Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP se nsitive Cl- channel that is defective in cystic fibrosis (CF). The most fre quent mutation, namely Delta F508-CFTR, accounts for 66% of CF. Here we sho w that cAMP-activation of CFTR occurs via at least two distinct pathways: a ctivation of CFTR molecules already present in the plasma membrane and prot ein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to th e plasma membrane and functional insertion into the membrane. We investigat ed the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorde d continuously membrane current (I-m), conductance (G(m)) and capacitance ( C-m), which is a direct measure of membrane surface area. Expression of CFT R alone did not change the plasma membrane surface area. However, activatio n of CFTR with cAMP increased I-m, G(m) and C-m while Delta F508-CFTR-expre ssing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in C-m wh ile increases of I-m and G(m) were still present. AT? or the xanthine deriv ative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFT R. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such a s primaquine, brefeldin A, nocodazole. From these data we conclude that cAM P activates CFTR by at least two distinct pathways: activation of CFTR alre ady present in the plasma membrane and exocytotic delivery of new CFTR mole cules to the oocyte membrane and functional insertion into it. (C) 2001 Els evier Science Inc. All rights reserved.