Figures and data
The YW motif on NaV1.2 and NaV1.6 increases activity-dependent GNE-4076 potency and subsequent channel inhibition
(A) Schematic depicting the fourth voltage sensing domain (VSD-IV) of NaV isoforms. The six transmembrane spanning regions have high sequence homology amongst different NaV isoforms, while linker regions display more sequence divergence. Orange box highlights extracellular S1-S2 loop where ASCs are stabilized by a tyrosine-tryptophan (YW) motif.
(B) Amino acid sequence within the S1-S2 loop of various NaV isoforms. Scn2a (NaV1.2) and Scn8a (NaV1.6) are the predominant channels expressed in mature, prefrontal pyramidal cells. Both isoforms share a conserved YW sequence that increases ASC potency. Knock-in mutations of Scn2a and Scn8a were generated by substituting the YW motif with a serine-arginine (SR) sequence present in Scn1a and Scn3a.
(C) Example NaV current traces (pA) of cells expressing either YW wildtype channels or SR knock-in mutant chimeras in the presence of 1μM GNE-4076. To activate exogenously expressed NaV channels, cells were held at -80 mV and stepped to 0 mV for 20 ms.
(D) Dose response curves for exogenously expressed Scn2a (HEK cells) or Scn8a (ND7/LoNaV) in immortalized cell lines. IC50 was measured for both YW wildtype channels and SR knock-in mutant chimeras. YW®SR knock-in mutations reduced GNE-4076 potency by about 400-to 500-fold relative to wildtype channels. Circles represent normalized mean NaV current amplitude ± SEM.
(E) Activation and steady-state inactivation curves for both YW wildtype channels and SR knock-in mutant chimeras. Scn2a or Scn8a YW®SR mutations alter efficacy of GNE-4076 without affecting biophysical properties of either isoform. Circles represent mean normalized NaV current amplitude ± SEM.
(F) Example current amplitude response graphs for NaV1.2 (red) and NaV1.6 (blue) expressed in HEK cells. Cells were perfused with increasing concentrations of GNE-4076 throughout the recording. Individual current response recordings from HEK cells expressing Scn2a were robust (3.2 nA), and recordings were reproducible for both YW wildtype channels (red) and SR knock-in mutant chimeras (transparent red). Current responses from cells expressing Scn8a were variable with only a few cells exhibiting channel conductance (400 pA). In select cells expressing Scn8a, current amplitude (blue) also decreases substantially with 30 nM GNE-4076 and completely with 1 μM.
(G) Transgenic mouse lines generated with the YW−SR knock-in mutation present on both ScnXa alleles. Scn2aSR/SR mice were crossed with Scn8aSR/SR mice to generate a dual 8a/2aSR/SR knock-in mouse.
(H) Overview of the various transgenic (or wildtype) mouse lines used throughout this study. Application of 200 nM GNE-4076 selectively inhibits NaV isoforms only containing the YW motif.
Activity-dependent onboarding of GNE-4076 to NaV channels alters action potential firing properties in layer 5b, thick-tufted excitatory neurons
(A) Representative AP firing response to 300 pA current injection for 10 sec in wildtype or 8a/2aSR/SR cells with or without 200 nM GNE-4076.
(B) Phase plane of AP traces shown in (A). Plots represent AP velocity by taking the first derivative (dV/dt, y-axis) versus the membrane potential (mV, x-axis). To represent changes with phase plane relative to time, a rainbow color spectrum is used with warmer colors representing more time lapsed.
(C) Delta threshold (mV), delta peak dV/dt (V/s) and delta instantaneous firing frequency (Hz) binned in 1 sec increments normalized to the initial 500 ms of current injection (binned time – initial 500 ms). Circles represent mean Δ value ± SEM. Two-way ANOVA, Holm-Šídák multiple comparisons test.
(D) Summary data for the final sec in (C). Delta values are normalized to the initial 500 ms of the stimulus (binned time – initial 500 ms). Boxplots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=12; wildtype + GNE-4076, n=12; 8a/2aSR/SR + GNE-4076, n=12). One-way ANOVA, Holm-Šídák multiple comparisons test. **p<0.01, ***p<0.001, ****p<0.0001.
Selective inhibition of NaV1.6 depolarizes AP threshold markedly while blocking both NaV1.6 and NaV1.2 reduces AP speed
(A) Representative AP firing response to 300 pA current injection for 10 sec in Scn2aSR/SR or Scn8aSR/SR cells with 200 nM GNE-4076 to selectively inhibit NaV1.6 or NaV1.2, respectively.
(B) Phase plane of AP traces shown in (A). Plots represent AP velocity by taking the first derivative (dV/dt, y-axis) versus the membrane potential (mV, x-axis). To represent changes with phase plane relative to time, a rainbow color spectrum is used with warmer colors representing more time lapsed.
(C) Delta threshold (mV), delta peak dV/dt (V/s) and delta instantaneous firing frequency (Hz) binned in 1 sec increments normalized to the initial 500 ms of current injection (binned time – initial 500 ms). Circles represent mean Δ value ± SEM. Average Δ value ± SEM for 8a/2aSR/ SR + GNE-4076 and wildtype + GNE-4076 from Fig. 2D are represented.
