The Nav1.2 channel is regulated by GSK3.

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 GSK3ß 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 GSK3ß 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.

The fibroblast growth factor 14·voltage-gated sodium channel complex is a new target of glycogen synthase kinase 3 (GSK3).

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na(+) (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na(+) currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.

Decreased sexual motivation and heightened anxiety in male Long-Evans rats are correlated with the memory for a traumatic event.

Individuals suffering from posttraumatic stress disorder (PTSD) frequently report disturbances in sexual functioning in addition to alterations in their affective behaviors. Notably, maladaptive cognitions and dysfunctional behaviors are perpetuated by the emergence of the intrusive thoughts that characterize the disorder. In rats, reminders of a traumatic event designed to simulate intrusive thoughts are associated with impairments in affective, social, and sexual behaviors. The current study examined the relationship between the memory for a traumatic event and changes in sexual and affective behaviors in male Long-Evans rats (N = 36). The trauma featured a combination stressor consisting of simultaneous exposure to a footshock and the odor of soiled cat litter. Memory for the trauma was reactivated by re-exposures to the context of the trauma in the absence of stressors and confirmed by assessing the percentage of time spent freezing. Following the second and final reminder, traumatized males exhibited reduced sexual motivation and increased anxiety, signified by longer latencies to achieve their first mount on a post-stress test of sexual behavior, and longer latencies to begin feeding in a novel environment, respectively. Correlational analyses revealed that decreased sexual motivation and heightened anxiety were predicted by the memory for the trauma as indicated by the time spent freezing during the re-exposures. The findings from the current study have implications for understanding the relationship between stress and sexual functioning and indicate that the impairments in sexual behavior that often occur in individuals with PTSD may be impacted by their memory for the trauma.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway.

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.