cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA.
The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in stage V/VI Xenopus oocytes by injection of cRNA transcribed in vitro from a pBluescript vector containing 6.2-kb wild-type cDNA. This clone was also used for the preparation of antisense RNA. Double-electrode voltage clamp was employed to measure transmembrane currents. In sense RNA-injected oocytes, cAMP depolarized the membrane potential (Vm) from -52 to -31 mV and increased membrane conductance (Gm) 10-fold. However, cAMP had no effect on Vm or Gm in uninjected oocytes or in oocytes injected with antisense RNA. The endogenous Ca-activated Cl currents of control oocytes were abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50 microM) or bath Cl replacement. In contrast, the cAMP-stimulated currents of CFTR-expressing oocytes were DIDS insensitive and were inhibited only approximately 50% when bath Cl was replaced by gluconate or glutamate. In addition, the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 50 microM) and diphenylamine-2-carboxylic acid (DPC; 3 mM) reduced the cAMP-evoked currents by only approximately 10%. The stimulated currents of CFTR-expressing oocytes were reduced approximately 30% by 10 mM Ba, suggesting that the Cl-independent current component is due to an increase in K conductance. Our results indicate that expression of CFTR in Xenopus oocytes produces a cAMP-activated Cl current. The Cl-independent current may represent a regulatory action of CFTR on K conductance pathways or a secondary response of the oocyte membrane to the high Cl conductance induced by CFTR expression.
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.
S A
SA
Cunningham
R T
RT
Worrell
D J
DJ
Benos
R A
RA
Frizzell
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D012313:RNA / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014982:Xenopus laevis
C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012313:RNA; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium