Heterozygous expression of a Kcnt1 gain-of-function variant has differential effects on SST- and PV-expressing cortical GABAergic neurons

Abstract

More than twenty recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (K_Na) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Y777H) on K_Na currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1-Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold K_Na currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.

Keywords: ADNFLE; DEE; GABAergic; GOF variant; KCNT1 channel; KNa current; PV; SST; Slack; VIP; electrophysiology; epilepsy; glutamatergic; potassium channel.

Figure 1.. Heterozygous Kcnt1-Y777H expression alters AP shape and generation in NFS GABAergic neurons.

(A₁-A₃) On the left, representative responses to step currents are shown for glutamatergic, and FS and NFS GABAergic, WT (black) and YH-HET (colors) neurons (top to bottom), illustrating the input resistance (in response to a depolarizing step) and the rheobase (the first trace with an AP in response to a hyperpolarizing step) for each neuron type. On the right, bar graphs show quantification and mean ± SEM of the membrane properties and AP parameters for each neuron type for WT (grey) and YH-HET (colors) groups, with individual neuron measurements overlaid in scatter plots. The p-values are shown on each graph where p < 0.05. (B₁-B₃) Representative traces are shown at low, medium, and high current steps for glutamatergic, and FS and NFS GABAergic, WT (black) and YH-HET (colors) neurons (left to right). The line graphs below show the number of APs (mean ± SEM) per current injection step in WT (black) and YH-HET (colors) neurons. Statistical significance was tested using Generalized Linear Mixed Models, and p-values are shown on each graph where p < 0.05.

Figure 2.. Heterozygous Kcnt1-Y777H expression increases subthreshold K_Na currents in NFS GABAergic neurons.

(A₁-A₃) Representative traces in control (top), 0.5 μM TTX (middle), and the difference current (bottom) calculated by subtracting the membrane current response to voltage steps (−80 to +50 mV) from a holding potential of −70 mV in TTX from the response in control external solution in glutamatergic, and FS and NFS GABAergic, WT (black) and YH-HET (colors) neurons. To include all of the representative traces in the figures, and prevent overlap of the traces, the large inward sodium currents were removed from each set of traces using the masking tool in Adobe Illustrator. (B₁-B₃) Summary data shows the K_Na current (mean ± SEM) for each voltage step in glutamatergic, and FS and NFS GABAergic, WT (black and gray) and YH-HET (colors) neurons. The p-values are shown on each graph where p < 0.05, and the n values are the number of neurons recorded for each group. (C₁-C₃) Plots of the K_Na current (mean ± SEM) for each voltage step from -80 to 0 mV in WT (black and gray) and YH-HET (colors) neurons to illustrate the values that are too small to be seen on the graphs in B₁-B₃. The shaded red area in C₃ indicates the subthreshold voltage range with significantly higher K_Na currents (red voltages along x-axis indicate steps where p < 0.05) in YH-HET relative to WT neurons. The p-values are shown on each graph where p < 0.05, and the n values are the number of neurons recorded for each group. Statistical significance for I-V plots was tested using Generalized Linear Mixed Models with genotype and voltage step as fixed effects followed by pairwise comparisons at each level.

Figure 3.. Heterozygous Kcnt1-Y777H expression does not alter synaptic connectivity or the excitation-inhibition balance.

(A₁-A₄) Evoked postsynaptic currents (PSCs) were recorded from neuron pairs [glutamatergic (excitatory, E) and GABAergic (inhibitory, I)] by stimulating the neuron type indicated on the left and recording the response in the neuron type indicated on the right (WT, gray; YH-HET E, green; YH-HET I, red). Bar graphs below each recorded neuron pair schematic show summary data (mean ± SEM) of the connection probability (left graph; numbers on bars represent connected pair number/recorded pair number) and peak evoked PSC amplitude (right graph; dots represent individual evoked responses) between each motif. (B₁) Bar graphs with overlaid individual neuron measurements and mean ± SEM show the spontaneous EPSC (sEPSC) or IPSC (sIPSC) frequency onto E neurons (WT, gray; YH-HET, green). (B₂) Bar graphs with overlaid individual neuron measurements and mean ± SEM show the sEPSC or sIPSC frequency onto I neurons (WT, gray; YH-HET, red). (B₃) Scatter plots show individual E/I ratio measurements onto E neurons (WT, gray; YH-HET, green) and I neurons (WT, gray; YH-HET, red). The p-values are shown on each graph where p < 0.05.

Figure 4.. Heterozygous Kcnt1-Y777H expression differentially affects the intrinsic excitability of SST- and PV-expressing GABAergic neurons.

