Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

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.

Given their essential role in regulating physiology throughout the body, Nav channels have historically been a traditional target for drug development. Unfortunately, current therapeutics targeting Nav channels bind to structural motifs of the α subunit that display high amino acid sequence homology among the nine Nav channels isoforms, which results in these therapeutics lacking isoform selectivity and giving rise to deleterious off-target side effects due to modulation of offtarget Nav channel isoforms (Catterall and Swanson, 2015). To address this challenge, the identification of novel Nav channel drug-binding sites is a necessary pre-requisite to identify therapeutics with improved selectivity (Dvorak et al., 2021).
Among structural components of the Nav channel that could be pharmacologically targeted to achieve improved selectivity, C-terminal domains (CTD) of Nav channels stand out as promising surfaces to target, as they display amino acid sequence divergence among isoforms that enables structurally and functionally specific protein:protein interactions (PPI) with auxiliary proteins (Lou et al., 2005;Laezza et al., 2007Laezza et al., , 2009Tseng et al., 2007;Wang et al., 2011;Pitt and Lee, 2016;Effraim et al., 2019;Lu et al., 2020). In the central nervous system (CNS), one salient example of such a PPI occurs between the CTD of Nav1.6 and its auxiliary protein fibroblast growth factor 14 (FGF14) (Liu et al., 2003;Lou et al., 2005;Goetz et al., 2009;Laezza et al., 2009;Ali et al., 2016Ali et al., , 2018Hsu et al., 2016;Singh et al., 2020;Wadsworth et al., 2020). Specifically, this PPI regulates the transient and resurgent Na+ currents of neurons through a mechanism thought to depend upon the N-terminus of FGF14 (Yan et al., 2014;White et al., 2019), as well as the action potential (AP) firing of neurons in clinically relevant brain regions, including the nucleus accumbens (NAc) (Ali et al., 2018) and hippocampus (Hsu et al., 2016). Translationally, perturbation of this PPI is increasingly being associated with a myriad of neurologic and neuropsychiatric disorders (Di Re et al., 2017;Paucar et al., 2020), highlighting its potential clinical relevance as a pharmacological target.
To guide drug discovery efforts targeting the PPI interface between FGF14 and the CTD of Nav1.6, we previously developed and interrogated a homology model of the PPI interface to identify putative clusters of amino acids central to assembly of the complex (Ali et al., 2014(Ali et al., , 2016. These investigations identified the Phe-Leu-Pro-Lys (FLPK) and Pro-Leu-Glu-Val (PLEV) motifs on the β12 sheet of FGF14, and the Glu-Tyr-Tyr-Val (EYYV) motif on the β8-β9 loop of FGF14, as being putatively essential for FGF14:Nav1.6 complex assembly (Ali et al., 2014(Ali et al., , 2016. To investigate if short peptides derived from these clusters of amino acids could exert functionally relevant modulation of the Nav1.6 channel macromolecular complex, we previously reported our pharmacological evaluation of the FLPK tetrapeptide . In that work, we showed that FLPK inhibited FGF14:Nav1.6 complex assembly, reversed FGF14-mediated regulatory effects on Nav1.6 channel activity and affected neuronal excitability of MSNs of the NAc . Based upon this premise, we sought in the current work to investigate the modulatory effects of PLEV and EYYV on the Nav1.6 channel macromolecular complex. By employing the split-luciferase complementation assay (LCA) and whole-cell patch clamp electrophysiology, we show that these two short peptides derived from FGF14 confer inhibitory effects on FGF14:Nav1.6 complex assembly. Correspondingly, both peptides confer functionally relevant modulation of Nav1.6 channel activity in a manner dependent on the N-terminal domain of FGF14. Overall, this study demonstrates that short peptides derived from "hot spot" (London et al., 2010(London et al., , 2013 of PPI interfaces could serve as innovative probes to guide drug discovery efforts.

Homology Model-Based Docking of PLEV and EYYV to FGF14
The homology model-based docking was run with Schrödinger Small-Molecule Drug Discovery Suite using the FGF14 chain of a previously described FGF14:Nav1.6 homology model (Ali et al., 2016;Singh et al., 2020). The structure for protein was prepared by using Protein Prepared Wizard and peptide fragments (PLEV or EYYV containing N-terminal acetylation and C-terminal amidation) were prepared with LigPrep and further initial lowest energy conformation was obtained. The grid box coordinates, grid generation, docking employment, docking poses were analyzed as previously described .

