Actin filament organization is required for proper cAMP-dependent activation of CFTR.
Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.
Prat
A G
AG
Renal Unit, Massachusetts General Hospital East, Charlestown 02129, USA.
Cunningham
C C
CC
Jackson
G R
GR
Borkan
S C
SC
Wang
Y
Y
Ausiello
D A
DA
Cantiello
H F
HF
D000199:Actins / Q000502:physiology*; D000838:Anions / Q000493:pharmacokinetics; D001966:Bromine / Q000493:pharmacokinetics; D002713:Chlorine / Q000493:pharmacokinetics; D003285:Contractile Proteins / Q000235:genetics / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology*; D003956:Dialysis; D064448:Filamins; D015870:Gene Expression / Q000502:physiology; D005942:Gluconates / Q000493:pharmacokinetics; D006801:Humans; D007455:Iodine / Q000493:pharmacokinetics; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008545:Melanoma; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008840:Microfilament Proteins / Q000235:genetics / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000201:enzymology
D000199:Actins; D000838:Anions; C505032:CFTR protein, human; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D064448:Filamins; D005942:Gluconates; D008840:Microfilament Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002713:Chlorine; D007455:Iodine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D001966:Bromine