Data are reported as absolute values or the absolute difference from baseline (delta, Δ). For Δ values, data were normalized either to the initial 500 ms of the stimulus (Figures 2 and 3) or baseline spiking (Figures 4 and 5). Time course graphs are represented as a mean ± standard error. Summary graphs are represented with box plots showing the median, quartiles, and 90% tails or with violin plots. All summary graphs overlay individual datapoints and represent recordings from single cells (reported n) for all electrophysiology experiments. Data were obtained from 5 to 9 animals (both sexes) per condition, which are standard group sample sizes used in the field. Group sizes were determined based on sample sizes previously used for similar studies. Power analysis was not required for this study. No attrition applies to our study, and all data collected is included. Analysis was performed blind to genotype ± drug. Statistical analysis was performed using Prism 10 (GraphPad Software). Quantified mean ± standard error and statistical test used is noted in figure legends. Significance was set at an alpha value of 0.05, and ‘ns’ indicates no significance.

ASCs exhibit high affinity for an extracellular region within voltage sensing domain IV (VSD IV) of select Na_V channels with a conserved tyrosine–tryptophan (YW) motif (Ahuja et al., 2015; Figure 1A and B). This YW motif found on Na_V1.2, Na_V1.6, and Na_V1.7 allows for ASC stabilization following channel inactivation (Figure 1B). By contrast, an SR motif present in Na_V1.1 and Na_V1.3 limits binding appreciably (Ahuja et al., 2015). Thus, we hypothesized that converting either Na_V1.2 or Na_V1.6 channels from those that contain the YW motif to those that contain an SR motif would alter ASC binding to those channels significantly.

To test for the effects of this motif substitution, we first examined currents generated by wildtype and YW→SR mutated channels expressed in immortalized cell lines (Figure 1C). HEK cells were primarily used for most experiments. But due to the known low transfection efficiency of Na_V1.6 in HEK cells, a subset of experiments was performed using an ND7/23 cell line engineered to lack most native Na_V conductance (ND7/LoNa_V) (Vanoye et al., 2024). Both Na_V1.2 and Na_V1.6 wildtype channels expressed in cell lines were inhibited markedly by 1 μM GNE-4076 (Figure 1C). By contrast, SR knock-in mutants continued to flux sodium in the presence of 1 μM GNE-4076. Dose–response curves revealed an IC₅₀ of 5.1 nM for wildtype Na_V1.2 that increases 365-fold to 1861 nM for mutant SR channels (Figure 1D). For Na_V1.6 channels, GNE-4076 IC₅₀ increased 475-fold from 184 nM to 87 μM with the SR mutant (Figure 1D). To assess whether SR mutations affect channel gating properties in the absence of GNE-4076 binding, steady-state activation and inactivation curves were assessed for wildtype or mutant channels (Figure 1E). Consistent with prior work studying a similar mutation in Na_V1.7 (Ahuja et al., 2015), the YW to SR mutation had no effect on slope factor for both voltage-dependent activation or inactivation for either Na_V1.2 or Na_V1.6 (Table 2); however, both voltage-dependent activation and inactivation hyperpolarized by approximately 2 mV for Na_V1.6, but not Na_V1.2.

While GNE-4076 binds potently to either Na_V1.2 (Figure 1D, F) or Na_V1.7 expressed in HEK cells (Roecker et al., 2017), its affinity was lower for Na_V1.6 expressed in ND7/LoNa_V cells. To test if this was due to reductions in affinity imposed by the ND7/23 cell line, we examined current in the few HEK cells in which Na_V1.6 current could be measured (Figure 1F). While peak currents were small (400 pA vs. 3.2 nA for Na_V1.2 transfected using identical protocols), we found that 30 nM GNE-4076 exhibited strong block of Na_V1.6 (Figure 1F), but due to limitations in expression efficiency in HEK cells, we were unable to collect full dose–response curves to determine IC₅₀.

