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

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Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, and Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, California 95616, and Department of Physiology, Kyung Hee University School of Medicine, Seoul 130-701, Korea
* To whom correspondence should be addressed. Dr. James S. Trimmer, Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, 196 Briggs Hall, University of California, One Shields Avenue, Davis, CA 95616-8519. E-mail: [email protected]
†Department of Neurobiology, Physiology and Behavior, University of California.
#These authors contributed equally.
§Department of Physiology, Kyung Hee University School of Medicine.
‡Department of Physiology and Membrane Biology, University of California.
Cite this: J. Proteome Res. 2010, 9, 4, 1976–1984
Publication Date (Web):February 5, 2010
Copyright © 2010 American Chemical Society

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    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 α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α 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 α 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 α 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.

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    Figures of peptide spectra for the Nav1.2 phosphopeptides, Mascot scores for the Nav1.1 and Nav1.2 phosphopeptides, summary of individual experiments, figures of peptide spectra for the Nav1.1 phosphopeptides, and results from analysis of Nav1.2 with consensus phosphorylation site prediction algorithms. This material is available free of charge via the Internet at

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