@article {Trebak558, author = {Mohamed Trebak and Nadine Hempel and Barbara J. Wedel and Jeremy T. Smyth and Gary St. J. Bird and James W. Putney, Jr.}, title = {Negative Regulation of TRPC3 Channels by Protein Kinase C-Mediated Phosphorylation of Serine 712}, volume = {67}, number = {2}, pages = {558--563}, year = {2005}, doi = {10.1124/mol.104.007252}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {TRPC3 is a nonselective cation channel member of the {\textquotedblleft}canonical{\textquotedblright} 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 Ser712 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 Ser712 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 Ca2+ 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.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/67/2/558}, eprint = {https://molpharm.aspetjournals.org/content/67/2/558.full.pdf}, journal = {Molecular Pharmacology} }