Rab3a is a small GTP binding protein associated with presynaptic vesicles that is thought to regulate vesicle targeting to active zones. Although this rab3a function implies that vesicle docking and action potential-evoked release might be inhibited in rab3a gene-deleted synapses, such inhibition has never been demonstrated. To investigate vesicle docking at the neuromuscular junction of rab3a gene-deleted (rab3a(-)) mice, we performed electron microscopy analysis of the diaphragm slow-fatigue (type I) synapses. We found a significant (26%) reduction in the number of vesicles docked to the presynaptic membrane in rab3a(-) terminals, although intraterminal vesicles were not affected. Aiming to detect possible changes in quantal release due to rab3a gene deletion, we minimized the variability between preparations employing focal recordings of synaptic responses from visualized type I endplates. We found a significant decrease in both evoked (27% reduction in quantal content) and spontaneous (28% reduction in mini frequency) quantal release. The decrease in the evoked release produced by rab3a deletion was most pronounced at reduced extracellular Ca(2+) concentrations (over 50% decrease at 0.5 and 0.2 mM Ca(2+)). By manipulating extracellular calcium, we demonstrated that calcium cooperativity is not altered in rab3a(-) synapses, however calcium sensitivity of quantal release is affected. Thus, we demonstrated that rab3a positively regulates docking and basal quantal release at the mouse neuromuscular junction. This result is consistent with the proposed role of rab3a in trafficking and targeting vesicles to the active zones.

译文

:Rab3a是与突触前囊泡相关的一种小GTP结合蛋白,被认为可调节将囊泡靶向活性区域。尽管此rab3a功能暗示在rab3a基因缺失的突触中囊泡对接和动作电位诱发的释放可能受到抑制,但这种抑制作用从未得到证实。若要研究囊泡停泊在rab3a基因缺失的(rab3a(-))小鼠的神经肌肉接头处,我们进行了隔膜慢疲劳(I型)突触的电子显微镜分析。我们发现停泊在rab3a(-)终端突触前膜的囊泡数量显着减少(26%),尽管终端内囊泡不受影响。为了检测由于rab3a基因缺失而导致的定量释放的可能变化,我们使用可视化I型终板突触反应的聚焦记录,将制剂之间的变异性最小化。我们发现诱发的(数量含量减少27%)和自发的(最小频率减少28%)定量释放均显着降低。 rab3a删除产生的诱发释放的减少在降低的细胞外Ca(2)浓度下最为明显(在0.5和0.2 mM Ca(2)处降低了50%以上)。通过操纵细胞外钙,我们证明了rab3a(-)突触中钙的协同作用并没有改变,但是钙对定量释放的敏感性受到影响。因此,我们证明了rab3a积极调节小鼠神经肌肉连接处的对接和基础定量释放。此结果与rab3a在贩运和将囊泡靶向活性区域中的拟议作用一致。

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