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

Voltage-gated Na+ (Na_v) channels are the primary determinants of the action potential in excitable cells. Na_v channels rely on a wide and diverse array of intracellular protein-protein interactions (PPIs) to achieve their full function. Glycogen synthase kinase 3β (GSK3β) has been previously identified as a modulator of Na_v1.6-encoded currents and neuronal excitability through PPI formation with Na_v1.6 and phosphorylation of its C-terminal domain (CTD). Here, we hypothesized that GSK3β functions as a scaffold in a regulatory PPI complex with the Na_v1.6 CTD. Mutagenesis screening using the split-luciferase complementation assay indicated that the axin-binding domain (ABD) of GSK3β (262-299) is necessary for complex formation between the Na_v1.6 CTD and GSK3β, and that residues within this domain are drivers of GSK3β-mediated regulation of the channel. Overexpression of an ABD-GFP fusion construct in human embryonic kidney 293 cells stably expressing Na_v1.6 significantly reduced Na_v1.6 nanocluster density compared with GFP alone. In addition, overexpression of the ABD-GFP fusion construct ablates GSK3β-mediated potentiation of Na_v1.6-encoded currents and alters channel kinetics. Finally, in vivo AAV-mediated overexpression of the ABD-GFP construct in the CA1 hippocampal region induced a reduction in maximal action potential firing and an increase in action potential current threshold in a manner resembling previously reported effects of GSK3β silencing in neurons. Taken together, these results not only suggest that GSK3β-mediated regulation of Na_v1.6 extends beyond transient phosphorylation but also implicates the ABD as a critical regulatory domain that facilitates GSK3β's functional effects on Na_v1.6 and neuronal excitability.