Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.
Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.
Wagner
J A
JA
Department of Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, CA 94305.
McDonald
T V
TV
Nghiem
P T
PT
Lowe
A W
AW
Schulman
H
H
Gruenert
D C
DC
Stryer
L
L
Gardner
P
P
D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D018118:Chloride Channels; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007473:Ion Channels / Q000187:drug effects*; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*
C505032:CFTR protein, human; D018118:Chloride Channels; D007473:Ion Channels; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D015759:Ionomycin; D000242:Cyclic AMP; D002118:Calcium