Axin-binding domain of glycogen synthase kinase 3β facilitates functional interactions with voltage-gated Na+ channel Nav1.6

Figures

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  Figure 1. Mutagenesis screening reveals ABD as a critical motif for GSK3β–Nav1.6 complex assembly.A, three-dimensional protein structure of GSK3β. Structural location representation of the GSK3β domains using X-ray crystallography (Protein Data Bank code: 1J1C), illustrating the surface locations of deletion aa 262 to 299 (red), activation loop aa 200 to 226 (purple), and CTD aa 344 to 388 (green). B, sequence alignment of the Nav1.6 CTD (1952–1976) with GSK3β-binding proteins axin (406–483) and GSKIP (117–139). C, schematic of GSK3β sequence indicating position of truncation mutants. D, real-time LCA output of GSK3β–Nav1.6 complex formation in HEK293 cells transfected with LCA constructs CLuc-GSK3β and CD4-Nav1.6-NLuc; d-luciferin was added at time 0. E, LCA results of Nav1.6 and GSK3β mutant LCA pairs (normalized to controls) reveal a key role of the ABD in mediating GSK3β–Nav1.6 complex formation. F, real-time LCA output of CD4-Nav1.6 Nluc and CLuc-GSK3β or CLuc-GSK3βΔABD treated with vehicle (0.5% DMSO) or 2 μM CHIR99021 (CHIR). G, bar-graph representation of the LCA conducted in F. H, Western blot illustrating expression of GSK3β mutant constructs. Data are mean ± SEM. In E; n = 32…

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  Figure 2. ABD overexpression decreases GSK3β–Nav1.6 complex assembly and alters Nav1.6 distribution patterns.A, schematic of GSK3β sequence indicating the location of the ABD construct. B, real-time LCA output of GSK3β–Nav1.6 complex assembly in cells overexpressing GFP (black) or ABD-GFP (blue). C, column representation of the LCA in (B); each point represents an individual replicate (n = 16 for each condition) (D–I) confocal images of HEK-Nav1.6 cells stably expressing Nav1.6 that were transiently transfected with either control (GFP, D–H) or ABD-GFP (I–M). Nav1.6 puncta were visualized with an Alexa-647-conjugated anti-rabbit Nav1.6 antibody. D–F, show overlays of GFP and Nav1.6 (D), GFP (E), Nav1.6 (F), puncta masks (G), and merged images (H) in HEK-Nav1.6 cells transiently transfected with GFP control. Panels i–iii indicate insets of F (i), E (ii), and H (iii). I–M, show overlays of GFP and Nav1.6 (I), GFP (J), Nav1.6 (K), puncta masks (L), and merged images (M) in HEK-Nav1.6 cells expressing ABD-GFP. Panels iv–vi indicate insets of K (iv), J (v), and M (vi). N, quantification of total puncta/cell in HEK-Nav1.6 cells expressing GFP (black) or ABD-GFP (blue). O, quantification of total…

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  Figure 3. Effects of GSK3β inhibition and overexpression on Nav1.6 puncta distribution.A, laser-scanning confocal images of HEK293 cells overexpressing GFP, ABD-GFP, or GSK3β-IRES-GFP (green) and treated with vehicle (0.5% DMSO) or 2 μM CHIR99021 (CHIR). DNA was visualized using 4′,6-diamidino-2-phenylindole (blue). Nav1.6 puncta were visualized using an Alexa-647-conjugated anti-rabbit Nav1.6 antibody (red). B, quantification of total puncta/cell in the indicated experimental groups. C, quantification of total puncta per unit/area (μm2) in the indicated experimental groups. Data are represented as mean ± SEM. Significance was assessed using an ordinary one-way ANOVA with multiple comparisons test. In (B and C), each point represents an individual cell. (GFP+DMSO, n = 33; GFP+CHIR, n = 32; ABD+DMSO, n = 33; ABD+CHIR, n = 30; GSK3β+DMSO, n = 27). In (B), GFP-DMSO versus GFP+CHIR, p = 0.0002; GFP+DMSO versus ABD+DMSO, p < 0.0001; GFP+DMSO versus GSK3β+DMSO, p = 0.0025; ABD-DMSO versus ABD+CHIR, p = 0.9709). In (C), GFP-DMSO versus GFP+CHIR, p = 0.0109; GFP+DMSO versus ABD+DMSO, p = 0.0090; GFP+DMSO versus GSK3β+DMSO, p = 0.0054; ABD-DMSO versus ABD+CHIR, p = 0.9944. ∗p < 0.05; ∗∗p < 0.01;…

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  Figure 4. Functional effects of ABD overexpression on Nav1.6-mediated currents in heterologous cells.A, representative traces of transient INa elicited by HEK-Nav1.6 cells transfected with GFP (black) or ABD-GFP (blue). B and C, comparison of peak INa + density (B) and tau (C) between the experimental groups described in (A). D, normalized conductance plotted as a function of voltage to characterize effects of experimental conditions on the voltage dependence of activation of Nav1.6-mediated INa+.E, bar graph representation of the voltage dependence of activation among indicated experimental groups. F, normalized current plotted as a function of voltage to characterize the voltage dependence of steady-state inactivation among experimental groups. G, bar graph comparison of the voltage dependence of steady-state inactivation among indicated experimental groups. Data are individual replicates with SEM error bars (n = 8–12 cells/group). Significance was assessed using an unpaired t test. ∗p < 0.05; ∗∗p < 0.01. ABD, axin-binding domain; HEK, human embryonic kidney cell line.

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  Figure 5. ABD overexpression reduces excitability and Nav1.6-encoded currents in CA1 pyramidal neurons.A, representative traces of evoked action potentials of CA1 cells from mice treated with either GFP (black) or ABD-GFP (blue) in response to the depicted current-clamp protocol. B, number of action potentials fired by CA1 cells of mice treated with GFP (black) or ABD-GFP (blue) across a range of injected current stimuli (0–220 pA). C, maximum number of action potentials evoked from CA1 cells in mice treated with GFP (black) or ABD-GFP (blue) across a range of injected current stimuli (0–220 pA). D–G, comparison of current threshold (D), voltage threshold (E), input resistance (F), and resting membrane potential (G) between the indicated experimental groups. H, representative traces of INa+ elicited by CA1 neurons from mice treated with either GFP (black) or ABD-GFP (blue) in response to the depicted protocol. I, current–voltage relationship of INa+ elicited from CA1 neurons treated with GFP (black) or ABD-GFP (blue). J, Bar-graph comparison of peak current density from the indicated experimental groups. Significance was assessed using an unpaired t test. Data shown are individual…