by
Gudio Veit;
Radu G. Avramescu;
Annette N. Chiang;
Scott A. Houck;
Zhiwei Cai;
Kathryn W. Peters;
Jeong S. Hong;
Harvey B. Pollard;
William B. Guggino;
William E. Balch;
William R. Skach;
Garry R. Cutting;
Raymond A. Frizzell;
David N. Sheppard;
Douglas M. Cyr;
Eric Sorscher;
Jeffrey L. Brodsky;
Gergely L. Lukacs
More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, δF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for δF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.
by
Annette Ehrhardt;
Wook Chung;
Louise C. Pyle;
Wei Wang;
Krzysztof Nowotarski;
Cory M. Mulvihill;
Mohabir Rannjeesingh;
Jeong Hong;
Sadanandan E. Velu;
Hal A. Lewis;
Shane Atwell;
Steve Aller;
Christine E. Bear;
Gergely L. Lukacs;
Kevin L. Kirk;
Eric Sorscher
In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.