Multisite Phosphorylation of Voltage-Gated Sodium Channel alpha Subunits from Rat Brain

作者：Berendt Frank J; Park Kang Sik; Trimmer James S^*

来源：Journal of Proteome Research, 2010, 9(4): 1976-1984.

DOI：10.1021/pr901171q

• voltage-gated sodium channels
• brain
• phosphorylation
• tandem mass spectrometry
• immunopurification
• monoclonal antibody
• nanoflow liquid chromatography

Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage, gated sodium or Nay channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary or a subunit of Nay channels, affecting diverse aspects of channel function. Previous studies of Nay a subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in mediating diverse aspects of Nay channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nay a subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nay a subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nay channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nay channels and other important membrane proteins in mammalian brain.

• 出版日期2010-4