P030 Ivacaftor responsiveness of two cystic fibrosis mutations that affect the same residue in the twelfth transmembrane segment of CFTR

Abstract

Many residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel are the site of more than one CFTR variant. As part of the effort to interpret the disease implications of CFTR2 variants, we investigated two rare missense substitutions of the serine at codon1159 in the twelfth transmembrane segment, which contributes to the channel pore. S1159F replaces the polar serine residue by the non-polar residue phenylalanine, whereas S1159P introduces the helix breaker proline. When expressed in recombinant airway epithelial cells, S1159F and S1159P are substantially rescued by ivacaftor and lumacaftor, with most function restored using both drugs together. To understand their effects on individual CFTR Cl- channels, we performed single-channel patch-clamp recordings on excised inside-out membrane patches from Chinese hamster ovary cells that were incubated at 27degreeC for >=7 days to promote the plasma membrane expression of CFTR variants. Both variants formed Cl- channels activated by PKA-dependent phosphorylation and gated by ATP. However, their single-channel current amplitudes were reduced by 27% (S1159F) and 24% (S1159P) at -50 mV (n > 20) when compared with that of wild-type CFTR. Although both variants retained the bursting pattern of channel gating characteristic of wild-type CFTR, the frequency of channel openings and their duration were reduced greatly with the result that open probability (Po) was decreased by 88% (S1159F) and 71% (S1159P) in ATP (1 mM) (n > 20) compared to that of wild-type CFTR. Consistent with these data, both variants severely reduced the affinity and efficacy of CFTR for ATP. Ivacaftor (10-100 nM) doubled the Po of both variants, but did not restore wild-type levels of channel activity to them. In Conclusion, S1159F and S1159P cause CF by altering both the conduction and gating properties of CFTR, but each responds robustly to ivacaftor-lumacaftor combination therapy.