Department of Anatomy and Cell Biology and Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical School, Kansas City, Kansas 66160, USA.
J Neurosci. 2011 Jan 12;31(2):512-25. doi: 10.1523/JNEUROSCI.3771-10.2011.
Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC β1b or β4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin β2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin β2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin β2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin β2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.
突触形成需要将突触前活性区（即突触囊泡释放位点）精确地排列在突触后神经递质受体簇附近；然而，负责这些过程的分子机制仍不清楚。在这里，我们发现 P/Q 型和 N 型电压依赖性钙通道（VDCCs）作为支架蛋白，在突触前活性区的组织中发挥着重要作用。P/Q 型和 N 型 VDCC 双敲除小鼠的神经肌肉接头显示出正常的大小，但活性区和停泊囊泡的数量显著减少，并且活性区蛋白 Bassoon、Piccolo 和 CAST/Erc2 的表达减弱。与这种表型一致，通过免疫沉淀证实了 VDCC β1b 或 β4 亚基与活性区特异性蛋白 Bassoon 或 CAST/Erc2 之间的直接相互作用。由于缺乏突触前 VDCC，活性区数量的减少类似于自身免疫性神经肌肉疾病 Lambert-Eaton 肌无力综合征中观察到的病理情况。在双敲除小鼠的突触裂隙中，我们还观察到突触组织者层粘连蛋白 β2 蛋白的减少，层粘连蛋白 β2 是 P/Q 型和 N 型 VDCC 的细胞外配体。然而，双敲除小鼠的层粘连蛋白 β2 转录水平并没有降低，这表明层粘连蛋白 β2 蛋白在突触中的积累需要与突触前 VDCC 的相互作用。这些结果表明，突触前 VDCC 将靶源性突触组织者层粘连蛋白 β2 与活性区蛋白联系起来，并作为支架蛋白将活性区蛋白锚定在突触前膜上。
