Abstract
Background and Purpose Cystic fibrosis (CF) is a life-limiting disease caused by mutations in the human CFTR gene, encoding an anion-selective channel. Because CF-causing mutations affect both CFTR permeation/gating and biogenesis, multi-assay approaches have been implemented in drug development, sequentially screening for channel function and membrane density. Here we present the first assay capable of simultaneous assessment of both CFTR characteristics.
Experimental approach Images of live HEK293 cells co-expressing a soluble and a CFTR-tagged fluorescent protein are automatically acquired and analysed to quantify both CFTR membrane density and ion channel function. We monitor F508del-CFTR, the most common disease-causing mutant. Furthermore we characterize a panel of 62 CF-causing mutations and profile effects of acute treatment with approved drug VX-770, mapping potentiation on CFTR structures.
Key Results We validate our assay by confirming F508del-CFTR rescue by incubation at low temperature, treatment with CFTR-targeting drugs and introduction of second-site revertant mutation R1070W. Measurements using the rare mutations panel also correlate well with published results.
Conclusions and Implications Mapping of VX-770 potentiation of mutants suggests that by increasing flexibility around the gate, the drug allows an alternative protein conformation at domain interfaces around site 1.
The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing more accurate inferences on gating/permeation properties than can be obtained by measuring cellular conductance alone. Finally, by providing a two-dimensional molecular characterization of individual mutant CFTR proteins, our assay can better inform development of single-drug and combination therapies addressing the root cause of CF disease.
What is already known
CFTR is an anion-selective channel, normally present in the plasma membrane of epithelial cells.
Hundreds of different mutations affect CFTR biogenesis and/or function causing cystic fibrosis (CF).
What this study adds
We present and validate an assay that simultaneously measures CFTR biogenesis and function.
Profiling a panel of CF-causing mutants suggests hypotheses on how approved drug VX-770 works.
Clinical Significance
The integrated assay boosts potential for discovery of more effective therapies, simultaneously repairing both defects.
Footnotes
- Text and Figure 6 have been updated, taking into consideration paper describing cryo-EM structure of VX-770-bound CFTR [Liu, F., Zhang, Z., Levit, A., Levring, J., Touhara, K. K., Shoichet, B. K., & Chen, J. (2019). Structural identification of a hotspot on CFTR for potentiation. Science, 364(6446), 1184-1188. doi:10.1126/science.aaw7611]. - A new panel has been added to Figure 6, to better represent overall profiling of VX-770 potentiation.
Abbreviations
- ABC
- ATP-binding cassette
- CF
- Cystic Fibrosis
- CFTR
- Cystic Fibrosis Transmembrane Conductance Regulator
- FYFP membrane
- average normalized YFP fluorescence intensity within the membrane zone
- FmCherry cell
- average normalized mCherry fluorescence over the entire cell
- GCFTR
- CFTR conductance
- Gtrans
- transient anion conductance
- IRES
- internal ribosome entry site
- NBD
- nucleotide binding domain
- PDL
- poly-D-lysine
- PO
- open probability
- σ
- CFTR membrane density
- SSR
- sum of squared residuals
- τtrans
- time constant of the transient anion conductance
- VM
- membrane potential
- WT
- wild type
- YFP
- yellow fluorescent protein