Results in heterologous expression systems indicate that YW→SR substitution reduces GNE-4076-binding affinity markedly. Given these results, we constructed transgenic mice containing the YW→SR mutation. Two distinct lines, termed Scn8a^SR/SR and Scn2a^SR/SR, were generated by mutating both alleles of each sodium channel gene (Figure 1G). SR/SR mutations were validated by PCR (see Methods), and mice were bred to be homozygous for the mutations. Comparisons were then made between wildtype mice, mice with YW→SR mutations into Na_V1.2 or Na_V1.6 alone (2a^SR/SR or 8a^SR/SR, respectively), or YW→SR mutations in both Na_V1.6 and Na_V1.2 (dual Scn8a/2a^SR/SR). In principle, one could determine a concentration of GNE-4076 in which native channels are blocked potently, sparing SR variant channels (Figure 1H). Thus, these mice should enable selective, acute manipulations of Na_V1.2- and Na_V1.6-dependent aspects of neuronal excitability using a chemical genetics approach.

Given the disparate binding results in heterologous cell lines, we first tested the effects of a dose that should potently block WT channels but not affect SR variant channels to verify that native channels behave similarly to those expressed in HEK cells. Neocortical pyramidal cell somata are thought to express similar levels of Na_V1.2 and Na_V1.6 on their membranes (Spratt et al., 2021; Spratt et al., 2019; Hu et al., 2009). Thus, excised patches from this region can provide insight into the block of both isoforms. Acute coronal slices containing mPFC were prepared from WT or Scn8a/2a^SR/SR mice, and nucleated patches were excised from layer 5 pyramidal cells. After establishing a stable baseline of Na_V-mediated current evoked from a holding voltage of –80 mV, a value comparable to the resting membrane potential studied in later current-clamp experiments, cells were pulsed to –12 mV for 10 s to achieve near-complete block of NaV isoforms, then returned to –80 mV (Figure 1I). In untreated WT conditions, currents recovered to near-baseline levels within 5 s (Figure 1J). A similar quick recovery was observed in cells from Scn8a/2a^SR/SR mice in the presence of 200 nM GNE-4076. By contrast, WT neurons exposed to the same dosing were inhibited markedly, with no appreciable recovery within 25 s (Figure 1I, J). Taken together, these results show that the YW to SR mutation reduces ASC binding and inhibition of Na_V chimeras both in immortalized cell lines and pyramidal neurons.

We also asked whether neuronal AP properties were affected by these mutations in Scn8a/2a^SR/SR mice by assessing both threshold and peak dV/dt in the absence of GNE-4076 (Figure 1—figure supplement 1). Compared to wildtype cells, there were no detectable changes to peak dV/dt in Scn8a/2a^SR/SR neurons (Figure 1—figure supplement 1B). However, the AP threshold was hyperpolarized in Scn8a/2a^SR/SR neurons, likely due to small changes in the voltage dependence of activation observed for heterologously expressed Na_V1.6 (Figure 1—figure supplement 1A, Table 2). Application with GNE-4076 in either wildtype or Scn8a/2a^SR/SR neurons had no further effect on threshold or peak dV/dt compared to controls recording in the absence of GNE-4076, suggesting minimal drug binding occurs without marked depolarization or spiking activity (Figure 1—figure supplement 1, Table 3).

Na_V1.2 and Na_V1.6 are differentially distributed in neuronal arbors and are thought to contribute to different aspects of AP initiation and propagation. In mature neocortical pyramidal cells, Na_V1.6 is enriched in the distal AIS, the site where APs initiate (Spratt et al., 2021; Hu et al., 2009; Katz et al., 2018; Royeck et al., 2008). Following initiation, APs forward propagate along the axon via Na_V1.6-enriched nodes of Ranvier, but also backpropagate into the soma through a region enriched with a mix of Na_V1.2 and Na_V1.6 (Hu et al., 2009). Several studies have sought to identify specific roles for each isoform by conditional deletion of either isoform (Spratt et al., 2021; Katz et al., 2018). Unfortunately, in such conditions, the residual isoform compensates for loss to some degree, making interpretation of individual isoform roles difficult. We therefore leveraged ASC-based blocks to study acute, differential inhibition of Na_V isoforms to better understand the individual roles of Na_V1.2 and Na_V1.6 in AP excitability.

Changes to the AP waveform were visualized with phase plane plots, which plot membrane voltage versus membrane voltage velocity (Figure 2B, Figure 3—figure supplement 1A). Within phase plots, spike threshold appears as a sudden deviation from rest and is defined as the voltage at which voltage velocity (dV/dt) first exceeds 15 V/s (Figure 3—figure supplement 1A, right). Following spike threshold, a neuron’s voltage transits through two components of Na_V recruitment, first in the AIS and then in the soma (Baranauskas et al., 2013; Kole and Stuart, 2008). These result in characteristic ‘humps’ in the depolarizing aspect of the phase plot (Shu et al., 2007; Yu et al., 2008). Thus, these different components of the phase plot can aid one’s understanding of effects on specific components of Na_V-mediated excitability.

To provide a priori predictions of potential ASC-based effects on excitability, we constructed a compartmental model in which each channel’s density could be modulated. Channels were distributed based on predictions from empirical anatomical and physiological studies (Spratt et al., 2021; Nelson et al., 2024; Hu et al., 2009; Katz et al., 2018; Kole et al., 2008). Na_V1.6 was enriched in the distal AIS, Na_V1.2 was enriched in the proximal AIS, both channels were expressed at equal levels in the soma and the first 20 µm of dendrite closest to the soma, and Na_V1.2 was expressed exclusively in all other dendrites (Figure 2A). Within this model, each channel’s density was modulated in 10% increments, from 100 to 0% (Figure 2B).

In WT conditions (100% density of both channels), models generated APs with a threshold and AP kinetics comparable to empirical baseline observations (Figure 2B, C). Within this model, reducing Na_V1.2 or Na_V1.6 density had clear, dissociable effects. Progressive reduction of Na_V1.6 produced a progressive depolarization of AP threshold and a corresponding decrement in dV/dt throughout the entire rising phase of the AP. Na_V1.2 reduction, by contrast, had no effect on AP threshold or the initial velocity of the AP initiated in the distal AIS. Instead, components of the AP related to backpropagation and recruitment of somatic Na_V channels were impaired only, with a decrement in peak dV/dt and peak AP membrane potential. To validate the accuracy of the parameters used in our model, we also performed sensitivity analysis (Figure 2—figure supplement 1). Here, we adjusted the crossover point of Na_V1.2 and Na_V1.6 within the AIS as well as varied the density ratio of both channels (Figure 2—figure supplement 1A, B). Increasing the Na_V1.6 ratio to 100% consistently hyperpolarized AP threshold across all AIS crossover positions (Figure 2—figure supplement 1B, C). However, dV/dt was highly variable and random when either the AIS crossover point is shifted or Na_V ratios are altered.

While these models provide clues to the differential roles of Na_V1.2 and Na_V1.6, we needed to design an experimental strategy that increased the overall degree of Na_V blockade using ASCs in mice where either Na_V1.6 or Na_V1.2 was mutated to be insensitive to 200 nM GNE-4076 binding (Figures 1G and 3A). Since it is nearly impossible to achieve full block with ASCs under physiological firing, we developed a hybrid current- and voltage-clamp experiment to study the effects of more complete isoform blockade (Figure 3B). In this protocol, neurons were held to –80 mV in current clamp with constant bias current (if necessary), and baseline APs were elicited with brief somatic current injection (300 ms, amplitude adjusted to evoke ~4–5 APs). We then promoted Na_V activation and inactivation by voltage-clamping neurons to –12 mV for 30 s (Figure 3A). Following voltage-clamp, neurons were returned to current-clamp with the same bias current as used in baseline conditions. We then evoked APs, first at an interstimulus interval of 2 s, then at successively increasing intervals of 5–60 s, allowing for somewhat consistent sampling on a log-base timescale that aligns well with both channel recovery from inactivation and recovery from GNE-4076 block (Figure 3D, F). For clarity and simplicity, we will describe studies based on which channel is inhibited rather than which channel was rendered insensitive to GNE-4076. For example, a study in the Scn2a^SR/SR/Scn8a^+/+ animal is a case where Na_V1.6 can be blocked.

