Trebak Mohamed, Hempel Nadine, Wedel Barbara J, Smyth Jeremy T, Bird Gary St J, Putney James W
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Mol Pharmacol. 2005 Feb;67(2):558-63. doi: 10.1124/mol.104.007252. Epub 2004 Nov 8.
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.
TRPC3是“典型”瞬时受体电位（TRPC）家族的一种非选择性阳离子通道成员，该家族成员由磷脂酶C偶联受体激活。TRPC3可被二酰基甘油类似物1-油酰基-2-乙酰基-sn-甘油（OAG）以不依赖蛋白激酶C的方式激活。另一方面，佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）抑制OAG介导的TRPC3通道激活，这表明蛋白激酶C介导的TRPC3磷酸化是受体介导的负反馈机制。在此，我们展示了PMA在体内诱导的TRPC3通道磷酸化。我们通过定点诱变证明，在TRPC家族所有成员中保守的一个包含Ser（712）的单一位点对于蛋白激酶C介导的TRPC3负调控至关重要。在表达TRPC3突变体（其中Ser（712）被丙氨酸取代（S712A））的人胚肾293细胞中，PMA未能阻断通道激活，而野生型TRPC3活性则被完全抑制。S712A TRPC3突变体的磷酸化在PMA处理后未被刺激。此外，乙酰甲胆碱激活后，S712A TRPC介导的Ca（2+）内流显著高于野生型TRPC3。这些发现证明了磷脂酶C产生的二酰基甘油的双重作用，它既是TRPC3激活的信号，也是通过蛋白激酶C介导的磷酸化进行负反馈的信号。
