Disease-causing Slack potassium channel mutations produce opposite effects on excitability of excitatory and inhibitory neurons

Abstract

The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, K_Na1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na⁺-dependent K⁺ (K_Na) and voltage-dependent sodium (Na_V) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of Na_V channel subunits, particularly that of Na_V1.6, is upregulated and that the length of the axon initial segment and of axonal Na_V immunostaining is increased in both neuron types. Our study on the coordinate regulation of K_Na currents and the expression of Na_V channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.

Keywords: ADNFLE; CP: Neuroscience; EIMFS; KCNT1; Na(V)1.6; Slack channel; epilepsy; gain of function; neuronal excitability; voltage-gated sodium (Na(V)) channel.

Figure 1.. Slack channels are highly expressed in cortical glutamatergic and GABAergic neurons

(A) Immunostaining of Slack channels with GluR1 (biomarker for glutamatergic neurons), GAD67 (biomarker for GABAergic neurons), and MAP2 (biomarker for soma and dendrites) in cultured cortical neurons on DIV 14. Scale bar, 50 μm. The rightmost panels show live light microscopy images (400× magnification) of recorded cortical neurons. (B) Immunostaining of Slack channels in cerebral cortex of 2-month-old WT mice. The top center panel shows distinct cortical layers identified based on previous studies.^, Scale bar, 50 μm. (C) Immunostaining of Slack channels with GluR1 and GAD67 in cerebral cortex of 2-month-old WT mice. Scale bar, 50 μm. (D) Representative western blotting and quantitative analysis of protein levels of Slack, GluR1, and GAD67 in cultured cortical neurons on DIV 14. Neuronal cultures were obtained from WT, heterozygous (WT/R455H, Slack^+/R455H), and homozygous (R455H/R455H, Slack^R455H/R455H) littermates. Data are shown as mean ± SEM (n = 4 cultures; data are not significant by 1-way ANOVA). (E) Representative western blotting and quantitative analysis of protein levels of Slack, GluR1, and GAD67 in cerebral cortex of 2-month-old WT and Slack^+/R455H mice. Data are shown as mean ± SEM (n = 4 mice; not significant by Student’s t test). See also Figure S1.

Figure 2.. K_Na currents, I_Na and I_NaP are increased in both glutamatergic and GABAergic neurons expressing the Slack-R455H mutation

(A–C) Representative current traces from whole-cell voltage-clamp recordings in physiological extracellular medium with 140 mM Na⁺ ions (left) and without Na⁺ ions (right) in response to voltage steps (−90 to +50 mV) in WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) neurons. Summary data (bottom) show the K_Na current, which was calculated by subtracting the trace obtained without Na⁺ from the trace with Na⁺, for each voltage step in glutamatergic neurons (A), FS-GABAergic neurons (B), or NFS-GABAergic neurons (C). Data are shown as mean ± SEM (n = 8–12 neurons, 2-way ANOVA). (D) Maximal K_Na current density at +50 mV for each genotype and neuron type. Data are shown as mean ± SEM (n = 8–12 neurons, 1-way ANOVA). (E) Maximal I_Na current density for each genotype and neuron type. Data are shown as mean ± SEM (n = 8–12 neurons, 1-way ANOVA). (F and I) I_NaP traces evoked by a slow voltage ramp protocol (30 mV/s from −80 to +10 mV) in glutamatergic neurons (F) and GABAergic neurons (I) obtained from WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) embryos under control conditions and in the presence of 0.5 μM TTX (gray trace). (G and J) Summary data show the I_NaP current, which was calculated by subtracting the trace obtained without TTX from the trace with TTX, at 5-mV intervals for each genotype in glutamatergic neurons (G) and GABAergic neurons (J). Data are shown as mean ± SEM (n = 8–14 neurons, 2-way ANOVA). (H and K) Maximal I_NaP density for each neuron type and genotype. Data are shown as mean ± SEM (n = 8–14 neurons, 1-way ANOVA). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S1.

Figure 3.. The Slack-R455H mutation enhances the excitability of glutamatergic neurons

(A) Representative traces from whole-cell current-clamp recordings from glutamatergic neurons in response to current step injections from WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) littermates. To compare the electrophysiological properties, neurons were injected with 200-ms square current pulses in 20-pA step increments, starting at −20 pA. To compare the maximum number of APs, 1.5-s square current pulses in 20-pA steps were injected until the number of APs per stimulus reached a plateau phase. (B–F) AP amplitude (B), AP half-width (C), AHP (D), rheobase (E), and input resistance (F) of recorded neurons from each genotype. Data are shown as mean ± SEM (n = 15–20 neurons, 1-way ANOVA). (G and H) Example traces and summary data showing the frequency of APs per current injection step in WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) neurons. Data are shown as mean ± SEM (n = 15–20 neurons, 2-way ANOVA). (I) Maximal firing rate for each genotype. Data are shown as mean ± SEM (n = 15–20 neurons, Kruskal-Wallis test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S2.