Empirical data at various time points post –12 mV hold reveals that recovery from ASC-dependent block increases channel availability from approximately 0–10% to about 90–95% of the baseline spike when more time has elapsed (Figures 2C and 3B, C). This recovery in available channels mimics changes observed to phase plots in compartmental models based on overall channel expression (Figure 2B).

We then focused our analysis on the recovery of threshed or peak dV/dt across all genotypes (Figure 3). In untreated WT cells, AP threshold following voltage-clamp recovered to baseline values immediately (Figure 3C, D). By contrast, peak somatic dV/dt recovered more slowly (Figure 3C, F), likely reflecting recovery from slow inactivation of channels in the perisomatic region (Colbert et al., 1997; Park et al., 2013; Toib et al., 1998). Peak dV/dt recovered to within 87.12 ± 0.86% by 12–20 s of voltage-clamp offset (Figure 3F, G). Subsequent recovery of the residual peak dV/dt was slower, taking another 15–30 s to recover the next 5% of peak dV/dt (Figure 3F). Identical results were observed in Scn8a/2a^SR/SR cells treated with 200 nM GNE-4076, suggesting that any effect observed in single knock-in recordings will be due to block of GNE-4076 sensitive channels (Figure 3B–G). We therefore focused on the 12–20 s after voltage-clamp offset for subsequent analysis, as it is a period in which most channel-intrinsic recovery has occurred, but also a period in which we would still expect significant block from GNE-4076.

When individual channel isoforms were blocked more completely with voltage steps to –12 mV, dramatic changes in AP threshold and peak dV/dt were observed. When Na_V1.6 was blocked, threshold depolarized by 5.8 ± 0.7 mV (Figure 3E). When Na_V1.2 was blocked instead, threshold was unaffected (∆ V_m: –0.1 ± 0.2). Thus, AP threshold and AP initiation appear to be initiated in an Na_V1.6-rich region in control conditions; but when Na_V1.6 is inhibited, APs can occur at more depolarized potentials, likely mediated predominately by Na_V1.2.

To examine these relative contributions further, we analyzed the rising phase of APs more closely (Figure 3—figure supplement 1A). As described above, the first component of the rising phase of the AP reflects recruitment of Na_V channels localized to the AIS (Figure 3—figure supplement 1A, middle). Based on changes in voltage acceleration (second derivative) within this period, the AIS component can be further divided into initiation and AIS backpropagation components (Hu et al., 2009; Favero et al., 2018; Figure 3—figure supplement 1A, right). These periods were divided based on a time point during the AIS phase where voltage acceleration first peaked (AIS inflection point) or afterwards decreased, creating a trough in an acceleration versus time graph (AIS max; Figure 3—figure supplement 1B). The dV/dt at the AIS inflection point was affected only by Na_V1.6 block (Figure 3—figure supplement 1C), whereas the dV/dt at the trough was affected by block of either isoform (Figure 3—figure supplement 1D, E). This reinforces the model where APs are initiated via the Na_V1.6-enriched distal AIS, and where the depolarization produced by these distally localized channels recruits Na_V1.2 in the more proximal AIS. Absolute values are also reported for threshold, peak dV/dt, and AIS max for all recorded cells in recovery experiments (Figure 3—figure supplement 2A–F).

Previously, we showed that conditional knockout of Scn2a increased AP output (Spratt et al., 2021). We hypothesized that this was due to the lack of Na_V1.2 in dendrites. When they are absent from this compartment, the dendrite does not depolarize as effectively, leading to a corresponding decrease in the recruitment of dendrite-localized potassium channels (K_V). Consequently, neurons repolarize less between APs, making it easier to evoke the next AP in the Na_V1.6-enriched AIS.

