Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.
Webe
W M
WM
Laboratory of Physiology, K U Leuven, Belgium. michael.weber@med.kuleuven.ac.be
Segal
A
A
Vankeerberghen
A
A
Cassiman
J J
JJ
Van Driessche
W
W
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D005260:Female; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives* / Q000494:pharmacology; D014982:Xenopus laevis
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C053907:8-cyclopentyl-1,3-dimethylxanthine; D000255:Adenosine Triphosphate; D013806:Theophylline; D000242:Cyclic AMP