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CFTR potentiators partially restore channel function to A 561 E ‐CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F 508del‐ CFTR
Author(s) -
Wang Yiting,
Liu Jia,
Loizidou Avgi,
Bugeja Luc A,
Warner Ross,
Hawley Bethan R,
Cai Zhiwei,
Toye Ashley M,
Sheppard David N,
Li Hongyu
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12791
Subject(s) - ivacaftor , potentiator , cystic fibrosis transmembrane conductance regulator , cystic fibrosis , chemistry , furin , mutant , microbiology and biotechnology , mutation , pharmacology , biochemistry , medicine , biology , gene , enzyme
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 A 561 E , a CF mutation found frequently in P ortugal. Like the most common CF mutation F 508del, A 561 E 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 A 561 E ‐ CFTR protein to the cell surface. Like F 508del‐ CFTR , low temperature‐rescued A 561 E ‐ CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A 561 E ‐ CFTR also exhibited thermoinstability, losing function more quickly than F 508del‐ 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 A 561 E ‐ CFTR . Interestingly, ivacaftor restored wild‐type levels of channel activity (as measured by open probability) to single A 561 E ‐ and F 508del‐ CFTR Cl − channels. However, it accentuated the thermoinstability of both mutants in cell‐free membrane patches. Conclusions and Implications Like F 508del‐ CFTR , A 561 E ‐ CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature‐rescued A 561 E ‐ CFTR . Transformational drug therapy for A 561 E ‐ CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.