Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G.
There are two known phosphorylation-mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr-11 and Ser-263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser-712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG-expressing construct and then transiently transfected with a TRPC3-expressing construct. Addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG), elicited a TRPC3-mediated [Ca2+]i rise in these cells. This OAG-induced rise in [Ca2+]i could be inhibited by phorbol 12-myristate 13-acetate (PMA), an agonist for PKC, in a dose-dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A-S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 microM) or H8 (10 microM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG-overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG-induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8-BrcGMP and S-nitroso-N-acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG-induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 microM) or DT-3 (500 nM), suggesting an important role of PKG in the PMA-induced inhibition of TRPC channels in native endothelial cells.
Kwan
Hiu-Yee
HY
Li Ka Shing Institute of Health Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Hong Kong, China.
Huang
Y
Y
Yao
Xiaoqiang
X
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D012694:Serine / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D004075:Diglycerides; D008840:Microfilament Proteins; D020030:Nitric Oxide Donors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C110881:S-nitro-N-acetylpenicillamine; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000098662:Vasodilator-Stimulated Phosphoprotein; C016276:8-bromocyclic GMP; D012694:Serine; C038237:1-oleoyl-2-acetylglycerol; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010396:Penicillamine; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate