CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization.
The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl(-) currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.
Ganeshan
Radhika
R
Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Blvd., MCLM 985, Birmingham, AL 35294, USA.
Nowotarski
Krzysztof
K
Di
Anke
A
Nelson
Deborah J
DJ
Kirk
Kevin L
KL
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D003106:Colon / Q000166:cytology / Q000187:drug effects; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D011412:Propanolamines / Q000494:pharmacology*; D053778:Thiazolidines / Q000494:pharmacology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000037:antagonists & inhibitors*
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002227:Carbazoles; D011412:Propanolamines; D053778:Thiazolidines; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C489942:wiskostatin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; C037068:latrunculin B