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The Nav1.2 channel is regulated by GSK3

James, Thomas F; Nenov, Miroslav N; Wildburger, Norelle C; Lichti, Cheryl F; Luisi, Jonathan; Vergara, Fernanda; Panova-Electronova, Neli I; Nilsson, Carol L LU ; Rudra, Jai S and Green, Thomas A, et al. (2015) In Biochimica et biophysica acta 1850(4). p.44-832
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... (More)

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.

CONCLUSION: These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail.

GENERAL SIGNIFICANCE: These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

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keywords
Amino Acid Sequence, Glycogen Synthase Kinase 3, HEK293 Cells, Humans, Molecular Sequence Data, NAV1.2 Voltage-Gated Sodium Channel, Phosphorylation, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
in
Biochimica et biophysica acta
volume
1850
issue
4
pages
13 pages
publisher
Elsevier
external identifiers
  • scopus:84922289434
ISSN
0006-3002
DOI
10.1016/j.bbagen.2015.01.011
language
English
LU publication?
no
id
c5bca94f-5a0f-4735-b2e3-f9e9228528c6
date added to LUP
2017-05-16 10:25:37
date last changed
2017-06-11 05:16:20
@article{c5bca94f-5a0f-4735-b2e3-f9e9228528c6,
  abstract     = {<p>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.</p><p>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.</p><p>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.</p><p>CONCLUSION: These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail.</p><p>GENERAL SIGNIFICANCE: These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.</p>},
  author       = {James, Thomas F and Nenov, Miroslav N and Wildburger, Norelle C and Lichti, Cheryl F and Luisi, Jonathan and Vergara, Fernanda and Panova-Electronova, Neli I and Nilsson, Carol L and Rudra, Jai S and Green, Thomas A and Labate, Demetrio and Laezza, Fernanda},
  issn         = {0006-3002},
  keyword      = {Amino Acid Sequence,Glycogen Synthase Kinase 3,HEK293 Cells,Humans,Molecular Sequence Data,NAV1.2 Voltage-Gated Sodium Channel,Phosphorylation,Journal Article,Research Support, N.I.H., Extramural,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {4},
  pages        = {44--832},
  publisher    = {Elsevier},
  series       = {Biochimica et biophysica acta},
  title        = {The Nav1.2 channel is regulated by GSK3},
  url          = {http://dx.doi.org/10.1016/j.bbagen.2015.01.011},
  volume       = {1850},
  year         = {2015},
}