Multisite phosphorylation of voltage-gated sodium channel alpha subunits from rat brain.
Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav 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 alpha subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav alpha subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in mediating diverse aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav alpha 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 Nav alpha subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav 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 Nav channels and other important membrane proteins in mammalian brain.
Berendt
Frank J
FJ
Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, School of Medicine, University of California, Davis, California 95616-8519, USA.
Park
Kang-Sik
KS
Trimmer
James S
JS
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002853:Chromatography, Liquid; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D036103:Nanotechnology; D009473:Neuronal Plasticity; D010766:Phosphorylation; D021122:Protein Subunits; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D021122:Protein Subunits; D015222:Sodium Channels