Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.
Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.
复制标题
DOI：
10.1074/jbc.m116.751537
复制
发表时间：
2017-03-03
期刊：
影响因子：
--
通讯作者：
Sheppard DN
中科院分区：
文献类型：
--
作者：
Meng X;Wang Y;Wang X;Wrennall JA;Rimington TL;Li H;Cai Z;Ford RC;Sheppard DN
囊性纤维化（CF）是由突变引起的，这些突变破坏了囊性纤维化跨膜传导调节因子（CFTR）Cl−通道的质膜表达、稳定性和功能。两种小分子，CFTR校正剂lumacaftor和增效剂ivacaftor，现在临床上用于治疗CF，尽管一些研究表明它们对CFTR稳定性具有抵消作用。在这里，我们研究了这些化合物对F508 del-CFTR（最常见的CF突变）不稳定性的影响。为了研究单个CFTR Cl−通道，我们进行了单通道记录，而为了评估整个CFTR群体，我们使用了纯化的CFTR蛋白和宏观CFTR Cl−电流。在37 °C下，低温拯救的F508 del-CFTR在无细胞膜贴片中更快地失去功能，并显示出改变的通道门控和通过开放通道的电流。与纯化的野生型CFTR相比，全长F508 del-CFTR的热稳定性低约10 °C。鲁玛卡托部分稳定了纯化的全长F508 del-CFTR，并略微延迟了单个F508 del-CFTR Cl−通道的失活。相比之下，依伐卡托进一步使全长F508 del-CFTR不稳定并加速通道失活。F508 del-CFTR表达细胞与鲁玛卡托和依伐卡托的慢性（延长）共孵育使宏观F508 del-CFTR Cl−电流失活。然而，在单通道水平，慢性共孵育大大增加了F508 del-CFTR通道的活性和时间稳定性，在大多数，但不是全部，无细胞膜补丁。我们得出结论，长期lumacaftor和ivacaftor联合治疗恢复了F508 del-CFTR Cl−通道的一小部分亚群的稳定性，但大多数仍然不稳定。更全面地了解这些影响和小F508 del-CFTR亚群的特征可能对CF治疗开发至关重要。
Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl− channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl− currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl− channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl− currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl− channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development.