Proteomic analyses of K(v)2.1 channel phosphorylation sites determining cell background specific differences in...

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• The K(v)2.1 potassium channel is crucial for neuronal membrane excitability.
• Its phosphorylation state is highly variable and regulates channel gating.
• Previous studies noted functional differences in K(v)2.1 between HEK293 and COS-1 cells, which disappeared after dephosphorylation.

Purpose of the Study:

• To investigate how K(v)2.1 phosphorylation differs between HEK293 and COS-1 cells.
• To understand the impact of these phosphorylation differences on K(v)2.1 channel gating.

Main Methods:

• Stable Isotope Labeling by Amino acids in cell culture (SILAC) was employed.
• Mass spectrometry was used to quantify K(v)2.1 phosphorylation sites.
• K(v)2.1 was purified from both HEK293 and COS-1 cells.

Main Results:

• Seven C-terminal phosphorylation sites on K(v)2.1 showed differential phosphorylation levels between HEK293 and COS-1 cells.
• Six sites were more phosphorylated in HEK293 cells, while one site was more phosphorylated in COS-1 cells.
• These differentially phosphorylated sites overlap with those targeted by calcineurin signaling.

Conclusions:

• Cell-specific differences in K(v)2.1 gating are attributed to differential phosphorylation at a subset of sites, not unique cell-specific sites.
• This explains basal differences in K(v)2.1 function between cell types and within the same cell type under varying conditions.