Stabilizing rescued surface-localized δf508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1

Abstract

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in CFTR, a plasma-membrane-localized anion channel. The most common mutation in CFTR, deletion of phenylalanine at residue 508 (ΔF508), causes misfolding of CFTR resulting in little or no protein at the plasma membrane. The CFTR corrector VX-809 shows promise for treating CF patients homozygous for ΔF508. Here, we demonstrate the significance of protein-protein interactions in enhancing the stability of the ΔF508 CFTR mutant channel protein at the plasma membrane. We determined that VX-809 prolongs the stability of ΔF508 CFTR at the plasma membrane. Using competition-based assays, we demonstrated that ΔF508 CFTR interacts poorly with Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) compared to wild-type CFTR, and VX-809 significantly increased this binding affinity. We conclude that stabilized CFTR-NHERF1 interaction is a determinant of the functional efficiency of rescued ΔF508 CFTR. Our results demonstrate the importance of macromolecular-complex formation in stabilizing rescued mutant CFTR at the plasma membrane and suggest this to be foundational for the development of a new generation of effective CFTR-corrector-based therapeutics.

Figure 1

ΔF508 CFTR interacts poorly with NHERF1. (A) Coomassie-stained gel showing purified Flag-WT CFTR (left) and Flag-ΔF508 CFTR (right) at 37 °C. El#1–2 refer to the number of eluted fractions of the purified proteins. (B) Protein-binding curve derived from the Alpha Screen competition assay to measure the affinity of GST-NHERF1-PDZ2 for Flag-WT CFTR (left panel) and Flag-ΔF508 CFTR (right panel) purified from HEK 293 cells overexpressing these proteins and maintained at 37 °C.

Figure 2

VX-809 potentiates the weaker interaction of ΔF508 CFTR with NHERF1. (A) and (B) Western blot data depicting the relative binding of Flag-WT CFTR (37 °C) and Flag-ΔF508 CFTR (37 or 28 °C) immunopurified proteins with NHERF1. The amounts of CFTR protein purified using Flag-beads are indicated. Flag-ΔF508 CFTR shows extremely weak binding with NHERF1 compared to WT-CFTR at 37 °C, as well as at 28 °C. (C) Western blots to demonstrate that VX-809 potentiates the interaction of Flag-ΔF508 CFTR with NHERF1. Bar graphs on the right side of each immunoblot represent the quantitation of NHERF1-CFTR interaction. Input refers to the cell lysates used prior to immunoprecipitation. (D) A competitive assay, in which GST-NHERF1-PDZ2 competes against endogenous NHERF1 to bind to Flag-ΔF508 CFTR in the presence or absence of VX-809, demonstrates that VX-809 improves CFTR-NHERF1 interaction affinity.

Figure 3

VX-809 potentiates ΔF508 CFTR function in mice organoids in a mechanism involving potentiation of CFTR-NHERF1 interaction at the plasma membrane. (A) Representative images of intestinal organoids isolated from Cftr (+/+), Cftr (+/ΔF508), and Cftr (ΔF508/ΔF508) male mice depicting Fsk-stimulated CFTR-dependent fluid secretion. (B) Bar graph representing luminal-size change after 30 min of Fsk treatment as a measure of fluid secretion in organoids. Addition of VX-809 stimulates CFTR-dependent fluid secretion in Cftr (ΔF508/ΔF508) organoids. Basal refers to the organoids which were not treated with Fsk. Data represents mean of luminal-size measurements of 3–12 organoids of various groups (***P < 0.001). (B) Confocal images of organoids isolated from Cftr (+/+) (upper panel) and Cftr (ΔF508/ΔF508) (lower panel) mice show proximity ligation assay signal in red (marked arrows) representative of CFTR-NHERF1 interaction at the plasma membrane. VX-809 potentiates CFTR-NHERF1 interaction in Cftr (ΔF508/ΔF508) organoids (right lower panel). neg, negative control (only CFTR antibody was used) and pos, positive control (both CFTR and NHERF1 antibodies were used) in Cftr (+/+) organoids. Cftr (+/+) or simply +/+ refers to homozygous for WT CFTR. Cftr (+/ΔF508) or simply +/Δ refers to one WT and one ΔF508 CFTR allele. Cftr (ΔF508/ΔF508) or simply Δ/Δ refers to homozygous for ΔF508 CFTR.

Figure 4

VX-809 restricts the mobility of ΔF508 CFTR at the plasma membrane in a PDZ-interaction-based mechanism. (A) Representative bright-field and quantum dot-labeled images of HEK-293 cells expressing no CFTR (referred to as HEK-P), Flag-WT CFTR, Flag-ΔF508 CFTR, and Flag-ΔF508 CFTR His 10. Flag-ΔF508 CFTR and Flag-ΔF508 CFTR His 10 were temperature rescued in the presence or absence of VX-809 for 48 h at 28 °C. (B) Representative mean square displacement curve to show the mobility kinetics of different groups of CFTR molecules. (C–G) Histograms representing the diffusion coefficients of a population of CFTR in various groups. 1000–4000 total objects were selected for analysis pooled from two to five independent experiments.

Figure 5

ΔF508 CFTR shows restricted trajectories in the presence of VX-809. Single-particle tracking images of HEK-293 cells (left panel) showing the overall mobility profile of (A) Flag-WT CFTR and (B) Flag-ΔF508 CFTR or (C) Flag-ΔF508 CFTR His 10 in the presence of DMSO or VX-809. Also shown are representative trajectories of individual CFTR molecules (right panel of each figure).