cAMP- and Ca2+-independent activation of cystic fibrosis transmembrane conductance regulator channels by phenylimidazothiazole drugs.
Patch-clamp, iodide efflux, and biochemical techniques were used to evaluate the ability of phenylimidazothiazoles to open normal and mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels and to investigate the mechanism of activation. As reported previously for bromotetramisole, levamisole activated wild-type CFTR channels stably expressed in Chinese hamster ovary cells in the absence of other secretagogues and without elevating intracellular cAMP or calcium. The protein kinase A (PKA) inhibitor N - (2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesul-fonamid e abolished activation by forskolin but only partially inhibited stimulation by levamisole, suggesting the involvement of other kinases. CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DeltaF508) all responded to phenylimidazothiazoles. Moreover, levamisole and bromotetramisole increased the activity of wild-type and mutant channels already exposed to PKA + MgATP, consistent with the inhibition of a constitutive, membrane-associated phosphatase activity. We conclude that phenylimidazothiazole drugs can open normal and mutated CFTR channels by stabilization of phosphoforms of CFTR that are produced by basal activity of PKA and alternative protein kinases. If similar stimulation is observed in humans in vivo, phenylimidazothiazoles may be useful in the development of pharmacological therapies for cystic fibrosis.
Becq
F
F
Department of Physiology, McGill University, 3655 Drummond Street, Montréal, Québec Canada H3G 1Y6.
Verrier
B
B
Chang
X B
XB
Riordan
J R
JR
Hanrahan
J W
JW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides; D013773:Tetramisole / Q000031:analogs & derivatives* / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
C505032:CFTR protein, human; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007546:Isoquinolines; D011994:Recombinant Proteins; D013449:Sulfonamides; D013844:Thiazoles; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D007978:Levamisole; D000001:Calcimycin; C006226:4-bromotetramisole; D000255:Adenosine Triphosphate; D013773:Tetramisole; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium