Mass spectrometric analysis of novel phosphorylation sites in the TRPC4[beta] 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 © 2012 John Wiley & Sons, Ltd. [PUBLICATION ABSTRACT]