Cell Culture
HEK293 cells were cultured in a 1:1 mixture of Dulbecco's Modified Eagle Medium (DMEM) with 1 g/L glucose and F-12 (Invitrogen, Carlsbad, CA, United States) that was additionally supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml streptomycin (Invitrogen). HEK293 cells stably expressing CLuc-FGF14 and CD4-Nav1.6-NLuc constructs were maintained similarly except for the addition of 500 μg/ml G418 and 100 ug/ml puromycin (Invitrogen) to maintain stable expression. This cell line was developed and characterized in previous studies and is hereafter coded as "Clone V" cells . Cells were grown at 37°C. The HEK293 cells stably expressing human Na v 1.6 channels have previously been described (Ali et al., 2016(Ali et al., , 2018Singh et al., 2020;Wadsworth et al., 2020). For transient transfections, the Lipofectamine 2000 protocol was followed (Invitrogen, Waltham, MA, United States), and the amount of cDNA used was 1 µg for each. For whole-cell, patch-clamp recordings HEK293-Nav1.6 cells were washed and replated at very low density prior to incubating the cells with peptides for recordings (Ali et al., 2016(Ali et al., , 2018Wadsworth et al., 2019Wadsworth et al., , 2020Singh et al., 2020).

Electrophysiology in Heterologous Cells
Whole-cell voltage-clamp recordings in heterologous cell systems were performed as previously described (Ali et al., 2016(Ali et al., , 2018Singh et al., 2020;Wadsworth et al., 2020). The HEK293 cells cultured as described above were dissociated using TrypLE and re-plated at very low density onto glass coverslips. HEK293 cells were then allowed at least 3-4 h for attachment before coverslips were transferred to the recording chamber. The recording chamber was filled with a freshly prepared extracellular recording solution comprised of: 140 mM NaCl; 3 mM KCl; 1 mM MgCl 2 ; 1 mM CaCl 2 ; 10 mM HEPES; and 10 mM glucose (pH 7.3; all salts purchased from Sigma-Aldrich, St. Louis, MO, United States). For control recordings, DMSO was added to the extracellular solution to obtain a final concentration of 0.1%. For the peptide conditions a PLEV and EYYV peptides were added to the extracellular solution to obtain their final concentrations. Cells were pre-incubated for at least 30 min in either DMSO or peptides containing extracellular solutions prior to the recordings. The pipettes were filled with an intracellular solution: 130 mM CH 3 O 3 SCs; 1 mM EGTA; 10 mM NaCl; and 10 mM HEPES (pH 7.3; all salts purchased from Sigma-Aldrich). The glass pipettes (Harvard Apparatus, Holliston, MA, United States) with a resistance of 3-5 MΩ were fabricated using a PC-100 vertical Micropipette Puller (Narishige International Inc., East Meadow, NY, United States). Recordings were obtained using an Axopatch 700B or 200B amplifier (Molecular Devices, Sunnyvale, CA, United States). Membrane capacitance and series resistance were estimated using the dial settings on the amplifier, and capacitive transients and series resistances were compensated by 70-80%. Data acquisition and filtering occurred at 20 and 5 kHz, respectively, before digitization and storage. Clampex 9.2 software (Molecular Devices) was used to set experimental parameters, and electrophysiological equipment interfaced to this software using a Digidata 1,200 analog-digital interface (Molecular Devices). Analysis of electrophysiological data was performed using Clampfit 9 software (Molecular Devices) and GraphPad Prism 7 software (La Jolla, CA, United States). After GΩ seal formation and entry into the whole-cell configuration, the voltage-clamp protocols were employed such as the currentvoltage (IV) protocol which entailed voltage-steps from −100 mV to +60 mV from a holding potential of −70 mV. The voltagedependence of steady-state inactivation was calculated using a paired-pulse protocol during which, from the holding potential, cells were stepped to varying test potentials between −20 mV and +20 mV prior to a test pulse to −20 mV. Current densities were obtained by dividing Na + current (I Na ) amplitude by membrane capacitance. Current-voltage relationships were then assessed by plotting current density as a function of applied voltage. Tau (τ) of fast inactivation was calculated by fitting the decay phase of currents at the -10 mV voltage step with a one-term exponential function. To assess voltage-dependence of activation, conductance (G Na ) was first calculated using the following equation: where I Na is the current amplitude at voltage V m , and E rev is the Na + reversal potential. Activation curves were then generated by plotting normalized G Na as a function of the test potential. Data was then fitted with the Boltzmann equation to determine V 1/2 of activation using the following equation: where G Na,max is the maximum conductance, V a is the membrane potential of half-maximal activation, E m is the membrane voltage, and k is the slope factor. For steady-state inactivation, normalized current amplitude (I Na /I Na,max ) at the test potential was plotted as a function of pre-pulse potential (V m ) and fitted using the Boltzmann equation: where V h is the potential of half-maximal inactivation, E m is the membrane voltage, and k is the slope factor.
