Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells
Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells
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DOI：
10.1007/s00441-015-2281-x
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发表时间：
2016-03-01
影响因子：
3.6
通讯作者：
Meier, Carola
中科院分区：
文献类型：
--
作者：
Beckmann, Anja;Grissmer, Alexander;Meier, Carola
Pannexins （Panx）是一种与连接蛋白具有相似膜拓扑结构的蛋白质，连接蛋白是间隙连接的整体膜蛋白。Panx1通道通常在大量系统和细胞过程中起重要作用，其功能已被彻底表征。相比之下，对通道结构和亚细胞分布知之甚少。因此，我们确定了Panx1通道在培养细胞中的亚细胞定位，并旨在体外鉴定通道形态。对过表达eyfp -Panx1的HEK 293细胞进行冷冻断裂复制免疫标记，在质膜上鉴定出大颗粒，用GFP或Panx1抗体进行免疫金标记。这些细胞的核膜中没有标记或颗粒，表明Panx1-EYFP通道定位于质膜。粒子的组装是不规则的，这与缝隙连接的规则模式形成对比。事实上，没有对应的细胞被鉴定出来，这将表明细胞间信号传导，支持Panx1通道在一个膜内的想法。对照细胞（仅转染EYFP，未转染）均无颗粒和免疫金标。总之，这项研究首次证明了Panx1通道在完整细胞中的形态和组装。将Panx1通道识别为质膜内的大颗粒，为今后的研究中识别组织中的Panx1通道提供了必要的知识。因此，这些结果为详细分析Panx1及其近邻如嘌呤能受体在体内的亚细胞定位开辟了新的途径。
Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.