Identification of the phosphorylation sites on intact TRPM7 channels from mammalian cells

► This study is the first MS-based analysis of the phosphorylation sites in intact TRPM7. ► We identified 14 phosphorylation sites of intact TRPM7, eight of which have not been previously reported. ► TRPM7 are phosphorylated at multiple sites, which may modulate its activity and functions. Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca2+ and Mg2+. TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca2+ and Mg2+ in mammalian cells. Although studies have shown that the kinase domain of TRPM7 is required for channel activation and can phosphorylate other target proteins, a systematic analysis of intact TRPM7 channel phosphorylation sites expressed in mammalian cells is lacking. We applied mass spectrometric proteomic techniques to identify and characterize the key phosphorylation sites in TRPM7 channels. We identified 14 phosphorylation sites in the cytoplasmic domain of TRPM7, eight of which have not been previously reported. The identification of phosphorylation sites using antibody-based immunopurification and mass spectrometry is an effective approach for defining the phosphorylation status of TRPM7 channels. The present results show that TRPM7 channels are phosphorylated at multiple sites, which serves as a mechanism to modulate the dynamic functions of TRPM7 channels in mammalian cells.