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.
|Keywords||Adenoviridae/genetics, Animals, Cell Line, Chloride Channels/*agonists, Cyclic GMP-Dependent Protein Kinases/genetics/*metabolism, Cyclic GMP/*pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator/*agonists, Gene Transfer Techniques, Isoenzymes/genetics/*metabolism, Patch-Clamp Techniques, Phosphorylation, Rats|
Vaandrager, A.B., Lohmann, S.M., de Jonge, H.R., Poller, W.C., Tilly, B.C., Smolenski, A., … Walter, U.. (1997). cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta. Journal of Biological Chemistry. Retrieved from http://hdl.handle.net/1765/8646