Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G.
Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca2+ stores and/or diacylglycerol and/or Ca2+/calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). We found that the overexpressed TRPC3 mediated store-operated Ca2+ influx and that this type of Ca2+ influx was inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels could be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolished the store-operated Ca2+ influx mediated by TRPC3 channels in HEK293 cells.
Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China.
Hiu-Yee
HY
Kwan
Yu
Y
Huang
Xiaoqiang
X
Yao
D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003331:Coronary Vessels; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D050052:TRPC Cation Channels; D014162:Transfection
D007473:Ion Channels; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D017869:Cyclic GMP-Dependent Protein Kinases; D002118:Calcium