title: The Nav1.2 channel is regulated by GSK3.
abstract: BACKGROUND
  Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels.
  
  
  METHODS
  We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues.
  
  
  RESULTS
  We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T(1966) at the C-terminal tail of Na(v)1.2.
  
  
  CONCLUSIONS
  These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail.
  
  
  CONCLUSIONS
  These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
authors.affiliation: Department of Pharmacology & Toxicology, USA; Neuroscience Graduate Program, USA.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: James
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Miroslav N
authors.initials: MN
authors.lastname: Nenov
authors.affiliation: Department of Pharmacology & Toxicology, USA; Neuroscience Graduate Program, USA.
authors.forename: Norelle C
authors.initials: NC
authors.lastname: Wildburger
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Cheryl F
authors.initials: CF
authors.lastname: Lichti
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Luisi
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Vergara
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Neli I
authors.initials: NI
authors.lastname: Panova-Electronova
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Nilsson
authors.affiliation: Department of Pharmacology & Toxicology, USA.
authors.forename: Jai S
authors.initials: JS
authors.lastname: Rudra
authors.affiliation: Department of Pharmacology & Toxicology, USA; Center for Addiction Research, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Green
authors.affiliation: Department of Mathematics, University of Houston, USA.
authors.forename: Demetrio
authors.initials: D
authors.lastname: Labate
authors.affiliation: Department of Pharmacology & Toxicology, USA; Center for Addiction Research, USA; Center for Biomedical Engineering, USA; Mitchell Center for Neurodegenerative Diseases, USA. Electronic address: felaezza@utmb.edu.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Laezza
mesh_terms: D000595:Amino Acid Sequence; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation
chemical_list: D062551:NAV1.2 Voltage-Gated Sodium Channel; D038362:Glycogen Synthase Kinase 3
keywords: Confocal microscopy; Glycogen synthase kinase 3; Patch-clamp electrophysiology; Protein–protein interactions; Sodium channel