The Nav1.2 channel is regulated by GSK3

Abstract

Background Phosphorylation plays an essential role in regulating the voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. Herein, 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 were 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-Ctail 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 modulate Nav channel function via its C-terminal tail. General Significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

“…As a result, these studies revealed a network of kinases centered on GSK–3 that modulate the FGF14:Nav channel complex bidirectionally (Hsu et al, 2015). As shown previously, pharmacological inhibition and/or silencing of GSK–3 suppresses the interaction of FGF14 with Nav1.6 and Nav1.2 channels, modulates the functional activity of FGF14 on Na + currents, and control the sub-cellular distribution and co-localization of the two proteins in the axon and the somatodendritic compartment, phenotypes that might contribute to the role of GSK–3 in regulating neuronal excitability in the brain (James et al, 2015; Shavkunov et al, 2013; Laezza et al, 2007, 2009). These studies highlight the potential of using protein complementation assays to assess signaling pathways, a method that has been extended in the current study through the identification of a novel pathway that controls FGF14 function.…”

“…Thus, we hypothesized that changes in phosphorylation of residues at the FGF14 C-tail could affect the interaction with Nav channels. Our previous research has shown that GSK–3, a multifunctional kinase important for neuronal survival, cellular signaling, and stress response (Jope et al, 2007; Jope & Roh, 2006; Wu et al, 2013; Chen et al, 2011; Kim & Snider, 2011) and dysregulated in a number of AD and psychiatric disorders (Emamian, 2012; Jope et al, 2007; Jope & Roh, 2006; Liu et al, 2013; Budni et al, 2012; Scala et al, 2015; Maqbool et al, 2016; Morris & Berk, 2016; Provensi et al, 2016; Avila et al, 2010) affecting cognition, including depression and bipolar disorder (Gould et al, 2004; Koros & Dorner-Ciossek, 2007; Omata et al, 2011), critically modifies the interaction, assembly, localization, and activity of FGF14 and Nav channels (Hsu et al, 2015; James et al, 2015; Shavkunov et al, 2013). Furthermore, we observed that the C-tail of FGF14 contains a consensus GSK–3 phosphorylation motif ( S/T )XXX(S/T), the first residue in this sequence corresponding to S226.…”

“…High resolution nanoLC-MS/MS analysis was performed using an Easy LC1000 (Proxeon Biosystems, Odense, Denmark) interfaced to a LTQ Orbitrap Elite (Thermo Fisher Scientific) as previously described (James et al, 2015). All LC gradient and MS data acquisition parameters were unchanged.…”

“…These phosphorylation sites control channel gating, expression, and sub-cellular localization, which impact Nav channel function and consequently affect excitability (Berendt et al, 2010; Hsu et al, 2015, 2016; Wildburger et al, 2015). Previous studies have identified alterations in the phosphoproteome concomitant with neurologic disease: studies in humans with AD as well as an early onset mouse model of AD reveal significant alterations in global phosphorylation profiles with critical alterations in multiple cellular signaling pathways (Wang et al, 2013; Zahid et al, 2012), and phosphorylation as well as other post-translational modifications of Nav1.2 channels (James et al, 2015), such as methylation, are reciprocally modulated in epilepsy (Baek et al, 2014). These observations suggest that PTMs on Nav channel complexes may have important implications in the screening and diagnosis of brain disorders.…”

“…Among these, fibroblast growth factor 14 (FGF14) stands out for its specific modulatory roles on neuronal Nav channels. Recent studies highlight the importance of FGF14, a multivalent accessory protein of the Nav channel, in animal as well as human studies (Hsu et al, 2015, 2016; James et al, 2015; Ali et al, 2016; Alshammari et al, 2016a, b, c; Tempia et al, 2015; Ali et al, 2014; Shavkunov et al, 2013). Through direct monomeric binding to the Nav channel C-terminal tail, FGF14 forms a complex with the channel that is required for proper gating, expression and trafficking of the channel to the AIS as well as the control of neuronal excitability (Ali et al, 2014, 2016; Alshammari et al, 2016a; Laezza et al, 2007, 2009; Lou et al, 2005; Wang et al, 2000, 2002; Xiao et al, 2007; Goetz et al, 2009).…”