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

被引:221
|
作者
Eckford, Paul D. W. [1 ,2 ]
Li, Canhui [1 ]
Ramjeesingh, Mohabir [1 ]
Bear, Christine E. [1 ,2 ,3 ]
机构
[1] Hosp Sick Children, Res Inst, Programme Mol Struct & Funct, Toronto, ON M5G 1X8, Canada
[2] Univ Toronto, Fac Med, Dept Biochem, Toronto, ON M5G 1X8, Canada
[3] Univ Toronto, Fac Med, Dept Physiol, Toronto, ON M5G 1X8, Canada
基金
加拿大健康研究院;
关键词
NUCLEOTIDE-BINDING DOMAIN; SMALL-MOLECULE MODULATOR; CL-CHANNEL; CHLORIDE CHANNEL; FUNCTIONAL RECONSTITUTION; IN-VITRO; RESCUE; G551D; PURIFICATION; DIMERIZATION;
D O I
10.1074/jbc.M112.393637
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:36639 / 36649
页数:11
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