cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta.
In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.
Vaandrager
A B
AB
Department of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands.
Tilly
B C
BC
Smolenski
A
A
Schneider-Rasp
S
S
Bot
A G
AG
Edixhoven
M
M
Scholte
B J
BJ
Jarchau
T
T
Walter
U
U
Lohmann
S M
SM
Poller
W C
WC
de Jonge
H R
HR
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D065101:Chloride Channel Agonists; D006152:Cyclic GMP / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists*; D018014:Gene Transfer Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D051381:Rats
D065101:Chloride Channel Agonists; D007527:Isoenzymes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP