Targeting the noncatalytic activity of GSK3β modulates neuronal excitability in medium spiny neurons via Nav1.6 interactions

Background and Purpose: Kinases phosphorylate ion channels, but their noncatalytic roles via protein–protein interactions (PPI) are less understood. Here, we identified the peptidomimetic ZL141 to characterize the PPI between GSK3β and Nav1.6, revealing a noncatalytic role for GSK3β in regulating Nav1.6 currents and neuronal excitability. Experimental Approach: To screen peptide inhibitors, the GSK3β/Nav1.6 C-terminal tail (CTD) PPI was reconstituted using split-luciferase complementation assay. Intrinsic fluorescence spectroscopy (IFS), surface plasmon resonance (SPR), kinase assays, and molecular docking were used to assess peptidomimetic binding and predict the PPI interface. ZL141's function was evaluated via patch-clamp electrophysiology in heterologous cells and nucleus accumbens (NAc) medium spiny neurons (MSNs), along with in vivo GSK3β silencing. Key Results: ZL141 disrupts GSK3β/Nav1.6 CTD complex formation without altering GSK3β's catalytic activity. AlphaFold modelling predicted the ZL141 binding site at the surface of the GSK3β/Nav1.6 CTD PPI interface. ZL141 has a micromolar affinity for GSK3β, suppresses Na⁺ currents, and affects Nav1.6 activation and inactivation kinetics. In vivo, silencing GSK3β in the NAc mimics the inhibitory effects of ZL141 on MSN firing and occludes any further pharmacological activity of the compound. Conclusions and Implications: These findings reveal a critical PPI role of GSK3β in transducing Nav1.6 function and contributing to MSN firing. Both GSK3β and Nav1.6 activities are dysregulated in neurological and neuropsychiatric disorders. Given that Nav1.6 hyperactivity is implicated in maladaptive reward signalling, PPI-targeted modulation using compounds like ZL141 may offer a precision pharmacological strategy for treating psychiatric disorders such as bipolar disorder or schizophrenia.