Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R.

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.