Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Centre National de la Recherche Scientifique, 40 Avenue du Recteur Pineau, Poitiers, France.
Am J Respir Cell Mol Biol. 2011 Jan;44(1):83-90. doi: 10.1165/rcmb.2009-0347OC. Epub 2010 Mar 4.
In cystic fibrosis (CF), abnormal control of cellular Ca(2+) homeostasis is observed. We hypothesized that transient receptor potential canonical (TRPC) channels could be a link between the abnormal Ca(2+) 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 Ca(2+) 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 Ca(2+) influx was also normalized. In CF cells, siRNA-TRPC6 reduced this abnormal Ca(2+) influx. In non-CF cells, siRNA-TRPC6 reduced the Ca(2+) 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 Ca(2+) 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 Ca(2+) influx is abnormal.
在囊性纤维化 (CF) 中，观察到细胞钙 (Ca(2+)) 稳态的异常控制。我们假设瞬时受体电位经典 (TRPC) 通道可能是 CF 细胞中异常 Ca(2+) 浓度与囊性纤维化跨膜电导调节因子 (CFTR) 功能障碍之间的联系。我们使用膜片钳和荧光探针测量 CF 和非 CF 人上皮细胞中的 TRPC 和 CFTR 活性（使用 Western blot 和共免疫沉淀测量相互作用）用特异性和乱序小干扰 RNA (siRNA) 处理。与非 CF 细胞相比，CF 中的 TRPC6 介导的 Ca(2+) 内流异常增加。在 CF 细胞中纠正异常 F508 缺失 (del)-CFTR 转运后，TRPC6 依赖性 Ca(2+) 内流水平也正常化。在 CF 细胞中，siRNA-TRPC6 降低了这种异常的 Ca(2+) 内流。在非 CF 细胞中，siRNA-TRPC6 降低了 Ca(2+) 内流和野生型 (wt)-CFTR 的活性。共免疫沉淀实验表明 CF 或非 CF 上皮细胞中存在 TRPC6/CFTR 和 TRPC6/F508 del-CFTR 相互作用。尽管 siRNA-CFTR 降低了非 CF 细胞中 wt-CFTR 的活性和校正 CF 细胞中 F508 del-CFTR 的活性，但它也增强了非 CF 细胞中 TRPC6 依赖性 Ca(2+) 内流，模拟了在 CF 细胞中获得的结果。最后，在从肺样本中新鲜分离的非 CF 纤毛人上皮细胞中也检测到 CFTR 和 TRPC6 之间的这种功能性和相互偶联。这些数据表明，在气道上皮人细胞中，TRPC6 和 CFTR 在分子复合物中具有功能性和相互偶联。由于这种功能偶联在 CF 细胞中丢失，因此 TRPC6 依赖性 Ca(2+) 内流异常。
