PhD Scientific Days 2024

Molecular Medicine III.

Rare, cystic fibrosis causing mutations of the CFTR chloride channel

Text of the abstract

Cystic fibrosis (CF) is a life-threatening disease caused by mutations of the CFTR anion channel, which is expressed in epithelial cells and plays a role in salt-water transport. For most patients, who carry frequent mutant alleles, available modulator therapy mitigates the symptoms of the disease. Nonetheless, CF is still an incurable disorder.
Recently, three rare, previously unobserved, cystic fibrosis causing missense mutations (G126D, T582I, D984V) were identified in the Hungarian CF population. Our goal is to better understand the functional consequences of these mutations and to propose individualised treatments for patients carrying such alleles.
First, we created the point mutations in the pGEMHE plasmid expression system. Linearised plasmids were transcribed in vitro into cRNA, then injected into Xenopus laevis oocytes. Expressing the channels in oocytes allowed us to study channel gating properties in macroscopic and single-channel inside-out patch clamp recordings.
Analysing the macroscopic currents of the mutants, we detected no significant changes in their ATP affinities compared to the wild type protein. Furthermore, the reversible component of activation caused by the binding of protein kinase A (PKA) was retained in all cases. Evaluation of single-channel recordings revealed a lower open probability (Po) in the case of the G126D construct compared to wild type CFTR, caused by a shortened mean burst duration. A decrease in the mutant channels’ conductance was also observed. For the T582I construct a lower Po, resulting from elongated mean interburst durations, was detected. Finally, for the D984V mutation, the mean burst and interburst durations both decreased and therefore the Po stayed similar to that of the wild type CFTR channel. Since all mutations affected channel gating, pharmacological studies were conducted, which showed that all constructs respond to the potentiator drugs available in CF therapy (Vx-770, Vx-445), with multifold increases in their currents.
In summary, we identified the main gating properties of three CF-associated mutants utilising macroscopic and microscopic patch-clamp measurements. Based on our pharmacological examination, we recommend the application of the potentiator drugs currently used in therapy for all the three cases.

Funding: Cystic Fibrosis Foundation CSANAD21G0 to László Csanády.