Quantitative localization of Cav2.1 (P/Q-type) voltage-dependent calcium channels in Purkinje cells: somatodendritic gradient and distinct somatic coclustering with calcium-activated potassium channels.
Quantitative localization of Cav2.1 (P/Q-type) voltage-dependent calcium channels in Purkinje cells: somatodendritic gradient and distinct somatic coclustering with calcium-activated potassium channels.
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DOI：
10.1523/jneurosci.2921-12.2013
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发表时间：
2013-02-20
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影响因子：
--
通讯作者：
Shigemoto R
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文献类型：
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作者：
Indriati DW;Kamasawa N;Matsui K;Meredith AL;Watanabe M;Shigemoto R
P/Q型电压依赖性钙通道在神经递质的释放、树突信号的整合、树突棘的产生和基因表达中起着关键作用。由这些通道产生的高细胞内钙浓度瞬变被限制在距离通道数十至数百纳米的范围内。因此，长期以来，人们一直在寻找这些通道沿质膜沿着的精确定位，以破译每个神经元细胞的功能是如何控制的。在这里，我们分析了Cav2.1亚基的P/Q型通道的分布，使用高灵敏度SDS消化冷冻断裂复制标记在大鼠小脑浦肯野细胞。标记效率是这样的，在每个平行的纤维活性区的免疫金颗粒的数量是可比的功能通道从以前的报告计算。Cav2.1在浦肯野细胞中有散在和聚集两种分布模式。散在的Cav2.1具有体树突梯度，免疫金颗粒的密度从索马到远端树突增加2.5倍。密度比分散颗粒高74倍的另一个群体被发现在索马和初级树突的P-面上的膜内颗粒簇内。Cav2.1的两个群体早在P3就被发现，并在出生后第二周增加到成熟水平。使用双免疫金标记，我们发现几乎所有的Cav2.1簇都与两种类型的钙激活钾通道BK和SK 2共定位，最近邻距离约为40 nm。通过Cav2.1通道的开放产生的钙纳米结构域可能激活限制去极化程度的两个通道。
P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Cav2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ~40 nm. Calcium nanodomain created by the opening of Cav2.1 channels likely activates the two channels that limit the extent of depolarization.