Though this hypothesis was supported by compartmental modeling demonstrating that acute block of Na_V1.2 mirrored empirical observations, we could not eliminate the possibility that some form of cellular compensation occurred in the weeks between conditional knockout induction and acute slice experiments (Miralles and Patel, 2022). Therefore, we leveraged GNE-4076 to test the effects of acute Na_V1.2 block (Figure 4A), eliminating the possibility of genetic or post-translational compensation. We further compared this manipulation to block of Na_V1.6 or both isoforms by examining overall AP output from protocols described in Figure 3A.

Remarkably, acute Na_V1.2 block mirrored conditional knockout and was the only condition in which AP output increased (Figure 4B) and was associated with a depolarization in afterhyperpolarization voltage (Figure 4C). In contrast, Na_V1.6 block decreased AP output, as did block of both channels (Figure 4B). This indicates that blocking Na_V1.2 alone can increase AP output, independent of compensatory changes to other channels that may occur with genetic manipulations.

During physiological activity, non-selective pharmacological inhibition of Na_V channels in neurons greatly reduces cellular excitability and AP firing (Tukker et al., 2023; Thouta et al., 2022). ASCs are unique in that they require prolonged bursts of activity or even neuronal hyperexcitability to stabilize more channels in the inactivated state (Ahuja et al., 2015; Goodchild et al., 2024; Johnson et al., 2024; Johnson et al., 2022). In contrast to the data presented above, where we observe near-complete block immediately following prolonged depolarization (Figure 3—figure supplement 2C, absolute values), neurons experiencing physiological levels of activity likely never reach high percentages of channel blockade. Thus, in normal spiking neurons, ASCs would therefore be predicted to exhibit use dependence, progressively blocking channels in proportion to a neuron’s activity rate.

To test this concept, we generated prolonged periods of activity in current-clamp by injecting 300 pA into the somatic pipette for 10 s (Figure 5A and B). Drug-naive WT neurons responded to somatic current injection with repetitive spiking at a rate of 14.7 ± 0.5 Hz. Neurons fired at a steady state after the first second, with stable AP threshold and instantaneous AP frequency (Figure 5C). Spiking characteristics were identical in Scn8a/2a^SR/SR cells treated with GNE-4076, indicating that, at 200 nM, neurons are insensitive to GNE-4076 when the YW→SR motif substitution is present (Figure 5C, D). However, in WT neurons treated with GNE-4076, threshold continued to depolarize after the first second, with corresponding decrements in instantaneous frequency and peak AP dV/dt throughout the stimulus (Figure 5C, D). This demonstrates that GNE-4076 suppresses firing of neurons with a baseline firing rate of ~15 Hz (Table 4).

We then repeated experiments described above for single mutated channels—where APs were generated with somatic depolarization over 10 s (Figure 6A, B). In cases where Na_V1.6 alone could be inhibited, AP threshold depolarized in response to GNE-4076 as much as in WT cases (Figure 6C, D). Furthermore, AP frequency and peak somatic AP dV/dt were reduced to levels observed in WT cells (Figure 6C, D). In cases where Na_V1.2 alone could be inhibited, changes in AP threshold and instantaneous frequency were no different than Scn8a/2a^SR/SR conditions. Indeed, the only change in AP properties was a decrease in peak AP dV/dt (Figure 6D). This suggests that AP threshold and instantaneous frequency are more sensitive to Na_V1.6 antagonism, whereas peak somatic dV/dt can be affected by antagonism of either isoform. Absolute values are also reported for threshold, peak dV/dt, and instantaneous frequency for all recorded cells in prolonged activity burst experiments (Figure 6—figure supplement 1A).

Epilepsy is often associated with aberrant, excessive excitability in neocortical networks, whether from direct hyperexcitability in pyramidal cells (Lopez-Santiago et al., 2017; Sanders et al., 2018) or disinhibition of pyramidal cells via alterations in inhibitory networks (Favero et al., 2018; Tran et al., 2020). Several use-dependent Na_V antagonists with structures similar to GNE-4076 are being developed, exhibiting differential selectivity for Na_V1.2 and Na_V1.6 (Goodchild et al., 2024). Use dependence may have advantages as anti-epileptics. In theory, they would have minimal effect on neurotypical network activity unless such networks were hyperactive enough to promote drug binding. This may occur preferentially in seizure states. But given the results above demonstrating that block of Na_V1.2 and Na_V1.6 can have different effects on overall AP output, it is critical to determine how block of either channel affects overall activity in seizure-like conditions.