Cumulative inactivation was examined by applying a 2 ms test pulse to −10 mV 20 times at frequency10 Hz from a holding potential of −80 mV. Current responses were normalized to the first recorded pulse and the currents at the 20 th pulses were compared Effraim et al., 2019).

Data Analysis
Results are expressed as mean ± standard error of the mean (SEM). Except where otherwise noted, statistical significance was determined using a Student's t-test or one way ANOVA Tukey's multiple comparisons test, comparing cells treated with vehicle (DMSO) or PLEV and EYYV, with p < 0.05 being considered statistically significant. The analysis was performed by using GraphPad Prism R (La Jolla, CA) software.

RESULTS
Homology Model-Based Docking of PLEV and EYYV to the Protein-Protein Interaction Interface Between FGF14 and the CTD of Nav1.6 The peptides used in this study were developed based upon segments of FGF14 that are known to be at the interface of the FGF14:Nav1.6 macromolecular complex. PLEV corresponds to an amino acid sequence on the β12 sheet of FGF14, and EYYV corresponds to a sequence on the β8-β9 loop of FGF14 (Ali et al., 2014). These peptides were docked to a homology model of the PPI interface between FGF14 and the CTD of Nav1.6. (Ali et al., 2016(Ali et al., , 2018Dvorak et al., 2020). In our previous study  we predicted that PLEV and EYYV were interacting toward the periphery of FGF14 at the β5 strand and N-terminus. We further showed the interactions of PLEV and EYYV with key residues at the FGF14:Nav1.6 PPI interface (modified Figure 1, Singh et al., 2020). The molecular docking of PLEV and EYYV revealed predicted interactions with residues at the FGF14:Nav1.6 PPI interface suggestive of the ability to disrupt FGF14:Nav1.6 complex assembly. For example, PLEV and EYYV displayed conserved interactions with R117 on the β5 strand of FGF14, a residue that is a crucial constituent of the core domain of FGF14 and is essential for enabling FGF14-mediated regulatory effects on transient I Na and resurgent I Na (Yan et al., 2014). Additionally, PLEV and EYYV displayed conserved interactions with K74 of the N-terminus of FGF14, which could be predictive of these peptides displaying functional activity due to the central role of the N-terminus of FGF14 in conferring modulatory effects on Nav channel inactivation and resurgent I Na (Laezza et al., 2007;Yan et al., 2014;White et al., 2019). Despite these conserved interactions, PLEV and EYYV also displayed divergent interactions with residues at the FGF14:Nav1.6 PPI interface, such as with P203, which could be suggestive of differential effects on Nav1.6 channel activity. Based upon these predicted interactions with residues at the FGF14:Nav1.6 PPI interface in silico, both PLEV and EYYV were further investigated in biological systems to interrogate potential modulatory effects on the Nav1.6 channel macromolecular complex.

In-Cell Testing of PLEV and EYYV Using the Split Luciferase Complementation Assay
To evaluate the hypothesis that PLEV and/or EYYV could modulate FGF14:Nav1.6 complex assembly, an in-cell assay was used with a HEK293 cell line (hereafter coded as "Clone V" cells) stably expressing both CLuc-FGF14 and CD4-Nav1.6-NLuc (Shavkunov et al., 2012;Shavkunov et al., 2013;Shavkunov et al., 2015;Ali et al., 2016Ali et al., , 2018Wadsworth et al., 2019Wadsworth et al., , 2020Dvorak et al., 2020Dvorak et al., , 2021Singh et al., 2020;Wang et al., 2020). In this assay, upon binding of FGF14 to the Nav1.6 C-terminal tail, there is reconstitution of the NLuc and CLuc halves of the luciferase enzyme, which produces luminescence in the presence of substrate D-luciferin (Figures 2A,D). Employing this assay in the current study, Clone V cells were seeded into 96 well plates and then treated with various concentrations (1-250 µM) of PLEV, EYYV, or 0.5% DMSO. After incubation, luciferin was dispensed, and the luminescence observed in each well was recorded. The max luminescence observed in each well was then normalized to the average max luminescence observed in per plate control wells. These investigations revealed that PLEV and EYYV displayed dosedependent inhibitory effects on FGF14:Nav1.6 complex assembly. Specifically, sigmoidal fitting of the dose response curve for PLEV and EYYV revealed IC 50 values of 41.1 ± 4.4 µM and 35.7 ± 4.7 µM, respectively. ( Figures 2B,C,E,F). Having demonstrated their inhibitory effects on FGF14:Nav1.6 complex assembly, the functional effects of these peptides on Nav1.6 channel activity were subsequently assessed using whole-cell patch-clamp electrophysiology in heterologous cells. Having determined the in-cell potency with which PLEV and EYYV inhibit FGF14:Nav1.6 complex assembly, we next used electrophysiology to evaluate whether PLEV or EYYV displayed modulatory effects on Nav1.6-mediated currents. These recordings were performed in HEK293 cells stably expressing Nav1.6 and transiently transfected with GFP (Nav1.6-GFP) or FGF14-GFP (Nav1.6-FGF14-GFP). The cells were incubated for at least 30 min with 0.1% DMSO or either one of the two tetrapeptides at 50 µM final concentration in a static bath before recording. After incubation, the modulatory effects of PLEV and EYYV on Nav1.6-mediated currents were assessed using whole-cell patch-clamp recordings (Figures 3, 4 and Table 1). In agreement with previous studies (Ali et al., 2016(Ali et al., , 2018Singh et al., 2020;Wadsworth et al., 2020), expression of FGF14-GFP suppressed the Nav1.6-mediated peak transient Na + current (I Na ) density (−20.58 ± 3.03 pA/pF, n 12 vs −59.63 ± 5.33 pA/pF, n 15; p < 0.001; one-way ANOVA Tukey's multiple comparison test; Figures 3A,B and Table 1).
In contrast to their conserved effects on Nav1.6 channel steady-state inactivation, PLEV and EYYV displayed divergent effects on long-term inactivation (LTI) and cumulative inactivation induced by applying a 2 ms test pulse to −10 mV 20 times at frequency of 10 Hz from a holding potential of −80 mV. Similar to results shown in Figure 3 and Figures 4A-D, both PLEV and EYYV displayed no effect on LTI or cumulative inactivation in the absence of FGF14 ( Figures 4E-H and Table 1). Additionally, EYYV displayed no effects on LTI or cumulative inactivation in the presence of FGF14 ( Figures 4E-H and Table 1). Notably, however, in the presence of FGF14, PLEV markedly altered the function of FGF14 as it pertains to its regulatory effects on LTI and cumulative inactivation. Specifically, treatment of HEK-Nav1.6 cells co-expressing FGF14 with PLEV resulted in an increased fraction of channels entering into LTI compared to HEK-Nav1.6 cells coexpressing FGF14 treated with vehicle ( Figures 4E,F and Table 1). Paradoxically, treatment of HEK293-Nav1.6 cells coexpressing FGF14 with PLEV resulted in an increased number of available channels after repetitive stimulation, whereas treatment of HEK293-Nav1.6 cells co-expressing FGF14 with DMSO (control) resulted in roughly the same number of available channels before and after repetitive stimulation ( Figures 4G,H and Table 1). Consistent with the results of the molecular modeling study shown in Figure 1, the results of these functional studies highlight conserved and divergent modulatory effects of PLEV and EYYV on the Nav1.6 channel macromolecular complex.

