Abstract
Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells. Structural analyses were also performed to study the molecular environment of the W361 residue. Western blot showed that W361R-CFTR was not efficiently processed to a mature band C, similar to F508del CFTR, but unlike F508del CFTR, it did exhibit significant transport activity at the cell surface in response to cAMP agonists. Importantly, W361R-CFTR also responded well to CFTR modulators: its maturation defect was efficiently corrected by VX-809 treatment and its channel activity further potentiated by VX-770. Based on these results, we postulate that W361R is a novel class-2 CF mutation that causes abnormal protein maturation which can be corrected by VX-809, and additionally potentiated by VX-770, two FDA-approved small molecules. At the structural level, W361 is located within a class-2 CF mutation hotspot that includes other mutations that induce variable disease severity. Analysis of the 3D structure of CFTR within a lipid environment indicated that W361, together with other mutations located in this hotspot, is at the edge of a groove which stably accommodates lipid acyl chains. We suggest this lipid environment impacts CFTR folding, maturation and response to CFTR modulators.
Highlights
Cystic fibrosis (CF) is one of the most common, lethal, autosomal recessive disease and is caused by mutations in the gene encoding the anion channel cystic fibrosis transmembrane conductance regulator (CFTR)
We have investigated the effects of the rare CFTR mutation W361R
We found a strong decrease in the levels of the fully glycosylated mature form compared to wild-type CFTR (WT-CFTR), a hallmark of class-2 CF mutations (Kerem, 2006)
Summary
Cystic fibrosis (CF) is one of the most common, lethal, autosomal recessive disease and is caused by mutations in the gene encoding the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). More than 2000 CF mutations have been identified to date, and these have been subdivided into six classes based on the functional defect they cause on CFTR mRNA or protein (Kerem, 2006). F508delCFTR exhibits a gating defect (Dalemans et al, 1991), and a reduced stability at the plasma membrane (Lukacs et al, 1993; Okiyoneda et al, 2010), typical defects of class-3 and class mutations, respectively. This mutation is present in 70% of homozygous patients and 90% of heterozygous patients [CFTR2 database1]. Patients carrying class-2 CFTR mutations on both alleles generally show severe disease with strong digestive and pulmonary defects
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