To test this, we mimicked synaptic input by repeatedly injecting a 60-s-long somatic current composed of Poisson-distributed EPSC and IPSC-like waveforms designed to elicit ~10 Hz spiking in baseline conditions (Figure 7A). Following this baseline, the PSC-like train was repeated, imposed upon a 400-pA standing current injection to increase spiking to ~30 Hz (Figure 7C), mimicking spike rates commonly observed during cortical seizure (Neckelmann et al., 1998; Timofeev and Steriade, 2004). After this seizure-like event, cells were returned to baseline voltages, and recovery was assessed with four more repetitions of the 60 s PSC stimulus (Figure 7A–C).

In the presence of 200 nM GNE-4076, baseline firing rates were mostly stable across all conditions (Figure 7—figure supplement 1A). Wildtype, no drug and Scn8a/2a^SR/SR conditions observed no change in firing rate, while dual or selective Na_V1.6 blockade saw a slight decrease by 60 s of postsynaptic activity (Figure 7—figure supplement 1B). Interestingly, we did see a slight increase in baseline firing by 60 s when Na_V1.2 was selectively targeted (Figure 7—figure supplement 1A, B), further highlighting a paradoxical shift to hyperactive neurons when Na_V1.2 availability decreases. By contrast, a slight depolarization in AP threshold was observed at baseline when both channels were sensitive to GNE-4076 (e.g., WT plus drug) and to a lesser extent with selective block of Na_V1.6 (Figure 7—figure supplement 1A, B). Given that this dose can completely block both Na_V1.2 and Na_V1.6 in HEK cells, a small amount of drug block was expected at baseline.

Seizure-like activity induction depolarized AP threshold markedly in all genotypes, and threshold depolarization persisted into the recovery phase for all neurons experiencing Na_V1.6 or dual inhibition (Figure 7C, D). Seizure-like activity increased spike rate across all genotypes at the onset of seizure-like activity induction (Figure 7C, E). All cells exhibited some degree of AP accommodation during this stimulus, including drug-naive controls and cases where both Na_V1.2 and Na_V1.6 were insensitive to GNE-4076 block. WT cells exposed to GNE-4076 accommodated markedly, returning to baseline firing rates at the end of the seizure-like stimulus (Figure 7C, E). A less dramatic effect was noted when Na_V1.6 was blocked alone (Figure 7E). By contrast, cells where Na_V1.2 could be blocked alone were not appreciably different than untreated controls (Figure 7E). Furthermore, some cells that remained hyperactive with selective Na_V1.2 block compared to control cells throughout the recovery phase. Together, these data suggest that use-dependent pharmacology that targets Na_V1.2 and Na_V1.6 may be most beneficial if designed for higher potency at Na_V1.6.

Electrophysiological recordings of AP propagation delays between somatic and axonal recordings demonstrate that APs initiate ~35–50 µm from the soma, in the distal AIS (Shu et al., 2007; Palmer and Stuart, 2006; Baranauskas et al., 2013; Hu et al., 2009). This region is enriched with Na_V1.6 (Hu et al., 2009; Tian et al., 2014; Royeck et al., 2008; Lorincz and Nusser, 2008; Akin et al., 2015). Consistent with this, we find that inhibition of Na_V1.6 alone alters AP initiation, with an increase in AP threshold and a decrease in total AP number from block of this isoform. Following distal AIS AP initiation, APs sequentially propagate across different neuronal compartments to influence local excitability (Shu et al., 2007; Palmer and Stuart, 2006; Baranauskas et al., 2013). Forward propagation was not examined here, but prior reports demonstrate that it is supported almost exclusively by Na_V1.6 in nodes of Ranvier and boutons (Tian et al., 2014; Caldwell et al., 2000; Kaplan et al., 2001). By contrast, backpropagation through the proximal AIS and soma recruits a mix of Na_V1.6 and Na_V1.2.

