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Updated: Jan 6, 2026
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
Identity, functional consequences, and context effects of amino acids inserted during suppression of CFTR nonsense
Kari Thrasher1, Jianguo Chen2, Lianwu Fu3
1Departments of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA.
Background:
Cystic fibrosis patients who carry a CFTR nonsense allele often express negligible CFTR protein, and thus, are unresponsive to CFTR modulators. Nonsense suppression (also called readthrough) is an emerging therapeutic approach for this patient subgroup that uses small molecules to suppress translation termination at in-frame premature termination codons (PTCs) and rescue full-length, functional CFTR protein. This study examines mechanistic aspects of readthrough at six CFTR PTCs commonly found in CF patients.
Methods:
CFTR expression and chloride conductance were used to assess the responsiveness of six different CFTR PTCs to G418-mediated readthrough. LC-MS/MS was used to identify the CFTR variant proteins generated by readthrough of PTCs in their native, local CFTR mRNA sequence context. For each CFTR variant protein identified, the abundance, processing, activity, and responsiveness to CFTR modulators were characterized.
Results:
CFTR expression and function varied widely among PTCs, with UGA generally being the most responsive to G418-mediated readthrough. The amino acids incorporated at PTCs during G418-induced readthrough also varied, depending on the PTC and its surrounding local mRNA context. Modulators stabilized and enhanced the abundance and activity of most CFTR variant proteins generated by PTC readthrough, with many variant proteins reaching WT CFTR activity.
Conclusions:
Nonsense suppression therapy shows promise as a treatment for CF patients who carry a PTC, especially when combined with current CFTR modulators. Mechanistic insights of readthrough gleaned from this study can be used to develop better therapeutic strategies for treating CF patients who carry a nonsense mutation.
Insights
Nonsense suppression therapy can restore CFTR protein function in cystic fibrosis patients with premature termination codons (PTCs). Combining readthrough with CFTR modulators shows promise for improved treatment strategies.
Area of Science:
- Molecular Biology
- Genetics
- Pharmacology
Background:
- Cystic fibrosis (CF) patients with CFTR nonsense alleles express little to no CFTR protein, rendering them unresponsive to CFTR modulators.
- Nonsense suppression therapy aims to restore functional CFTR protein by enabling translation readthrough of premature termination codons (PTCs).
Purpose of the Study:
- To investigate the mechanistic aspects of readthrough at six common CFTR PTCs.
- To assess the impact of readthrough on CFTR protein expression, function, and responsiveness to modulators.
Main Methods:
- Assessed CFTR expression and chloride conductance in response to G418-mediated readthrough for six CFTR PTCs.
- Utilized LC-MS/MS to identify CFTR variant proteins resulting from readthrough.
- Characterized abundance, processing, activity, and modulator responsiveness of identified CFTR variant proteins.
Main Results:
- Significant variation in CFTR expression and function was observed across different PTCs, with UGA showing highest responsiveness to G418.
- The amino acid incorporated during readthrough varied based on PTC and local mRNA context.
- CFTR modulators enhanced the stability and activity of most readthrough-generated variant proteins, with many achieving wild-type CFTR activity levels.
Conclusions:
- Nonsense suppression therapy, particularly when combined with CFTR modulators, holds significant therapeutic potential for CF patients with PTCs.
- Understanding readthrough mechanisms can guide the development of improved therapeutic strategies for CF patients with nonsense mutations.
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