Abstract

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe(508) (DeltaF508). The DeltaF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 degrees C, we found that maturation could be restored if Val(510) was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of DeltaF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

Highlights

  • Cystic fibrosis (CF)2 is a lethal inherited disorder caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein [1]

  • Our goal was to test whether correctors that promoted maturation of ⌬F508 CFTR such as corr-4a, VRT-325, and VRT-532 directly interacted with the protein

  • Position 510 appears to be important for CFTR maturation as introduction of the suppressor mutation V510D into ⌬F508 CFTR promoted maturation of the protein (Fig. 2D)

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Summary

Introduction

Cystic fibrosis (CF) is a lethal inherited disorder caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein [1]. The ⌬F508 CFTR protein undergoes rapid degradation because it is defective in folding. Deletion of the equivalent residue in human P-gp (⌬Y490) causes defective folding of P-gp and rapid degradation [2]. The quinazoline derivative VRT-325 was first identified that can promote maturation of both P-gpand CFTR-processing mutants [3, 5]. Further study showed that correctors could have an additive effect on maturation of CFTR-processing mutants [7]. It is not known, whether correctors directly bind to CFTR or have indirect effects on the cellular folding pathways. We tested whether correctors directly interacted with CFTR by assaying for inhibition of disulfide cross-linking between the two halves of the protein

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