Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis
Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis
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DOI：
10.1111/j.1600-0854.2009.00949.x
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发表时间：
2009-09-01
期刊：
影响因子：
4.5
通讯作者：
Pointis, Georges
中科院分区：
文献类型：
--
作者：
Carette, Diane;Gilleron, Jerome;Pointis, Georges
Connexin 33 （Cx33）是睾丸特异性的间隙连接蛋白。我们之前报道过，Cx33通过将Cx43隔离在支持细胞的早期核内体中，对间隙连接的细胞间通讯产生显性负向影响。然而，驱动这一过程的分子机制尚不清楚。本研究分析了：(1)转染Cx33- dsred2和Cx43-绿色荧光蛋白载体的野生型支持细胞中Cx33和Cx43的转运；（ii）异质Cx33/Cx43半通道的形成及其与间隙连接斑块的结合。荧光寿命成像显微镜-荧光共振能量转移和视频显微镜研究表明，Cx33和Cx43结合形成异质低聚物，沿微管运输到质膜。然而，含有Cx33的斑块没有功能。免疫沉淀实验显示，封闭带-1 （ZO-1）是一种支架蛋白，用于在细胞-细胞边界的间隙连接斑块中固定Cx，与睾丸提取物中的Cx33相关。在表达Cx33的细胞中，Cx33和ZO-1特异性地与P-1磷酸化和P-0未磷酸化的Cx43亚型相互作用，使ZO-1膜信号水平降低。提示Cx33改变Cx43/ZO-1的关联可能是Cx33对间隙连接斑块发挥显性负作用的机制之一。
Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell-cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P-1 phosphorylated and P-0 unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.