Functional Effects of PLEV and EYYV on Nav1.6 Channel Activity Are Dependent Upon the Presence of the N-terminal Domain of FGF14
The role of the N-terminal domain of FGF14 in conferring its functional regulation of Nav channel activity is widely recognized (Lou et al., 2005;Laezza et al., 2007;Yan et al., 2014;White et al., 2019). According to current models of iFGF-mediated regulation of Nav channel activity, it is proposed that the core domain of FGF14 interacts with the CTD of Nav1.6, which resultantly orients the N-terminal domain of FGF14 such that it can occlude the internal mouth of the pore (Yan et al., 2014;White et al., 2019). Resultantly, both mutating residues of the core domain of FGF14 that enable its PPI with the CTD of Nav1.6 and truncation of the N-terminal domain of FGF14 have been shown to reverse FGF14-mediated regulation of Nav1.6 channel activity (Yan et al., 2014). Based upon this model of FGF14mediated regulation of Nav1.6 channel activity, we next sought to investigate if PLEV and EYYV, which are derived from the core domain of FGF14, exert their modulatory effects on Nav1.6 channel activity through a mechanism dependent upon the N-terminal domain of FGF14. To do so, HEK293-Nav1.6 cells were transiently transfected with a cDNA construct corresponding to a FGF14 protein product with a truncated N-terminal domain (FGF14-ΔNT-GFP, 64-252 amino acid residues). Transiently transfected cells were then treated with 0.1% DMSO, 50 µM PLEV, or 50 µM EYYV, after which wholecell patch-clamp electrophysiology was performed as described for Figure 3 and Figure 4.
In these studies, we observed the previously identified (Laezza et al., 2007;Singh et al., 2020) phenotype in which co-expression   1, 2). Unlike the effects of PLEV on peak transient I Na density observed in the presence of FGF14 (see Figure 3), the peptide displayed no effects on this electrophysiological parameter when the   (Figures 5A-C). EYYV similarly displayed no effects on peak transient I Na density in the presence of FGF14-ΔNT ( Figures 5A-C). The lack of effects of both peptides on peak transient I Na density in the presence of FGF14-ΔNT were accompanied by lack of effects on τ of fast inactivation ( Figure 5D).    (13) 6.57 ± 0.37 (13) 1.11 ± 0.14 (13) Data are mean ± SEM; ns nonsignificant. Similar to their lack of effects on Nav1.6 channel activation in the presence of FGF14, PLEV and EYYV displayed no effects on this electrophysiological parameter in the presence of FGF14-ΔNT ( Figures 6A,B). Notably, however, whereas both PLEV and EYYV reversed FGF14-mediated regulatory effects on Nav1.6 channel steady-state inactivation ( Figures  4C,D), neither peptide displayed effects on this parameter in the presence of FGF14-ΔNT ( Figures 6C,D). Furthermore, the additional modulatory effects of PLEV on LTI and cumulative inactivation observed in the presence of FGF14 were also not observed in the presence of FGF14-ΔNT ( Figures 6E-H).
Overall, these studies provide strong evidence that these two peptides, despite being derived from the core domain of FGF14, exert their effects on Nav1.6 channel activity through a mechanism dependent upon the presence of the N-terminal domain of FGF14.

DISCUSSION
PLEV and EYYV represent previously identified tetrapeptides that correspond to clusters of amino acids on the β12 sheet and β8-β9 loop of FGF14, respectively, that are at the FGF14's PPI interface with the CTD of Nav1.6 (Ali et al., 2014(Ali et al., , 2016; Singh   , 2020). Given the primacy of these structural motifs of FGF14 in enabling FGF14:Nav1.6 complex assembly, we investigated if short peptides corresponding to these motifs of FGF14 could confer functionally relevant modulation of the Nav1.6 channel macromolecular complex. Given that perturbation of the PPI between FGF14 and the CTD of Nav1.6 gives rise to neural circuitry aberrations that are linked to neurologic and neuropsychiatric disorders (Di Re et al., 2017;Paucar et al., 2020), such peptides could represent promising "small-molecular inhibitor starting points (SMISP)" to develop PPI-targeting neuromodulators (Koes and Camacho, 2012a;2012b). To pharmacologically evaluate the PLEV and EYYV tetrapeptides as potential SMISPs, we employed an amalgam of complementary and orthogonal approaches including in silico molecular modeling, the LCA, and whole-cell patch-clamp electrophysiology. These results demonstrated that PLEV and EYYV both displayed functional modulation of the Nav1.6 channel macromolecular. Despite displaying mostly convergent modulatory effects on Nav1.6 channel activity, PLEV and EYYV displayed some divergent effects, consistent with their derivation from different structural motifs of FGF14. Overall, these studies provide strong evidence that PLEV and EYYV could serve as promising scaffolds for the development of chemical probes targeting the PPI interface between FGF14 and the CTD of Nav1.6. Additionally, this study further support the notion that short peptides derived from "hot spot" (London et al., 2010(London et al., , 2013 of PPI interfaces could serve as innovative probes to guide drug discovery efforts.
