Connexin 43 hemichannels are permeable to ATP
Connexin 43 hemichannels are permeable to ATP
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DOI：
10.1523/jneurosci.5048-07.2008
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发表时间：
2008-04-30
影响因子：
5.3
通讯作者：
Nedergaard, Maiken
中科院分区：
文献类型：
--
作者：
Kang, Jian;Kang, Ning;Nedergaard, Maiken
星形胶质细胞是电不可兴奋的细胞，其通过Ca 2+信号传导进行通信。长距离的细胞间Ca 2+波是由ATP的释放和附近细胞上的嘌呤能受体的激活启动的。以前的研究表明连接蛋白43（Cx43）参与ATP的释放，但ATP通过Cx43半通道的确切证据并不存在。在这里，通过几种替代方法，我们表明，ATP阴离子可以渗透通过Cx43半通道。首先，在表达Cx43的C6细胞中，在细胞附着和由内而外的斑片记录中检测到Cx43半通道的开口，但在表达Cx43-eGFP（增强型绿色荧光蛋白）或Cx43的C末端截短突变体的C6细胞中未检测到。第二，Cx43半通道开放被三种结构不同的缝隙连接通道阻断剂抑制，但不被P2 X（7）阻断剂Brilliant blue G。第三，ATP的生物发光成像结合单通道记录在由内而外的补丁配置表明，ATP流出符合通道开放，并缺席时，Cx43半通道关闭。第四，离子置换实验证实Cx43半通道对ATP是可渗透的。总之，这些观察结果提供了ATP通过Cx43半通道流出的第一个直接证据。此外，在急性制备的切片中，在海马星形胶质细胞的膜中鉴定出具有与C6细胞中的Cx43半通道相同的特征的推定的Cx43半通道。
Astrocytes are electrically nonexcitable cells that communicate by means of Ca2+ signaling. Long-distance intercellular Ca2+ waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here, through several alternative approaches, we show that ATP anions can permeate through Cx43 hemichannels. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X(7) blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single-channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In summary, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices.