Functional integrity of the vesicle transporting machinery is required forcomplete activation of CFTR expressed in Xenopus laevis oocytes

We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We per f ormed simultaneous and continuous recording of membrane current (I-m), cond uctance (G(m)) and capacitance (C-m), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxa nthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger C-m exh ibited larger currents. Expression of CFTR itself did not change the C-m of the oocytes. However, activation of CFTR with cAMP-cocktail increased I-m and G(m) 15- and 20-fold, respectively while membrane surface area increase d by about 7%, indicating the functional insertion of preformed CFTR into t he plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, I BMX alone, but not caffeine or theophylline, was sufficient to stimulate mo re than half of the increases in I-m and G(m) as observed with cAMP-cocktai l. Since C-m was not significantly stimulated by IBMX, we conclude that IBM X alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in t he activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages p revented the translocation of CFTR from intracellular pools to the plasma m embrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways includin g direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we sho w that complete activation of CFTR requires an intact protein trafficking m achinery.