Apical membrane insertion of CFTR is increased by protein kinase C activation

CFTR channels regulate ion and fluid transport across respiratory epithelium, however the signaling pathways that govern their translocation and insertion into the apical membrane are poorly understood. We tested the hypothesis that PKC phosphorylation, which modulates responsiveness of the CFTR channel to PKA, also influences its insertion into the apical membrane of polarized Calu‐3 cells. Monolayers were cultured on porous supports and biotinylated by apical exposure to sulfo‐NHS‐SS‐biotin before, or after, activation of PKC or PKA using PMA or forskolin, respectively. Apical membrane proteins were recovered on streptavidin beads and the amount of CFTR in the pulldowns was assessed by Western blotting. The physiological agonist VIP, which stimulates both PKA and PKC activities through a G protein coupled receptor, was also tested. PMA increased the amount of CFTR in the pulldowns whereas forskolin had no effect. Moreover, the increase in surface expression induced by PMA was blocked by the PKC inhibitor Bis I, and was correlated with larger short‐circuit current responses to forskolin+IBMX stimulation in parallel Ussing chamber studies. Bis I also reduced current responses to VIP. We conclude that PKC activation increases functional CFTR protein in the apical membrane as part of an integrated response to VIP and perhaps other secretagogues. Supported by the Breathe program of the Canadian CF Foundation and CIHR