RT Journal Article
SR Electronic
T1 Negative Regulation of TRPC3 Channels by Protein Kinase C-Mediated Phosphorylation of Serine 712
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 558
OP 563
DO 10.1124/mol.104.007252
VO 67
IS 2
A1 Trebak, Mohamed
A1 Hempel, Nadine
A1 Wedel, Barbara J.
A1 Smyth, Jeremy T.
A1 Bird, Gary St. J.
A1 Putney, James W.
YR 2005
UL http://molpharm.aspetjournals.org/content/67/2/558.abstract
AB 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 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.