Department of Molecular and Cellular Physiology, Stanford University, 265 Campus Drive, Stanford, CA 94305-5453, USA.
Neuron. 2011 Apr 28;70(2):244-51. doi: 10.1016/j.neuron.2011.03.011.
Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Doc2B lacking Ca(2+)-binding sites. Thus, our data suggest that Doc2s are modulators of spontaneous synaptic transmission that act by a Ca(2+)-independent mechanism.
突触结合蛋白和 Doc2 蛋白，令人感兴趣的是，Doc2 蛋白可能代表高亲和力的钙传感器，它通过缺失突触结合蛋白而被激活，从而解释了在突触结合蛋白缺失的突触中自发释放的增加。在这里，我们使用依赖 shRNA 的 Doc2 家族的所有四个钙结合蛋白的四重敲低，证实了 Doc2 缺失的突触中自发释放事件的频率明显降低。然而，在突触结合蛋白-1 缺失的突触中敲低 Doc2s，并不能降低这些突触的自发释放增加或诱发释放减少，这表明 Doc2s 不构成异步释放的钙传感器。此外，挽救实验表明，在野生型突触中，Doc2 敲低诱导的自发释放减少完全被缺乏钙结合位点的突变型 Doc2B 逆转。因此，我们的数据表明，Doc2 蛋白是通过钙独立机制发挥作用的自发突触传递的调节剂。
