Molecular and pharmacological evaluation of rare, cystic fibrosis-causing missense mutations of the CFTR channel.

Cystic fibrosis (CF) is a life-threatening condition caused by pathogenic mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. While most patients with frequent mutations are treated with a combination of corrector and potentiator drugs (elexacaftor-tezacaftor-ivacaftor, ETI), for many rare variants no such treatment is approved. Here we characterized the molecular pathologies of five rare CF-associated mutants (G126D, I336K, T465I, T582I and D984V), and evaluated their responsiveness to ETI treatment. Channel expression was investigated by western blot from HEK-293T cells transiently expressing mutant channels, with or without corrector drugs (elexacaftor and tezacaftor). Drug efficiency was assessed by the enhancement of channel glycosylation. Maturation was mildly (T582I, G126D) to severely (T465I) defective for all five mutants, but could be restored to wild-type levels by incubation with corrector drugs. Gating properties and responses to potentiator drugs (ivacaftor and elexacaftor) were assessed in macroscopic and single-channel inside-out patch-clamp recordings from mutant channels expressed in Xenopus laevis oocytes. Mutations G126D, T582I, I336K and T465I markedly decreased channel open probability, with the greatest reduction being caused by mutation T465I. Mutations G126D, I336K and T465I also slightly impaired unitary conductance. Applying potentiator drugs boosted gating for all variants, producing multifold increases in macroscopic currents. In conclusion, all five mutations impair channel maturation and gating to various degrees, but a normal glycosylation pattern can be restored with correctors, and channel gating can be enhanced with potentiators. These in vitro observations suggest that ETI treatment would be beneficial for CF patients carrying an allele with any of the five mutations. KEY POINTS: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel cause cystic fibrosis. Highly efficient modulator therapy has been approved for ∼90% of cystic fibrosis patients, but a large number of rare mutations are not yet eligible for treatment. Five rare CFTR mutations found in cystic fibrosis patients in Hungary and worldwide are shown here to produce complex channel pathologies that to differing degrees affect protein maturation, channel gating and anion permeation through the open pore. All five variants respond strongly to clinically employed modulator drugs that boost channel surface expression and gating. The results help us understand how these CFTR mutations lead to cystic fibrosis, and suggest that patients carrying any of the five variants would probably benefit from modulator therapy.