Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner
复制标题
DOI：
10.1074/jbc.m112.393637
复制
发表时间：
2012-10-26
影响因子：
4.8
通讯作者：
Bear, Christine E.
中科院分区：
文献类型：
--
作者：
Eckford, Paul D. W.;Li, Canhui;Bear, Christine E.
囊性纤维化跨膜传导调节因子（CFTR）作为上皮细胞顶膜上的通道。囊性纤维化基因中的致病突变可导致CFTR蛋白错误折叠，如F508 del突变和/或通道功能障碍的情况。最近，一种小分子VX-770（ivacaftor）在恢复携带G551 D突变的患者的肺功能方面显示出疗效，这与修复其通道门控缺陷有关。然而，这些研究并未详细揭示VX-770的作用机制。通常，CFTR通道活性受磷酸化、ATP结合和水解调节。因此，已经假设VX-770改变了这些代谢事件中的一个或多个。在这项研究中，我们使用纯化CFTR蛋白的重建系统检查VX-770活性，该系统能够控制已知的调节因子。我们研究了VX-770与CFTR相互作用的后果纳入平面脂质双层和脂蛋白体，使用一种新的通量为基础的测定。我们发现，纯化和磷酸化的CFTR在Mg-ATP存在下增强，表明VX-770直接结合CFTR蛋白，而不是相关的激酶或磷酸酶。有趣的是，我们还发现VX-770在名义上不存在Mg-ATP的情况下增强了纯化的和突变的CFTR的通道活性。这些发现表明，VX-770可以通过非传统的ATP依赖性机制引起CFTR通道开放。这项工作为将来研究使用VX-770作为探针介导CFTR门控的结构特性奠定了基础。
The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.