Neuroscience

Differential roles of Na_V1.2 and Na_V1.6 in neocortical pyramidal cell excitability

Joshua D Garcia
Chenyu Wang
Ryan PD Alexander
Emmie Banks
Timothy Fenton
Jean-Marc DeKeyser
Tatiana V Abramova
Alfred L George Jr.
Roy Ben-Shalom
David H Hackos
Kevin J Bender author has email address

Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, United States
Department of Neurology, University of California, San Francisco, San Francisco, United States
Department of Neuroscience, Genentech, Inc, South San Francisco, United States
Department of Neurology, MIND Institute, University of California Davis School of Medicine, Sacramento, United States
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, United States

https://doi.org/10.7554/eLife.105696.2

Open access
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Figures and data

The YW motif on Na_V1.2 and Na_V1.6 increases activity-dependent GNE-4076 potency and subsequent channel inhibition
(A) Schematic depicting the fourth voltage sensing domain (VSD-IV) of Na_V isoforms. The six transmembrane spanning regions have high sequence homology amongst different Na_V 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 Na_V isoforms. Scn2a (Na_V1.2) and Scn8a (Na_V1.6) 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 Na_V current traces (pA) of cells expressing either YW wildtype channels or SR knock-in mutant chimeras in the presence of 1 μM GNE-4076. To activate exogenously expressed Na_V channels, cells were held at −80 mV and stepped to 0 mV for 20 ms. GNE-4076 onboarding was performed by holding cells at 0 mV for 10 sec.
(D) Dose response curves for exogenously expressed Scn2a (HEK cells) or Scn8a (ND7/LoNa_V) 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 Na_V current amplitude ± SEM.
(E) Activation and steady-state inactivation curves for both YW wildtype channels and SR knock-in mutant chimeras (Scn2a^YW/YW, n=6; Scn2a^SR/SR, n=7; Scn8a^YW/YW, n=8; Scn8a^SR/SR, n=6). Scn2a or Scn8a YW->SR mutations alter efficacy of GNE-4076 while having minor effects on biophysical properties of either isoform. Circles represent mean normalized Na_V current amplitude ± SEM. Unpaired t-test with Welch’s correction. No significance detected between wildtype and mutant channels for both V1/2 of activation or inactivation.
(F) Example current amplitude response graphs for Na_V1.2 (red) and Na_V1.6 (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. Scn2a^SR/SR mice were crossed with Scn8a^SR/SR mice to generate a dual 8a/2a^SR/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 Na_V isoforms only containing the YW motif.
(I) Nucleated patch experiments from prefrontal pyramidal cells performed in wildtype or dual 8a/2a^SR/SR knock-in cells in the presence of 200 nM GNE-4076. Baseline conductance was measured by depolarizing cells from −80 mV to −12 mV every 2 sec for 10 pulses. Na_V channels were inactivated by holding nucleated patch at −12 mV for 10 sec. Test pulses were again acquired during recovery similar to baseline pulses.
(J) Summary graph of normalized current amplitude from nucleated patch experiments in (I). Baseline and recovery test pulses were acquired for at least 20 sec before and 25 sec after channel inactivation step. Solid line represents normalized mean Na_V current amplitude ± SEM. Graph also include wildtype, no drug control nucleated patch experiments (wildtype no drug, n=4; wildtype + GNE-4076, n=4; 8a/2a^SR/SR + GNE-4076, n=4).

Global Scn2a, Scn8a or dual loss in compartmental models distinctly impacts key AP properties
(A) Na_V1.6 and Na_V1.2 are equally expressed in soma and proximal dendrites. Expression pattern is more distinct in other regions with Na_V1.6 enriched in the distal AIS, axon and nodes of Ranvier, whereas Na_V1.2 is found exclusively in the proximal AIS and distal dendrites.
(B) Compartmental model representing changes to phase plots when Na_V isoform expression is reduced from 100% (warmer colors) to 0% (in 10% increments) based on known localization across distinct neuronal localities. Lower Na_V1.6 expression depolarized spike threshold and decreased both AIS and somatic AP velocity (dV/dt). Reduced Na_V1.2 expression largely impacts backpropagation and somatic AP velocity.
(C) Empirical observations of phase plot following near complete channel block with ASCs. Darker trace represents phase plot taken at baseline prior to −12 mV hold for 30 sec (see Fig. 3A). Colored traces represent recovery phase plots at different times post −12 mV hold for 30 sec with warmer colors depicting more time lapsed and increased channel recovery.

Recovery of AP firing properties is greatly diminished following dual Na_V1.6 and Na_V1.2 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 Na_V 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/2a^SR/SR + GNE-4076, n=12; Scn2a^SR/SR + GNE-4076, n=12; Scn8a^SR/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 (V_m) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For Δ V_m, baseline value is subtracted from individual timepoints throughout the recovery phase (Δ mV = recovery timepoint – baseline). Colors are matched to conditions represented in (B). Gray shaded bar represents recovery between 12-20 sec.
(E)Summary data for Δ V_m at 12-20 sec post GNE-4076 onboarding (time period represented as gray bar in (D)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm-Šídák multiple comparisons test. ****p<0.0001.

Acute inhibition of Na_V1.2 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/2a^SR/SR + GNE-4076, n=12; Scn2a^{SR/ SR} + GNE-4076, n=12; Scn8a^SR/SR. + GNE-4076, n=12). Dashed line represents V_m 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 velocity (dV/dt) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For Δ dV/dt, baseline value is subtracted from individual timepoints throughout the recovery phase (Δ V/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 Δ dV/dt at 12-20 sec post GNE-4076 onboarding (time period represented as gray bar in (F)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm-Šídák multiple comparisons test. ****p<0.0001.

Activity-dependent onboarding of GNE-4076 to Na_V 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/2a^SR/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 (Δ mV), delta peak dV/dt (Δ V/s) and delta instantaneous firing frequency (Δ 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). Box plots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=12; wildtype + GNE-4076, n=12; 8a/2a^SR/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 Na_V1.6 depolarizes AP threshold markedly while blocking both Na_V1.6 and Na_V1.2 reduces AP velocity
(A) Representative AP firing response to 300 pA current injection for 10 sec in Scn2a^SR/SR or Scn8a^SR/SR cells with 200 nM GNE-4076 to selectively inhibit Na_V1.6 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 (Δ mV), delta peak dV/dt (Δ V/s) and delta instantaneous firing frequency (Δ 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/2a^{SR/ 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). Box plots show median and 90% tails. Circles represent individual cells (8a/2a^SR/SR + GNE-4076, n=12; wildtype + GNE-4076, n=12; Scn2a^SR/SR + GNE-4076, n=10; Scn8a^SR/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.

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 (V_m) of 0 mV. Tick marks above traces represent detected spike defined as a change in V_m 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). Box plots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=7; wildtype + GNE-4076, n=7-8; Scn2a^SR/SR + GNE-4076, n=12; Scn8a^SR/SR + GNE-4076, n=12; 8a/2a^SR/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). Box plots show median and 90% tails. Circles represent individual cells (wildtype no drug, n=7; wildtype + GNE-4076, n=7-8; Scn2a^SR/SR + GNE-4076, n=12; Scn8a^SR/SR + GNE-4076, n=12; 8a/2a^SR/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.

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