PLEV and EYYV Disrupt FGF14:Nav1.6 Complex Assembly Through Predicted Interactions With Resides at the PPI Interface In our previous study , we pharmacologically evaluated all three tetrapeptides at a single concentration of 50 µM using the LCA. In the FGF14: Nav1.6 wild type condition, all three tetrapeptides displayed comparable single concentration activity. In conditions in which putative "hot spot" residues at the FGF14:Nav1.6 PPI interface were mutated, such as Y158 and V160 (Ali et al., 2016(Ali et al., , 2018, the three peptides displayed some divergent effects. For example, in the FGF14 V160A condition, all three peptides lost activity, whereas in the FGF14 Y158A condition, FLPK retained its inhibitory effects on FGF14:Nav1.6 complex assembly, whereas PLEV and EYYV were shown to increase FGF14:Nav1.6 complex assembly. Based upon these divergent effects of PLEV and EYYV compared to FLPK, we elected to further pharmacologically evaluate these two tetrapeptides in the present investigation. In dose-response analyses studies, PLEV and EYYV were shown to inhibit FGF14:Nav1.6 complex assembly with IC 50 values of 41.1 ± 4.4 µM and 35.7 ± 4.7 µM, respectively. These in-cell studies, considered collectively with the in silico molecular modeling studies, highlight potential residues of FGF14 that, when occupied by a ligand, inhibit FGF14:Nav1.6 complex assembly. For example, PLEV and EYYV have predicted interactions with residues of the core domain of FGF14 including R117 and E152, as well as predicted interactions with residues of the N-terminal domain of FGF14 including K74. Given these predicted modes of binding coupled with the inhibitory effects of PLEV and EYYV on FGF14:Nav1.6 complex assembly, these residues could represent potential "hot spots" for the development of small molecular modulators targeting the FGF14:Nav1.6 PPI interface.

PLEV and EYYV Modulates Nav1.6 Channel Activity
To test whether PLEV and EYYV affected Nav1.6-mediated currents, we employed whole-cell patch-clamp electrophysiology in heterologous cell systems. We used HEK cells stably expressing human Nav1.6 (HEK-Nav1.6), and transiently transfected with GFP (HEK293-Nav1.6-GFP) or FGF14-GFP (HEK293-Nav1.6-FGF14-GFP) and treated with 50 µM PLEV and EYYV or 0.1% DMSO (Figures 3, 4). Both peptides did not show measurable effect in the absence of FGF14 (Nav1.6-GFP); however, PLEV partially reversed the FGF14-mediated regulatory effects on peak transient I Na density (Figures 3A-C; Table 1). This effect was not observed due to treatment with EYYV, highlighting the divergent mechanisms of action of the two tetrapeptides. Unlike their differential modulation of FGF14-mediated regulatory effects on peak transient I Na density, PLEV and EYYV displayed conserved effects in terms of reversing FGF14-mediated regulatory effects on Nav1.6 channel steady-state inactivation (Figures 4C,D; Table 1). Whereas PLEV and EYYV displayed conserved modulatory effects on Nav1.6 channel steady-state inactivation, PLEV also displayed distinct effects on LTI and cumulative inactivation that were not observed due to treatment with EYYV. Specially, PLEV increased the fraction of Nav1.6 channels that entered into LTI in a FGF14-dependent fashion, indicating a complex mechanism of action where treatment with PLEV results in altered function of FGF14. PLEV similarly altered the function of FGF14 as it related to cumulative inactivation, as HEK293-Nav1.6-FGF14-GFP cells treated with PLEV displayed an increased number of available channels after repetitive stimulation, whereas the same cells treated with DMSO displayed no change in the number of available channels before and after repetitive stimulation. All together, these effects demonstrate that PLEV and EYYV display both convergent and divergent effects on Nav1.6 channel activity, which could be attributable to their derivation from different structural motifs of FGF14 and differential interactions with residues at the FGF14:Nav1.6 PPI interface.
