yang X;Südhof TC
络合蛋白是一种小的可溶性蛋白，在突触囊泡胞吐过程中与SNARE复合物结合，进而介导神经递质释放。络合蛋白是自发“迷你”释放和启动和突触蛋白依赖的Ca2+触发诱发释放所必需的。哺乳动物基因组编码四种复杂蛋白，包括激活突触胞外作用的n端非结构化序列、抑制胞外作用的辅助α-螺旋、与SNARE复合物结合并发挥其所有功能的基本中心α-螺旋，以及功能尚不清楚的长且明显非结构化的c端序列。在这里，我们使用培养的小鼠神经元来证明复杂蛋白-1的c端序列不是其突触结合蛋白激活功能所必需的，而是其启动和箝位功能所必需的。野生型络合素-3不能抑制胞吐，但能充分启动和激活胞吐。引人注目的是，将络合蛋白1c末端交换为络合蛋白3c末端废除了夹紧，而将络合蛋白3c末端交换为络合蛋白1c末端则启用了夹紧。对络合蛋白-1 C端点突变的分析发现了两个单氨基酸取代，它们在不改变络合蛋白-1激活功能的情况下破坏了夹紧。刺激序列诱导的释放检查显示，钳夹缺陷的c端络合蛋白突变体产生适度的相对延迟释放增加。总的来说，我们的研究结果表明，相对较大的c端络合蛋白1序列在启动和箝制突触胞涌出中起作用，并且表明络合蛋白3的箝制功能并不保守，可能是因为其不同的c端序列。
Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous “mini” release and for the priming and synaptotagmin-dependent Ca2+ triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory α-helix that clamps exocytosis, an essential central α-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.