Zimmermann Johannes, Trimbuch Thorsten, Rosenmund Christian
Neuroscience Research Center and NeuroCure Cluster of Excellence, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Neuroscience Research Center and NeuroCure Cluster of Excellence, Charité-Universitätsmedizin Berlin, Berlin, Germany
J Neurophysiol. 2014 Sep 15;112(6):1559-65. doi: 10.1152/jn.00340.2014. Epub 2014 Jun 18.
The core machinery of synaptic vesicle fusion consists of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, the two t-SNAREs at the plasma membrane (SNAP-25, Syntaxin 1) and the vesicle-bound v-SNARE synaptobrevin 2 (VAMP2). Formation of the trans-oriented four-α-helix bundle between these SNAREs brings vesicle and plasma membrane in close proximity and prepares the vesicle for fusion. The t-SNAREs are thought to be necessary for vesicle fusion. Whether the v-SNAREs are required for fusion is still unclear, as substantial vesicle priming and spontaneous release activity remain in mammalian mass-cultured synaptobrevin/cellubrevin-deficient neurons. Using the autaptic culture system from synaptobrevin 2 knockout neurons of mouse hippocampus, we found that the majority of cells were devoid of any evoked or spontaneous release and had no measurable readily releasable pool. A small subpopulation of neurons, however, displayed release, and their release activity correlated with the presence and amount of v-SNARE synaptobrevin 1 expressed. Comparison of synaptobrevin 1 and 2 in rescue experiments demonstrates that synaptobrevin 1 can substitute for the other v-SNARE, but with a lower efficiency in neurotransmitter release probability. Release activity in synaptobrevin 2-deficient mass-cultured neurons was massively reduced by a knockdown of synaptobrevin 1, demonstrating that synaptobrevin 1 is responsible for the remaining release activity. These data support the hypothesis that both t- and v-SNAREs are absolutely required for vesicle priming and evoked release and that differential expression of SNARE paralogs can contribute to differential synaptic coding in the brain.
突触小泡融合的核心机制由三种可溶性 N - 乙基马来酰亚胺敏感因子附着受体（SNARE）蛋白组成，即位于质膜上的两种靶标 SNARE（SNAP - 25、Syntaxin 1）以及与小泡结合的囊泡 SNARE 突触融合蛋白 2（VAMP2）。这些 SNARE 之间形成的反式排列的四螺旋束使小泡与质膜紧密靠近，并为小泡融合做好准备。一般认为靶标 SNARE 对小泡融合是必需的。囊泡 SNARE 是否为融合所必需仍不清楚，因为在哺乳动物大量培养的突触融合蛋白/细胞融合蛋白缺陷神经元中仍保留有大量的小泡预充能和自发释放活性。利用从小鼠海马体的突触融合蛋白 2 基因敲除神经元建立的自突触培养系统，我们发现大多数细胞没有任何诱发或自发释放，并且没有可测量的易释放池。然而，一小部分神经元显示出释放，并且它们的释放活性与所表达的囊泡 SNARE 突触融合蛋白 1 的存在和量相关。在拯救实验中对突触融合蛋白 1 和 2 的比较表明，突触融合蛋白 1 可以替代另一种囊泡 SNARE，但在神经递质释放概率方面效率较低。通过敲低突触融合蛋白 1，突触融合蛋白 2 缺陷的大量培养神经元中的释放活性大幅降低，这表明突触融合蛋白 1 负责剩余的释放活性。这些数据支持这样的假说，即靶标 SNARE 和囊泡 SNARE 对于小泡预充能和诱发释放都是绝对必需的，并且 SNARE 旁系同源物的差异表达可能有助于大脑中不同的突触编码。
