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Missense mutation (G480C) in the CFTR gene associated with protein mislocalization but normal chloride channel
L S Smit1, T V Strong, D J Wilkinson
1Department of Human Genetics, University of Michigan, Ann Arbor 48109, USA.
Abstract:
We have identified a novel CFTR missense mutation associated with a protein trafficking defect in mammalian cells but normal chloride channel properties in a Xenopus oocyte assay. The mutation, a cysteine for glycine substitution at residue 480 (G480C), was detected in a pancreatic insufficient, African-American, cystic fibrosis (CF) patient. G480C was found on one additional CF chromosome and on none of 220 normal chromosomes, including 160 chromosomes from normal African-American individuals. Western blot analysis and immunofluorescence studies revealed that, in 293T cells, the encoded mutant protein was not fully glycosylated and failed to reach the plasma membrane, suggesting that the G480C protein was subject to defective intracellular processing. However, in Xenopus oocytes, a system in which mutant CFTR proteins are less likely to experience an intracellular processing/trafficking deficit, expression of G480C CFTR was associated with a chloride conductance that exhibited a sensitivity to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) that was similar to that of wild-type CFTR. This appears to be the first identification of a CFTR mutant with a single amino acid substitution in which the sole basis for disease is mislocalization of the protein.
Insights
A novel cystic fibrosis transmembrane conductance regulator (CFTR) mutation, G480C, causes protein mislocalization and disease. Despite normal channel function in oocytes, the mutant CFTR protein fails to reach the cell surface in mammalian cells.
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- Cystic Fibrosis (CF) is a genetic disorder caused by mutations in the CFTR gene.
- Understanding CFTR mutation mechanisms is crucial for developing targeted therapies.
Purpose of the Study:
- To identify and characterize a novel CFTR mutation.
- To investigate the functional consequences of the G480C mutation on CFTR protein trafficking and function.
Main Methods:
- Genetic analysis of CF patients.
- Mammalian cell expression studies (Western blot, immunofluorescence) to assess protein trafficking.
- Xenopus oocyte electrophysiology to evaluate chloride channel activity.
Main Results:
- A novel CFTR missense mutation, G480C, was identified in a CF patient.
- G480C-CFTR exhibited defective intracellular processing and failed to reach the plasma membrane in mammalian cells.
- G480C-CFTR displayed normal chloride channel activity and forskolin/IBMX sensitivity in Xenopus oocytes.
Conclusions:
- The G480C mutation leads to cystic fibrosis solely through protein mislocalization.
- This finding highlights the importance of protein trafficking in CF pathogenesis.
- G480C represents a unique CFTR mutant where mislocalization is the primary disease mechanism.