Rescue of functional CFTR-DF508 protein by overexpression of the NHE-RF protein in A549 cells

Amal Robay, Gilles Toumaniantz, Jean Merot, Chanta Gauthier

INSERM U533, Nantes, France

In cystic fibrosis (CF), the anterograde trafficking of CFTR-DF508 (DF508) from the endoplasmic reticulum to the plasma membrane is inefficient. Identification of strategies for increasing delivery of DF508 to the apical membrane is important for the treatment of CF. Recent studies have demonstrated that PDZ domains play an essential role in determining protein trafficking and polarised targeting. The NHERF protein, which contains two PDZ domains, can bind the C-terminus of CFTR. In our study, we have hypothesised that overexpression of NHE-RF protein might increase apical membrane expression of DF508. For this, the plasmids encoding DF508 and NHE-RF were intranuclearly co-injected in A549 cells. DF508 activity was functionally assayed using SPQ fluorescent probe. The DF508 stimulation was performed by the application of 10 mM forskolin (Fsk). Cells injected with DF508 alone presented very low chloride permeability. Conversely, the overexpresion of NHE-RF protein increased the basal DF508 permeability to halides by two-folds. The activation of DF508 by Fsk was also increased by six-folds (ns19, P-0.001). Activation of DF508 by Fsk was abolished by injection of antisens oligonucleotides of NHE-RF mRNA. The specificity of this antisens was checked by the use of a sens oligonuclotide of NHE-RF mRNA that didn't affected the stimulation of DF508 activity by Fsk in the presence of an overexpression of NHE-RF. Similar results were obtained in polarised cells, MDCK. These studies showed that a modification in expression level of CFTRpr oteins partners, like NHE-RF, resulted in a rescue of DF508 activity.