Negative regulation of TRPC3 channels by protein kinase C-mediated phosphorylation of serine 712.
TRPC3 is a nonselective cation channel member of the "canonical" transient receptor potential (TRPC) family whose members are activated by phospholipase C-coupled receptors. TRPC3 can be activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) in a protein kinase C-independent manner. On the other hand, phorbol 12-myristate 13-acetate (PMA) inhibits OAG-mediated TRPC3 channel activation, suggesting that phosphorylation of TRPC3 by protein kinase C is a mechanism of receptor-mediated negative feedback. Here, we show PMA-induced phosphorylation of TRPC3 channels in vivo. We demonstrate by site-directed mutagenesis that a single site containing Ser(712) and conserved among all members of the TRPC family is essential for protein kinase C-mediated negative regulation of TRPC3. In human embryonic kidney 293 cells expressing a TRPC3 mutant in which Ser(712) was replaced by alanine (S712A), PMA failed to block channel activation, whereas wild-type TRPC3 activity was completely inhibited. Phosphorylation of the S712A TRPC3 mutant was not stimulated in response to PMA treatment. Furthermore, S712A TRPC3 mutant-mediated Ca(2+) entry after methacholine activation was significantly greater than that of wild-type TRPC3. These findings demonstrate a dual role for phospholipase C-generated diacylglycerol, which serves as a signal for TRPC3 activation as well as a signal for negative feedback via protein kinase C-mediated phosphorylation.
Trebak
Mohamed
M
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. trebak@niehs.nih.gov
Hempel
Nadine
N
Wedel
Barbara J
BJ
Smyth
Jeremy T
JT
Bird
Gary St J
GS
Putney
James W
JW
D000595:Amino Acid Sequence; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D011493:Protein Kinase C / Q000502:physiology*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D050052:TRPC Cation Channels; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007473:Ion Channels; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D012694:Serine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate