Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis.

KEY POINTS: Electrophysiological characterization of Q1412X-CFTR, a C-terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X-CFTR suggest that the reduced open probability in Q1412X-CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C-terminal region of NBD2 reveal the importance of the last two beta-strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically-approved drugs (VX-770 and VX-809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. ABSTRACT: Cystic fibrosis (CF) is caused by loss-of-function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-activated but ATP-gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease-associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C-terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X-CFTR, a severe-form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po ). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP-binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta-strands in NBD2 and the whole C-terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C-terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild-type channels. However, the deletion of the last beta-strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically-approved VX-809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX-770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X-CFTR provides the conceptual basis for the treatment of patients carrying this mutation.