Transient Receptor Potential Canonical Channel 6 Links Ca2+ Mishandling to Cystic Fibrosis Transmembrane Conductance Regulator Channel Dysfunction in Cystic Fibrosis
Transient Receptor Potential Canonical Channel 6 Links Ca2+ Mishandling to Cystic Fibrosis Transmembrane Conductance Regulator Channel Dysfunction in Cystic Fibrosis
复制标题
DOI：
10.1165/rcmb.2009-0347oc
复制
发表时间：
2011-01-01
影响因子：
6.4
通讯作者：
Vandebrouck, Clarisse
中科院分区：
文献类型：
--
作者：
Antigny, Fabrice;Norez, Caroline;Vandebrouck, Clarisse
在囊性纤维化（CF）中，观察到细胞Ca2+稳态的异常控制。我们假设瞬时受体电位规范（TRPC）通道可能是CF细胞中Ca2+浓度异常和囊性纤维化跨膜传导调节剂（CFTR）功能障碍之间的联系。我们用特异性和混乱的小干扰RNA（siRNA）处理CF和非CF人上皮细胞，测量了TRPC和CFTR的活性（使用膜片钳和荧光探针）和相互作用（使用Western blotting和共免疫沉淀）。与非CF细胞相比，CF中trpc6介导的Ca2+内流异常增加。在纠正CF细胞中异常的F508缺失(del)-CFTR运输后，trpc6依赖性Ca2+内流水平也正常化。在CF细胞中，siRNA-TRPC6减少了这种异常的Ca2+内流。在非cf细胞中，siRNA-TRPC6减少了野生型(wt)-CFTR的Ca2+内流和活性。共免疫沉淀实验显示TRPC6/CFTR和TRPC6/F508 del-CFTR在CF或非CF上皮细胞中相互作用。尽管siRNA-CFTR在非CF细胞中降低了wt-CFTR的活性，在校正后的CF细胞中降低了F508 del-CFTR的活性，但它也增强了非CF细胞中trpc6依赖性Ca2+内流，与CF细胞中获得的结果相似。最后，CFTR和TRPC6之间的功能和互反偶联也在新鲜分离的肺样本非cf纤毛人上皮细胞中检测到。这些数据表明，TRPC6和CFTR在气道上皮细胞的分子复合体内功能上相互偶联。因为这种功能偶联在CF细胞中丢失，trpc6依赖性Ca2+内流是异常的。
In cystic fibrosis (CF), abnormal control of cellular Ca2+ homeostasis is observed. We hypothesized that transient receptor potential canonical (TRPC) channels could be a link between the abnormal Ca2+ concentrations in CF cells and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We measured the TRPC and CFTR activities (using patch clamp and fluorescent probes) and interactions (using Western blotting and co-immunoprecipitation) in CF and non-CF human epithelial cells treated with specific and scrambled small interfering RNA(siRNA). The TRPC6-mediated Ca2+ influx was abnormally increased in CF compared with non-CF cells. After correction of abnormal F508 deletion (del)-CFTR trafficking in CF cells, the level of TRPC6-dependent Ca2+ influx was also normalized. In CF cells, siRNA-TRPC6 reduced this abnormal Ca2+ influx. In non-CF cells, siRNA-TRPC6 reduced the Ca2+ influx and activity wild-type (wt)-CFTR. Co-immunoprecipitation experiments revealed TRPC6/CFTR and TRPC6/F508 del-CFTR interactions in CF or non-CF epithelial cells. Although siRNA-CFTR reduced the activity of wt-CFTR in non-CF cells and of F508 del-CFTR in corrected CF cells, it also enhanced TRPC6-dependent Ca2+ influx in non-CF cells, mimicking the results obtained in CF cells. Finally, this functional and reciprocal coupling between CFTR and TRPC6 was also detected in non-CF ciliated human epithelial cells freshly isolated from lung samples. These data indicate that TRPC6 and CFTR are functionally and reciprocally coupled within a molecular complex in airway epithelial human cells. Because this functional coupling is lost in CF cells, the TRPC6-dependent Ca2+ influx is abnormal.