Mass spectrometric analysis of novel phosphorylation sites in the TRPC4β channel

Abstract

Rationale: The transient receptor potential canonical (TRPC) channel 4β is a non-selective cation channel that is regulated by intracellular Ca(2+) and G protein-coupled receptors. Tyrosine phosphorylation of TRPC4β is important in mediating the activity and membrane expression of this channel protein. However, studies of TRPC4β Ser/Thr phosphorylation are lacking.

Methods: To investigate the phosphorylation sites involved in regulating the diverse functions of TRPC4β in mammalian cells, we used nano-liquid chromatography/tandem mass spectrometry to identify key phosphorylation sites in TRPC4β that was immunopurified from HEK293 cells with monoclonal anti-TRPC4β antibody.

Results: We identified four phosphorylation sites in the C-terminus of TRPC4β, none of which had been previously reported. Our data show that TRPC4β 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 TRPC4β channels in mammalian cells.

Conclusions: These novel phosphorylation sites on TRPC4β may play a potential role in the phosphorylation-mediated regulation of TRPC4β channel activity and function in mammalian cells.