Prior work has suggested that Na_V1.2 channels localized to the proximal AIS are critical for backpropagation, with modeling suggesting that APs would fail to effectively backpropagate into the soma in the absence of proximal AIS Na_V1.2 channels (Hu et al., 2009). While some efforts have been made to test this empirically with conditional Scn2a knockout, such experiments are imperfect, as Na_V1.6 redistributes in the AIS of cells that lack Na_V1.2 (Spratt et al., 2021). Here, we were able to test the role of Na_V1.2 in backpropagation without compensatory effects, using acute pharmacological inhibition. Backpropagation failures were not observed, consistent with results from conditional Scn2a knockout (Spratt et al., 2021). However, we did observe a reduction in the velocity at which the AIS component of the AP depolarized when Na_V1.2 was blocked selectively (Figure 3—figure supplement 1B, C). This suggests that proximal AIS-localized Na_V1.2 does aid in boosting backpropagating APs as they transit from the distal AIS to the soma. Nevertheless, Na_V1.2 channels are still dispensable for recruitment of somatic Na_V channels, at least in mouse mPFC layer 5b pyramidal cells. Whether similar effects are observed in other cells, including those with larger somata that may be more difficult to depolarize in the absence of Na_V1.2, remains to be tested.

Na_V1.2 expression in pyramidal cell dendrites appears to have two roles in neuronal excitability. Intuitively, these channels provide local inward current that boosts dendritic excitability, leading to a recruitment of voltage-gated calcium channels (Spratt et al., 2019; Nelson et al., 2024). But this depolarization also appears to be critical for the recruitment of dendritically localized voltage-gated potassium channels that contribute to AP repolarization and net membrane potential between spikes (Spratt et al., 2021). Indeed, conditional knockout of Na_V1.2 paradoxically increases AP output, and we suggested previously that this was due in large part to loss of interactions between dendritic Na_V channels and K_V channels. But given observed changes in Na_V1.6 function as well as potential for other compensatory changes in non-Na_V ion channels, it was difficult to ascribe hyperexcitability purely to the loss of Na_V1.2 alone (Miralles and Patel, 2022). Here, we showed that identical increases in excitability—associated with depolarization of membrane potential between APs—could be observed with acute Na_V1.2 block. This indicates that such effects can be due purely to the interplay between dendritic Na_V1.2 and K_V channels, and that modifications to potassium channel distribution or function are not necessary for such effects.

Similar to Na_V1.2 conditional knockout, where Na_V1.6 is upregulated, knockout of Na_V1.6 results in an increase in Na_V1.2 expression, at least in the AIS. In pyramidal cells from mice constitutively lacking Na_V1.6, Na_V1.2 occupies the entirety of the AIS rather than just the region proximal to the soma (Ye et al., 2018; Katz et al., 2018; Royeck et al., 2008). Thus, it has not been possible to evaluate the role of Na_V1.6 in its normal distribution using genetic manipulations. Here, we find that acute Na_V1.6 block reduces AP output with a concomitant increase in AP threshold. This contrasted markedly with block of Na_V1.2, which had no effect on threshold. Of note, this distinction can be leveraged to assess the specificity of other Na_V-targeting pharmacology that has been suggested to have specificity at select isoforms, as any change in threshold indicates that drugs are interacting with Na_V1.6 (Ye et al., 2018; Filipis et al., 2023).

Epilepsy can arise from genetic and non-genetic factors, often with unexplained etiology (Lindy et al., 2018; Deng et al., 2014). Seizure onset is broadly classified as an electrical imbalance of cellular and network activity that favors hyperexcitability (Agbo et al., 2023), whether it be cell intrinsic, synaptic, or due to complex network effects (Greenfield, 2013; Yin et al., 2013). Thus, seizure suppression can be targeted at multiple levels. Nevertheless, proper dosing can be difficult, as one aims to provide drug concentrations that temper excess activity but limit side effects like sedation associated with elevated drug concentrations.

