The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR—one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)—have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

中文翻译：

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用于同时定量 CFTR 离子通道功能和质膜邻近性的荧光测定。


囊性纤维化跨膜电导调节器（CFTR）是质膜阴离子通道，在控制跨上皮液体运动中发挥关键作用。过度激活会导致分泌性腹泻期间肠道液体流失，而 CFTR 突变则导致囊性纤维化 (CF)。阴离子渗透性取决于 CFTR 通道的工作情况（渗透/门控）以及膜上存在的数量。最近，针对 CFTR 的两类药物治疗——一种增强离子通道功能（增强剂），另一种增加质膜密度（校正剂）——为 CF 患者带来了显着的健康益处。在这里，我们提出了一种基于图像的荧光测定，可以快速同时估计 CFTR 离子通道功能和蛋白质与膜的接近程度。我们监测最常见的 CF 致病变异 F508del-CFTR，并确认通过低温、CFTR 靶向药物和第二位点回复突变 R1070W 进行挽救。此外，我们还描述了一组 62 个 CF 引起的突变。我们的测量结果与已发表的数据（电生理学和生物化学）很好地相关，进一步证实了该测定的有效性。最后，我们分析了经批准的增强剂药物 VX-770 进行急性治疗对罕见突变组的影响。绘制 CFTR 结构上的增强曲线提出了药物作用的机制假设，表明 VX-770 可能允许具有域界面替代排列的开放通道构象。该测定是研究 CFTR 分子机制的宝贵工具，可以准确推断门控/渗透。 此外，通过提供 CFTR 蛋白的二维表征，它可以更好地为解决 CF 疾病根本原因的单一药物和精准疗法的开发提供信息。