TY - JOUR
T1 - The Nav1.2 channel is regulated by GSK3
AU - James, Thomas F.
AU - Nenov, Miroslav
AU - Wildburger, Norelle C.
AU - Lichti, Cheryl
AU - Luisi, Jonathan
AU - Vergara, Fernanda
AU - Panova-Electronova, Neli I.
AU - Nilsson, Carol
AU - Rudra, Jai Simha
AU - Green, Thomas A.
AU - Labate, Demetrio
AU - Laezza, Fernanda
N1 - Publisher Copyright:
© 2015 Published by Elsevier B.V.
PY - 2015/4
Y1 - 2015/4
N2 - Background Phosphorylation plays an essential role in regulating voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 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 Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-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 Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2 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 T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulates Nav channel function via its C-terminal tail. General significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
AB - Background Phosphorylation plays an essential role in regulating voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 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 Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-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 Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2 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 T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulates Nav channel function via its C-terminal tail. General significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
KW - Confocal microscopy
KW - Glycogen synthase kinase 3
KW - Patch-clamp electrophysiology
KW - Protein-protein interactions
KW - Sodium channel
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U2 - 10.1016/j.bbagen.2015.01.011
DO - 10.1016/j.bbagen.2015.01.011
M3 - Article
C2 - 25615535
AN - SCOPUS:84922289434
SN - 0304-4165
VL - 1850
SP - 832
EP - 844
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 4
ER -