Received for publication September 14, 2004.
Revised October 22, 2004.
Accepted for publication November 8, 2004.

Negative Regulation of TRPC3 Channels by Protein Kinase C-mediated Phosphorylation of Serine 712

Abstract

TRPC3 is a non-selective 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 analogue, 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 HEK293 cells expressing a TRPC3 mutant in which Ser⁷¹² was replaced by alanine (S712A), PMA failed to block channel activation, while 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 following 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.

Key words: Muscarinic cholinergic, Ion channel regulation, Protein Kinase C, Ca imaging, Mutagenesis/Chimeric approaches