Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses
Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses
复制标题
DOI：
10.1073/pnas.1101818108
复制
发表时间：
2011-08-23
影响因子：
11.1
通讯作者：
Klingauf, Jurgen
中科院分区：
文献类型：
--
作者：
Sinha, Raunak;Ahmed, Saheeb;Klingauf, Jurgen
突触囊泡在快速突触传递过程中的胞吐作用是由N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)家族的三个成员卷绕而成的复合体所介导的：囊泡SNARE蛋白、突触短缩蛋白2(Syb2)以及突触前膜SNARE-1a和SNAP-25。然而，对于SV启动和融合最少需要多少诱捕复合体还存在争议。为了量化这一有效数量，我们测量了在培养的缺乏内源性Syb2的海马神经元中，表达PHluorin(PHL)的单个融合囊泡的荧光反应。PHL是GFP的一种对pH敏感的变体，与囊泡圈套Syb2的管腔域(Sph)融合。荧光响应是定量的，单一信号精确地对应于单个PHluorin分子。采用这种方法，我们发现每个SV两个拷贝的SPH完全挽救了诱发性融合，而只表达一个SPH的SVS不能在刺激下快速融合。因此，两个syb2分子和可能的两个SNARE复合体对于快速突触传递过程中的SV融合是必要的和充分的。
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.