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The voltage‐gated sodium (Nav) channel complex is comprised of pore‐forming α subunits (Nav1.1–1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C‐terminal tail binds directly to FGF14 and this interaction modifies Nav1.6‐mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14 V160 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14 V160 to probe for the FGF14 interaction with the Nav1.6 C‐terminal tail and to evaluate the activity of the peptide on Nav1.6‐mediated currents. In silico docking predicts FLPK to bind to FGF14 V160 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split‐luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole‐cell patch‐clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14‐dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N‐terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na + currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.

The voltage-gated Na+ (Nav) channel is a primary molecular determinant of the initiation and propagation of the action potential. Despite the central role of the pore-forming α subunit in conferring this functionality, protein:protein interactions (PPI) between the α subunit and auxiliary proteins are necessary for the full physiological activity of Nav channels. In the central nervous system (CNS), one such PPI occurs between the C-terminal domain of the Nav1.6 channel and fibroblast growth factor 14 (FGF14). Given the primacy of this PPI in regulating the excitability of neurons in clinically relevant brain regions, peptides targeting the FGF14:Nav1.6 PPI interface could be of pre-clinical value. In this work, we pharmacologically evaluated peptides derived from FGF14 that correspond to residues that are at FGF14’s PPI interface with the CTD of Nav1.6. These peptides, Pro-Leu-Glu-Val (PLEV) and Glu-Tyr-Tyr-Val (EYYV), which correspond to residues of the β12 sheet and β8-β9 loop of FGF14, respectively, were shown to inhibit FGF14:Nav1.6 complex assembly. In functional studies using whole-cell patch-clamp electrophysiology, PLEV and EYYV were shown to confer differential modulation of Nav1.6-mediated currents through mechanisms dependent upon the presence of FGF14. Crucially, these FGF14-dependent effects of PLEV and EYYV on Nav1.6-mediated currents were further shown to be dependent on the N-terminal domain of FGF14. Overall, these data suggest that the PLEV and EYYV peptides represent scaffolds to interrogate the Nav1.6 channel macromolecular complex in an effort to develop targeted pharmacological modulators.

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