Figure 4.. The Slack-R455H mutation suppresses the excitability of FS-GABAergic and NFS-GABAergic neurons

(A) Representative traces from whole-cell current-clamp recordings from FS-GABAergic neurons in response to current step injections from WT (black), Slack^+/R455H (blue), and Slack^R455H//R455H (red) littermates. (B–F) AP amplitude (B), AP half-width (C), AHP (D), rheobase (E), and input resistance (F) of recorded neurons from each genotype. Data are shown as mean ± SEM (n = 12–18 neurons, 1-way ANOVA). (G and H) Example traces and summary data showing the frequency of APs per current injection step in WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) neurons. Data are shown as mean ± SEM (n = 12–18 neurons, 2-way ANOVA). (I) Maximal firing rate for each genotype. Data are shown as mean ± SEM (n = 12–18 neurons, Kruskal-Wallis test). (J) Representative traces from whole-cell current-clamp recordings from NFS-GABAergic neurons in response to current step injections from WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) littermates. (K–O) AP amplitude (K), AP half-width (L), AHP (M), rheobase (N), and input resistance (O) of recorded neurons from each genotype. Data are shown as mean ± SEM (n = 13–17 neurons, 1-way ANOVA). (P and Q) Example traces and summary data showing the frequency of APs per current injection step in WT (black), Slack^+/R455H (blue), and Slack^R455H/R455H (red) neurons. Data are shown as mean ± SEM (n = 13–17 neurons, 2-way ANOVA). (R) Maximal firing rate for each genotype. Data are shown as mean ± SEM (n = 13–17 neurons, Kruskal-Wallis test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S2.

Figure 5.. The Slack-R455H mutation upregulates Na_V channel subunit expression

(A) Representative western blotting and quantitative analysis of protein levels of Na_V1.1, Na_V1.2, and Na_V1.6 in cultured cortical neurons on DIV 14 obtained from WT, Slack^+/R455H, and Slack^R455H/R455H littermates. Data are shown as mean ± SEM (n = 4 cultures, 1-way ANOVA). (B) Representative western blotting and quantitative analysis of protein levels of Na_V1.1, Na_V1.2, and Na_V1.6 in cerebral cortex of 2-month-old WT and Slack^+/R455H mice. Data are shown as mean ± SEM (n = 4 mice, Student’s t test). *p < 0.05.

Figure 6.. Subcellular localizations of Slack and Na_V1.6 channels

(A) Immunostaining of Slack and Na_V1.6 with the dendrite (MAP2) and AIS (AnkG) markers in cultured cortical neurons on DIV 14. Glutamatergic and GABAergic neurons were differentiated according to their morphological features. Scale bar, 50 μm. (B) Immunostaining of Slack and Na_V1.6 with MAP2 and AnkG in cerebral cortex of 2-month-old mice (top). Glutamatergic (center) and GABAergic (bottom) neurons were differentiated by coimmunostaining with VGlut1 and GAD67. Scale bar, 25 μm. See also Figures S3 and S4.

Figure 7.. The Slack-R455H mutation alters the AIS length of both glutamatergic and GABAergic neurons

(A) Representative high-magnification images of a single neuron for quantification of the AIS length, AIS diameter, and gap length. Dashed line: AIS length; solid line: gap length. Scale bar, 10 μm. (B) Representative images of single neurons and their AISs (green, AnkG) for cortical glutamatergic neurons (top) and GABAergic neurons (bottom) on DIV 14. Scale bar, 50 μm. (C) Representative images of neurons and their AISs (green, Na_V1.6 staining) for pyramidal cells (red, VGlut1) and interneurons (red, GAD67) from layer II/III of the frontal cortex of 2-month-old mice. Scale bar, 25 μm. (D–F) Quantification of overall AIS length (D), AIS diameter (E), and gap length (F) in excitatory glutamatergic neurons (yellow) and inhibitory GABAergic neurons (green). Data are shown as mean ± SEM (n = 27 AISs, 1-way ANOVA for different genotypes within each neuron type and Student’s t test for different neuron types within each genotype). Each symbol represents an individual AIS. (G–I) Quantification of overall AIS length (G), AIS diameter (H), and gap length (I) in excitatory pyramidal (yellow) and interneuron (green) populations. Data are shown as mean ± SEM (n = 27 AISs, Student’s t test). Each symbol represents an individual AIS. (J and K) Representative western blotting and quantitative analysis of protein levels of AnkG and MAP2 in cultured cortical neurons on DIV 14 (J, n = 4 cultures, 1-way ANOVA) and in cerebral cortex of 2-month-old mice (K, n = 4 mice, Student’s t test). (L) Coimmunoprecipitation of Slack and Na_V channels from mouse cerebral cortex. Brain lysates were subjected to immunoprecipitation using anti-Slack antibody or chicken immunoglobulin Y, followed by western blotting with Slack, FMRP, PP1, Na_V1.1, Na_V1.2, and Na_V1.6 antibodies. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.