PhD Scientific Days 2025

Molecular Medicine V.

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

Name of the presenter

Závoti Olivér

Institute/workplace of the presenter

Department of Biochemistry

Authors

Dr. Olivér Závoti¹

1: Department of Biochemistry

Text of the abstract

Cystic fibrosis (CF) is a life-threatening disease caused by the pathogenic mutations of the CFTR anion channel. For most patients who carry frequent mutant alleles, modulator therapy combining corrector and potentiator drugs mitigates the symptoms of the disease. Still, for many rare variants, no such treatment is available.
Recently, five rare, previously unobserved, cystic fibrosis causing missense mutations (G126D, I336K, T465I, T582I, D984V) were identified in the Hungarian CF population. Our goal was to better understand the functional consequences of these mutations.
Channel expression and maturation were examined by Western blot using crude membrane preps of transiently transfected HEK-293 cell cultures. The mutant channels were also expressed in Xenopus laevis oocytes, and channel gating was studied using the inside-out patch clamp technique.
Channel glycosylation was impaired in each of the mutant constructs. While G126D and T582I produced a slight decrease in the amount of fully glycosylated CFTR, I336K, T465I, and D984V markedly affected maturation. Treating the cell cultures with corrector drugs (Vx-445 and Vx-661) completely restored glycosylation to wild-type levels.
Our earlier results pointed out that G126D, T582I and D984V all affect channel gating differently. Consequently, pharmacological studies were conducted, which showed that the three constructs respond to potentiator drugs (Vx-770, Vx-445) with multifold increases in their currents. The potentiators were now also tested on the I336K and T465I variants. Upon exposure to the potentiators, the two mutants exhibited even more robust increases in their currents, revealing that these constructs heavily impact channel gating.
In summary, we have identified the pathomechanism of five rare CF-causing mutations. Channel maturation was impaired in all cases and could be treated with the available corrector drugs. Channel gating was also affected for each variant and could be enhanced by the addition of the potentiator drugs used in therapy. Based on our findings, we recommend using the currently available combination of corrector and potentiator drugs for all five allele mutations.

Funding:
EU Horizon 2020 Research and Innovation Program 739593 to László Csanády.
Cystic Fibrosis Foundation CSANAD21G0 to László Csanády.