Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening

J Biomol Screen. 2015 Dec;20(10):1204-17. doi: 10.1177/1087057115605834. Epub 2015 Sep 18.


There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

Keywords: CFTR chloride channel; high-throughput functional screening; insertion/deletion mutagenesis; mutation theratype; rare cystic fibrosis mutations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminophenols / therapeutic use
  • Base Sequence
  • Cystic Fibrosis / drug therapy*
  • Cystic Fibrosis Transmembrane Conductance Regulator / chemistry
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / physiology
  • Drug Discovery*
  • HEK293 Cells
  • High-Throughput Screening Assays*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Polymerase Chain Reaction*
  • Quinolones / therapeutic use
  • Sequence Alignment


  • Aminophenols
  • CFTR protein, human
  • Quinolones
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • ivacaftor