Sites and Regulation of L-Type Ca2+ Channel Cav1.2 Phosphorylation in Brain.

Cav1.2 channel phosphorylation plays an important role in regulating neuronal plasticity by action potential-dependent Ca2+ entry. Most studies of Cav1.2 regulation by phosphorylation have been reported in heart and muscles. Here, we identified phosphorylation sites of neuronal Cav1.2 channel protein purified from rat brain using mass spectrometry. The functional characterization of these phosphorylation sites showed altered voltage-dependent biophysical properties of the channel, without affecting current density. These results show that neuronal Cav1.2 channel is regulated by phosphorylation in a complex mechanism involving multiple phosphorylation sites.

Src regulates membrane trafficking of the Kv3.1b channel.

The Kv3.1 channel plays a crucial role in regulating the high-frequency firing properties of neurons. Here, we determined whether Src regulates the subcellular distributions of the Kv3.1b channel. Co-expression of active Src induced a dramatic redistribution of Kv3.1b to the endoplasmic reticulum. Furthermore, co-expression of the Kv3.1b channel with active Src induced a remarkable decrease in the pool of Kv3.1b at the cell surface. Moreover, the co-expression of active Src results in a significant decrease in the peak current densities of the Kv3.1b channel, and a substantial alteration in the voltage dependence of its steady-state inactivation. Taken together, these results indicate that Src kinase may play an important role in regulating membrane trafficking of Kv3.1b channels.

Mass spectrometric analysis of novel phosphorylation sites in the TRPC4ß channel.

RATIONALE: The transient receptor potential canonical (TRPC) channel 4ß is a non-selective cation channel that is regulated by intracellular Ca(2+) and G protein-coupled receptors. Tyrosine phosphorylation of TRPC4ß is important in mediating the activity and membrane expression of this channel protein. However, studies of TRPC4ß Ser/Thr phosphorylation are lacking. METHODS: To investigate the phosphorylation sites involved in regulating the diverse functions of TRPC4ß in mammalian cells, we used nano-liquid chromatography/tandem mass spectrometry to identify key phosphorylation sites in TRPC4ß that was immunopurified from HEK293 cells with monoclonal anti-TRPC4ß antibody. RESULTS: We identified four phosphorylation sites in the C-terminus of TRPC4ß, none of which had been previously reported. Our data show that TRPC4ß 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 TRPC4ß channels in mammalian cells. CONCLUSIONS: These novel phosphorylation sites on TRPC4ß may play a potential role in the phosphorylation-mediated regulation of TRPC4ß channel activity and function in mammalian cells.

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

Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). 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 Ca(2+) and Mg(2+) 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.

Contribution of the delayed-rectifier potassium channel Kv2.1 to acute spinal cord injury in rats.

Recent studies have reported that delayed-rectifier Kv channels regulate apoptosis in the nervous system. Herein, we investigated changes in the expression of the delayed-rectifier Kv channels Kv1.2, Kv2.1, and Kv3.1 after acute spinal cord injury (SCI) in rats. We performed RT-PCR analysis and found an increase in the level of Kv2.1 mRNA after SCI but no significant changes in the levels of Kv1.2 and Kv3.1 mRNA. Western blot analysis revealed that Kv2.1 protein levels rapidly decreased and then dramatically increased from 1 day, whereas Kv3.1b protein levels gradually and sharply decreased at 5 days. Kv1.2 protein levels did not change significantly. In addition, Kv2.1 clusters were disrupted in the plasma membranes of motor neurons after SCI. Interestingly, the expressional changes and translocation of Kv2.1 were consistent with the apoptotic changes on day 1. Therefore, these results suggest that Kv2.1 channels probably contribute to neuronal cell responses to SCI.