Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels

Abstract

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.

The Nav1.2 channel is regulated by GSK3

“…To test this hypothesis, we expressed chimeric constructs carrying the single transmembrane domain protein CD4, depleted of its intracellular C-terminal tail (CD4-ΔC-tail), fused in frame with either the I–II loop (CD4-Na v 1.2-I-II loop), the II–III loop (CD4-Na v 1.2-II-III loop), or the C-terminal tail of the Na v 1.2 (CD4-Na v 1.2-C-tail), intracellular domains of the Na v 1.2 channel rich in trafficking motifs and phosphorylation sites [36–38]. These constructs were transiently expressed in COS-7 cells, and the CD4 surface pool was labeled using an Alexa 488-conjugated antibody against the extracellular domain of CD4 applied on live cells, followed by fixation and staining with HCS Cell Mask ™ Deep Red Plasma membrane stain to label the cell contour (Fig.…”

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

“…Thus, by impacting the affinity of the Nav channels for its chaperone protein, FGF14, S/T phosphorylation of FGF14 is likely to be a critical nexus in neuronal excitability. Interestingly, S/T phosphorylation sites that match a GSK–3 motif are also found on Nav; the T1966 residue in the C-tail of Nav1.2 has been validated in vivo and also lies in a putative GSK–3 phosphorylation motif (Baek et al, 2011; Farghaian et al, 2011). This residue is conserved in Nav1.6 and might also be a candidate for the effect of GSK–3 phosphorylation that we observed in this study.…”

“…Quantitative phosphoproteomics can be combined with state-of-the-art mass spectrometry to identify potential signaling pathways that regulate post-translational modifications of cellular components (Baek et al, 2011; Berendt et al, 2010; Olsen et al, 2006). Recent studies have identified highly clustered areas containing phosphorylation sites on the voltage-gated Na + (Nav) Nav1.2 isoform, particularly at the I–II linker and the C-terminal intracellular regions (Baek et al, 2011; Berendt et al, 2010).…”

“…Recent studies have identified highly clustered areas containing phosphorylation sites on the voltage-gated Na + (Nav) Nav1.2 isoform, particularly at the I–II linker and the C-terminal intracellular regions (Baek et al, 2011; Berendt et al, 2010). 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).…”

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)

“…Multisite phosphorylation has been shown to dynamically regulate the localization, activity, and expression of the voltagegated sodium channel and potassium channel in mammalian brain (41). In contrast, multiple phosphorylations on the C terminus of photoexcited rhodopsin are required for its inactivation (for a review, see Ref.…”