Abstract
CFTR, a plasma membrane anion channel, plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrhoeas, while 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 (reflecting folding, trafficking, metabolic stability). Recently, treatments with two drug classes targeting CFTR – one boosting ion-channel function (potentiators), 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 around site 1.
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
New Figure 6 added, with labels for individual mutations. This allows an immediate representation of how severe a defect each mutation causes in biogenesis (distance from WT-CFTR on x-axis) and/or in gating and permeation properties (vertical distance from blue dotted line). New Supplementary Figure 8 added. Results obtained here with the rare mutation panel are compared with published datasets (Van Goor et al., J Cyst Fibros. 2014; 13: 29-36; Yu et al., J Cyst Fibros. 2012; 11: 237-45; Sosnay et al. Nat Genet. 2013; 45: 1160-7).