Functional integrity of the vesicle transporting machinery is required for complete 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 performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (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 Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX 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 the 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 prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.
Weber
W M
WM
Laboratory of Physiology, KU Leugen, Belgium. Michael.Weber@med.kuleuven.ac.be
Segal
A
A
Simaels
J
J
Vankeerberghen
A
A
Cassiman
J J
JJ
Van Driessche
W
W
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D015870:Gene Expression; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011319:Primaquine / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*; D014982:Xenopus laevis
D000962:Antimalarials; D000970:Antineoplastic Agents; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D011500:Protein Synthesis Inhibitors; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020126:Brefeldin A; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004533:Egtazic Acid; D000242:Cyclic AMP; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D011319:Primaquine; D015739:Nocodazole; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine