cAMP-independent regulation of CFTR by the actin cytoskeleton.
Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.
Prat
A G
AG
Renal Unit, Massachusetts General Hospital East, Charlestown 02129, USA.
Xiao
Y F
YF
Ausiello
D A
DA
Cantiello
H F
HF
D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology*; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D064448:Filamins; D006801:Humans; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D000199:Actins; C505032:CFTR protein, human; D002352:Carrier Proteins; D018118:Chloride Channels; D003285:Contractile Proteins; D064448:Filamins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015638:Cytochalasin D; D000242:Cyclic AMP