Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14318-23. doi: 10.1073/pnas.1101818108. Epub 2011 Aug 15.
Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.
在快速突触传递过程中，突触小泡（SV）的胞吐作用是由可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的组装介导的，该复合物由三个成员的卷曲螺旋形成：囊泡 SNARE 蛋白、突触融合蛋白 2（syb2）以及突触前膜 SNAREs 突触融合蛋白 1A 和 SNAP-25。然而，对于 SV 引发和融合所需的最小 SNARE 复合物数量存在争议。为了定量这个有效数量，我们测量了在缺乏内源性 syb2 的培养海马神经元中表达 pH 敏感型 GFP 变体 pHluorin（pHl）的融合囊泡的荧光反应，该 pHl 融合到囊泡 SNARE syb2 的腔域。荧光反应是量子化的，单位信号精确对应于单个 pHl 分子。使用这种方法，我们发现每个 SV 有两个拷贝的 spH 可以完全挽救诱发融合，而只表达一个 spH 的 SV 则无法在刺激时快速融合。因此，在快速突触传递过程中，两个 syb2 分子和可能两个 SNARE 复合物对于 SV 融合是必要且充分的。
