Norez Caroline, Antigny Fabrice, Becq Frédéric, Vandebrouck Clarisse
Institut de Physiologie et Biologie Cellulaires CNRS UMR 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France.
Traffic. 2006 May;7(5):562-73. doi: 10.1111/j.1600-0854.2006.00409.x.
The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.
囊性纤维化最常见的突变F508del，会导致囊性纤维化跨膜传导调节蛋白（CFTR）滞留在内质网（ER）中。滞留依赖于伴侣蛋白，其中许多蛋白，如钙连蛋白，需要钙来实现最佳活性。在此，我们表明有限且维持的内质网钙水平足以抑制F508del-CFTR/钙连蛋白相互作用，并恢复cAMP依赖的CFTR氯离子转运，从而显示异常转运得到纠正。我们将蛋白质免疫印迹分析、碘外流和钙测量技术应用于人气道上皮囊性纤维化细胞系CF15（F508del/F508del）。用毒胡萝卜素、姜黄素、2,5-二叔丁基对苯二酚或环匹阿尼酸抑制内质网钙泵，可维持与内源性F508del-CFTR转运活性恢复相关的钙阈值水平。特别是，环匹阿尼酸可恢复对2-氨基乙氧基二苯硼酸敏感的F508del-CFTR转运，其半数有效浓度为915纳米。相比之下，1,4,5-三磷酸或IP3受体激活剂，即ATP和组胺，虽然会暂时清空内质网细胞内钙库，但并不影响F508del-CFTR的转运。我们的数据表明，降低内质网钙水平不足以恢复F508del-CFTR的缺陷转运，而降低并维持低内质网钙水平可纠正人气道上皮细胞中内源性F508del-CFTR缺陷的生物合成途径。