(D)Summary data for the final sec in (C). Delta values are normalized to the initial 500 ms of the stimulus (binned time – initial 500 ms). Boxplots show median and 90% tails. Circles represent individual cells (8a/2aSR/SR + GNE-4076, n=12; wildtype + GNE-4076, n=12; Scn2aSR/SR + GNE-4076, n=10; Scn8aSR/SR + GNE-4076, n=12). One-way ANOVA, Holm-Šídák multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Recovery of AP firing properties is greatly diminished following dual NaV1.6 and NaV1.2 inhibition compared to selective block of individual channels
(A) Protocol used to characterize recovery of AP firing properties. Baseline spiking is determined by injecting current for 300 ms to elicit 5-6 APs. To promote NaV inactivation and maximal GNE-4076 onboarding, neurons are held at -12 mV in voltage-clamp for 30 sec. Recovery of AP firing is evaluated by injecting same current stimulus defined during baseline spiking with an inter-stimulus interval starting at 2 sec, followed by 5,15, 30 and 60 sec.
(B) Overlaid waveform of 1st AP at baseline or 18 sec post GNE-4076 onboarding for all conditions (wildtype no drug, n=12; wildtype + GNE-4076, n=11; 8a/2aSR/SR + GNE-4076, n=12; Scn2aSR/SR + GNE-4076, n=12; Scn8aSR/SR. + GNE-4076, n=12).
(C) Overlaid phase plane of AP traces at baseline (100% transparency) or 18 sec post GNE-4076 onboarding (20% transparency) for each condition in (B). Plots represent the AP velocity by taking the first derivative (dV/dt, y-axis) versus the membrane potential (mV, x-axis). Colors are matched to conditions represented in (B).
(D) Recovery of AP threshold (Vm) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For ΔVm, baseline value is subtracted from individual timepoints throughout the recovery phase (ΔmV = recovery timepoint – baseline). Colors are matched to conditions represented in (B). Gray shaded bar represents recovery between 12-20 sec.
(E) Summary data for ΔVm at 12-20 sec post GNE-4076 onboarding (time period represented as gray bar in (D)). One-way ANOVA, Holm-Šídák multiple comparisons test. ****p<0.0001.
Acute inhibition of NaV1.2 increases AP excitability
(A) AP train over 300 ms at baseline (black) or 18 sec post GNE-4076 onboarding (color) for all conditions (wildtype + GNE-4076, n=10; 8a/2aSR/SR + GNE-4076, n=12; Scn2aSR/ SR + GNE-4076, n=12; Scn8aSR/SR. + GNE-4076, n=12). Dashed line represents Vm of last after-hyperpolarization (AHP).
(B) Summary data for Δ spike number at 12-20 sec post GNE-4076 onboarding. One-way ANOVA, Holm-Šídák multiple comparisons test. *p<0.05, **p<0.01, ****p<0.0001.
(C) Summary data for Δ last AHP at 12-20 sec post GNE-4076 onboarding. One-way ANOVA, Holm-Šídák multiple comparisons test. *p<0.05, **p<0.01.
(F) Recovery of AP peak speed (dV/dt) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For dV/dt, baseline value is subtracted from individual timepoints throughout the recovery phase (ΔV/s= recovery timepoint – baseline). Colors are matched to conditions represented in (B). Gray shaded bar represents recovery between 12-20 sec.
(G) Summary data for ΔdV/dt at 12-20 sec post GNE-4076 onboarding (time period represented as gray bar in (F)). One-way ANOVA, Holm-Šídák multiple comparisons test. ****p<0.0001.
GNE-4076 onboarding following seizure-like activity continually impacts neuronal firing into recovery
(A) Stimulation protocol and example firing trace of cell injected with fluctuating post-synaptic potentials (PSPs) randomly generated using a Poisson probability distribution function for 60 sec. PSPs were continuously applied to acquire baseline activity, seizure-like activity and recovery activity. During seizure-like activity, a 400 pA step was applied in addition to the PSP. Recovery was continuously recorded for up to 4 mins post seizure-like activity.
(B) Zoomed-in example traces for all genotypes at the Baseline ® Seizure transition, Seizure ® Recovery transition and start of 3-4 min recovery period (highlighted in (A)). Solid horizontal black bar represents membrane potential (Vm) of 0 mV. Tick marks above traces represent detected spike defined as a change in Vm of 15 V/s or greater.
(C) Threshold (mV) or instantaneous firing frequency (Hz) binned in 5 sec increments for all genotypes in (B). Solid lines represent mean value ± SEM. Timescale on x-axis mirrors activity presented in (A).
(D) Summary of threshold data for the final 5 sec of seizure-like activity or entire 3-4 min recovery time-point in (C). Delta values are normalized to baseline activity (either at final 5 sec or entire period). Boxplots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=7; wildtype + GNE-4076, n=7-8; Scn2aSR/SR + GNE-4076, n=12; Scn8aSR/SR + GNE-4076, n=12; 8a/2aSR/SR + GNE-4076, n=6). One-way ANOVA, Holm-Šídák multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
(E) Summary of instantaneous frequency data for the final 5 sec of seizure-like activity or entire 3-4 min recovery time-point in (C). Delta values are normalized to baseline activity (either at final 5 sec or entire period). Boxplots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=7; wildtype + GNE-4076, n=7-8; Scn2aSR/SR + GNE-4076, n=12; Scn8aSR/SR + GNE-4076, n=12; 8a/2aSR/SR + GNE-4076, n=6). One-way ANOVA, Holm-Šídák multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
