RT Journal Article
SR Electronic
T1 Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 631614
DO 10.1101/631614
A1 Stella Prins
A1 Emily Langron
A1 Cato Hastings
A1 Emily J. Hill
A1 Andra C. Stefan
A1 Lewis D. Griffin
A1 Paola Vergani
YR 2020
UL http://biorxiv.org/content/early/2020/05/05/631614.abstract
AB 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 StatementThe authors have declared no competing interest.