CFTR potentiators partially restore channel function to A561E-CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR

Br J Pharmacol. 2014 Oct;171(19):4490-503. doi: 10.1111/bph.12791. Epub 2014 Sep 5.


Background and purpose: Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease.

Experimental approach: Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques.

Key results: Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl(-) channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches.

Conclusions and implications: Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminophenols / pharmacology
  • Animals
  • Biotinylation
  • Cell Line
  • Cricetinae
  • Cystic Fibrosis / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / physiology*
  • Genistein / pharmacology
  • Iodides / metabolism
  • Ion Channel Gating / drug effects
  • Mutation
  • Quinolones / pharmacology
  • Temperature


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