FGF14-1b N-Terminus Domain Required to Modulate Nav1.6 Channel Activity The N-terminal domain of FGF14-1b, the splice variant of FGF14 studied in the present investigation, is essential for conferring FGF14-mediated regulation of Nav1.6 channel activity, as deletion of the N-terminus of FGF14-1b abolishes the regulatory effects of FGF14 on a myriad of electrophysiological properties of Nav1.6 channels (Laezza et al., 2007;Yan et al., 2014). In line with previous studies, FGF14-ΔNT potentiates Nav1.6 current densities (Figures  3A-C, 5A-C; Tables 1, 2), causes a depolarizing shift in the voltage-dependence of Nav1.6 channel activation, a hyperpolarizing shift in the voltage-dependence of Nav1.6 channel steady-state inactivation, while having no effects on LTI or cumulative inactivation of Nav1.6 channels (Figures 3, 5E-H; Tables 1, 2) (Laezza et al., 2007Ali et al., 2018;Singh et al., 2020). In contrast to the modulatory effects of PLEV and EYYV on Nav1.6 channel activity in the presence of FGF14, the peptides display no effects in the presence of FGF14-ΔNT. As such, while the peptides are derived from clusters of amino acids constituent to the core domain of FGF14, their mechanisms of action are nevertheless dependent upon the N-terminal domain of FGF14. Given the central role of the N-terminus of FGF14-1b in the generation of resurgent I Na and the repetitive firing of action potentials (Yan et al., 2014), these results have important implications for anticipating the functional effects of both tetrapeptides in the native system. As FGF14-1b is highly enriched in clinically relevant brain regions, including the nucleus accumbens (Ali et al., 2018) and hippocampus (Hsu et al., 2016), along with Nav1.6, these peptides that disrupt their complex assembly could represent promising scaffolds for their development of PPI-targeting neuromodulators (Dvorak et al., 2021).

CONCLUSION
We have studied the modulatory effects of the tetrapeptides PLEV and EYYV, which correspond to residues of FGF14 that are at its PPI interface with the CTD of the Nav1.6 channel, on FGF14: Nav1.6 complex assembly and the functional activity of the Nav1.6 channel macromolecular complex. We have shown that both peptides functionally modulate Nav1.6 channel activity in a manner dependent upon the N-terminal domain of FGF14. Whereas both tetrapeptides inhibited FGF14:Nav1.6 complex assembly and reversed the FGF14-mediated depolarizing shift in the voltage-dependence of Nav1.6 channel steady-state inactivation, PLEV exerted additional modulatory effects not observed due to treatment with EYYV. Specially, PLEV increased the fraction of Nav1.6 channels that entered into LTI and modulated the fraction of channels that re-open during repetitive stimulation in a FGF14-depedent manner, indicating a complex mechanism of action where treatment with PLEV results in altered function of FGF14. Consistent with the molecular modeling studies shown in Figure 1, these nonoverlapping modulatory effects on Nav1.6 channel activity could be attributable to the tetrapeptides being derived from different structural motifs of FGF14 and resultantly displaying divergent interactions with residues at the FGF14:Nav1.6 PPI interface.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
Conceptualization, AS and FL; formal analysis, AS; investigation, AS, and FL; data curation, AS, ND., CT, AM; technical, SA and ZB; writing-original draft preparation, AS; writing-review and editing, AS, ND and FL; visualization, AS, and FL; supervision, AS, and FL; docking, HC; resources, JZ and FL; project administration, JZ and FL; funding acquisition, JZ, and FL. All authors have read and agreed to the published version of the manuscript.