VIP-dependent increase in f508del-CFTR membrane localization is mediated by PKCε

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/ CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, β, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC₅₀ of 4.9 μM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC₅₀ = 5.7 μM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.