Mass spectrometric analysis of novel phosphorylation sites in the TRPC4β channel

RATIONALE The transient receptor potential canonical (TRPC) channel 4 beta is a non-selective cation channel that is regulated by intracellular Ca2+ and G protein-coupled receptors. Tyrosine phosphorylation of TRPC4 beta is important in mediating the activity and membrane expression of this channel protein. However, studies of TRPC4 beta Ser/Thr phosphorylation are lacking. METHODS To investigate the phosphorylation sites involved in regulating the diverse functions of TRPC4 beta in mammalian cells, we used nano-liquid chromatography/tandem mass spectrometry to identify key phosphorylation sites in TRPC4 beta that was immunopurified from HEK293 cells with monoclonal anti-TRPC4 beta antibody. RESULTS We identified four phosphorylation sites in the C-terminus of TRPC4 beta, none of which had been previously reported. Our data show that TRPC4 beta in mammalian cells is highly phosphorylated under basal conditions at multiple sites, and that a mass spectrometric proteomic technique combined with antibody-based affinity purification is an effective approach to define the phosphorylation sites of TRPC4 beta channels in mammalian cells. CONCLUSIONS These novel phosphorylation sites on TRPC4 beta may play a potential role in the phosphorylation-mediated regulation of TRPC4 beta channel activity and function in mammalian cells. Copyright (C) 2012 John Wiley & Sons, Ltd.