For genetically defined sodium channelopathies, attention has been directed to neuronal cell types that express each isoform (e.g., SCN1A: Na_V1.1, SCN2A: Na_V1.2, and SCN8A: Na_V1.6) (Encinas et al., 2020; Menezes et al., 2020). Seizures resulting from SCN1A loss of function limit inhibitory neuron excitability, thereby disinhibiting excitatory pyramidal cells (Favero et al., 2018; Tran et al., 2020; Strzelczyk and Schubert-Bast, 2022). SCN2A gain-of-function results in hyperexcitability in pyramidal cells, especially in early development when Na_V1.2 channels are the sole isoform expressed in the AIS (Jenkins and Bender, 2025; Gazina et al., 2015). Later in development, SCN8A-encoded Na_V1.6 channels are expressed more ubiquitously in the AIS of most cell classes, supporting AP initiation in both excitatory and inhibitory cells (Favero et al., 2018; Lopez-Santiago et al., 2017; Akin et al., 2015; Boiko et al., 2003).

With these cellular distributions and mechanisms of action, guidelines have emerged for treatment. For SCN1A loss-of-function, sodium channel blocking anti-epileptics are typically contraindicated (Kalume et al., 2013; Oakley et al., 2013). This is because currently prescribed sodium channel blockers are non-selective, with little preference for Na_V1.1, 1.2, or 1.6 (Bai et al., 2022). Thus, further blocking of the remaining Na_V1.1 leads to even more disinhibition of excitatory cells before any beneficial effects of blocking Na_V channels in excitatory cells are realized. Similarly, nonspecific sodium channel blockers are contraindicated for SCN2A loss-of-function seizures, as they tend to increase seizure severity. Instead, non-selective Na_V inhibitors are useful when channel gain-of-function affects excitatory cells, as is the case for both SCN2A and SCN8A gain-of-function cases.

ASCs may be useful within each of these domains, as their chemistry can be adjusted to bias binding to specific isoforms in an activity-dependent manner (Ahuja et al., 2015; Goodchild et al., 2024; Johnson et al., 2024; Johnson et al., 2022). Indeed, compounds similar to those used here that inhibit both Na_V1.2 and Na_V1.6 (but not other Na_V channels) are effective at suppressing chemoconvulsant-induced seizures in ex vivo models (Goodchild et al., 2024). This parallels our observations, where excess activity was dampened most effectively by dual block of Na_V1.2 and Na_V1.6 (Figure 7C). This effect appears due in large part to the block of Na_V1.6, since, ultimately, this is the isoform responsible for AP initiation. Consistent with this, Na_V1.6-preferring ASCs show promise in protecting from chemoconvulsant-induced seizures in vivo (Johnson et al., 2024; Johnson et al., 2022). Similar to our results, Johnson et al. also show that a selective ASC for Na_V1.6 (NBI-921352) preferentially targets cortical pyramidal cells, unlike carbamazepine that non-selectively targets many Na_V isoforms leading to impaired interneuron activity (Johnson et al., 2022). In fact, drug therapies using agents like carbamazepine, lamotrigine, and phenytoin that have little to no selectivity for Na_V isoforms can have adverse effects in certain situations (Agbo et al., 2023). Thus, it may be that preferential block of Na_V1.6 would be beneficial in multiple sodium channelopathy conditions, including those associated with channel loss, since network hyperexcitability is ultimately dictated by Na_V1.6 function in the AIS of excitatory neurons after the first months of life.

Beyond selectivity, there may also be advantages to ASC activity-dependent properties. The ASC-binding pocket is hidden from drug in a channel’s closed state. This feature, combined with on- and off-rate kinetics, results in an accumulation of block primarily for highly active neurons (Figures 3 and 7). Similar to on-demand, closed-loop electrical or optogenetic approaches for seizure intervention (Krook-Magnuson et al., 2013; Nagaraj et al., 2015; Ledri et al., 2023), ASCs are essentially biased to suppress prolonged, high-frequency activity, including the type of activity commonly observed during seizures. In this study, our focus centered mainly on seizure-like activity across individual neurons. Future studies can extend this approach to examining networks ex vivo and systems in vivo.