(A) A schematic diagram illustrates the strategy for generating fluorescently labeled GABAergic subtype-specific neurons. YH-HET mice were crossed to VIP-, SST-, or PV-Cre mice, and the resulting P0 WT and YH-HET littermate pups were used to isolate and culture cortical neurons. At DIV 1, neurons were infected with AAV-CamKII-GFP to label glutamatergic neurons, and AAV-hSyn-DIO-mCherry to label Cre-expressing neurons. At DIV 13–17, whole-cell, patch-clamp electrophysiology was performed on mCherry⁺/GFP⁻ neurons. (B₁-B₃) On the left, representative responses to step currents are shown for VIP-, SST-, and PV-expressing WT (black) and YH-HET (colors) neurons (top to bottom). For each neuron type, the superimposed dark traces illustrate the input resistance (in response to a depolarizing step) and the rheobase (the first trace with an AP in response to a hyperpolarizing step), and the light trace shows the first step current response to induce repetitive AP firing across the step. Above the superimposed traces, the first AP of each rheobase trace is shown (same vertical scale, increased horizontal scale). On the right, bar graphs show quantification of the membrane properties and AP parameters for each neuron type (VIP, SST, and PV, top to bottom) for WT (grey) and YH-HET (colors) groups, with individual neuron measurements overlaid in scatter plots. The p-values are shown above each graph where p < 0.05. (C₁-C₃) For VIP-, SST-, and PV-expressing neurons (left to right), representative traces are shown at low, medium, and high current steps, and the line graphs below show the number of APs (mean ± SEM) per current injection step in WT (black) and YH-HET (colors) neurons. Statistical significance was tested using Generalized Linear Mixed Models, and p-values are shown above each graph where p < 0.05.

Figure 5.. The Kcnt1-Y777H variant increases Kcnt1-mediated currents across subthreshold voltages in SST- and PV-expressing GABAergic neurons.

(A₁-A₃) Representative traces in control (top), 10 μM VU170 (middle), and the difference current (bottom) calculated by subtracting the membrane current response to voltage steps (−80 to +50 mV) from a holding potential of −70 mV in VU170 from the response in control external solution in VIP-, SST-, and PV-expressing, WT (black) and YH-HET (colors) neurons. To include all of the representative traces in the figures, and prevent overlap of the traces, the large inward sodium currents were removed from each set of traces using the masking tool in Adobe Illustrator. (B₁-B₃) Summary data shows the KCNT1 current (mean ± SEM) for each voltage step in VIP-, SST-, and PV-expressing, WT (black and gray) and YH-HET (colors) neurons. The p-values are shown on each graph where p < 0.05, and the n values are the number of neurons recorded for each group. (C₁-C₃) Plots of the K_Na current (mean ± SEM) for each voltage step from -80 to 0 mV in WT (black and gray) and YH-HET (colors) neurons to illustrate the values that are too small to be seen on the graphs in B₁-B₃. The shaded red areas in C₂ and C₃ indicate the subthreshold voltage range with significantly higher K_Na currents (p < 0.05) in YH-HET relative to WT neurons. Statistical significance for I-V plots was tested using Generalized Linear Mixed Models with genotype and voltage step as fixed effects followed by pairwise comparisons at each level.

Figure 6.. Compartmental models of KCNT1 GOF in SST, but not PV, neurons are consistent with experimental data.

(A) Activation curves for VIP, SST, and PV neurons used to model the KCNT1 channel in each neuron type. (B) Representative traces of the response of an outside-out membrane patch pulled from an SST neuron in control external solution (top) and after application of 10 μm VU170 (bottom). (C) Representative trace of the VU170-sensitive current in an outside-out patch pulled from an SST neuron with a single exponential fit (red curve) overlaid and summary data showing the time constant (mean ± SEM) obtained from these fits. (D) Simulated traces showing the Kcnt1-mediated current in model neurons at three levels of Na⁺-sensitivity (Con-40 mM, GOF-35 mM, and GOF-30 mM). (E₁, F₁, and G₁) Simulated traces from model VIP (E₁; purple), SST (F₁; blue), and PV (G₁; yellow) neurons in response to 500-ms current injections at three levels of Na⁺-sensitivity [control (Con), and two levels of KCNT1 GOF]. Representative traces are shown at increasing current steps from bottom to top for each level. (E₂, F₂, and G₂) Summary data from 10 model VIP (E₂; purple), SST (F₂; blue), and PV (G₂; yellow) neurons showing (from left to right) the number of APs at different current steps (F-I plot), the input resistance, and rheobase. The bar graphs (mean ± SEM) are overlaid with scatter plots of individual neuron measurements. Statistical significance was tested using a Repeated Measures ANOVA. Asterisks indicate where p < 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Figure 7.. The persistent Na⁺ current is increased by the Kcnt1-Y777H variant in PV, but not SST, neurons.

(A) Representative traces of WT (left) and YH-HET (right) SST neurons in response to slow voltage ramp in control (dark gray) and 7 seconds after application of 0.5 nM TTX (light gray) with the difference current (I_NaP) plotted on the same scale. (B) I_NaP I-V curves of WT (gray) and YH-HET (blue) SST neurons constructed from averaging the ramp-evoked difference current (mean ± SEM) at 5-mV intervals (left), and a bar graph with overlaid scatter plot showing the peak negative value for each neuron (right). (C) Representative traces of WT (left) and YH-HET (right) PV neurons in response to slow voltage ramp in control (dark gray) and 7 seconds after application of 0.5 nM TTX (light gray) with the difference current (I_NaP) plotted on the same scale. The upper traces and difference currents are plotted on different scales. (D) I_NaP I-V curves of WT (gray) and YH-HET (orange) PV neurons constructed from averaging the ramp-evoked difference current (mean ± SEM) at 5-mV intervals (left), and a bar graph with overlaid scatter plot showing the peak negative value for each neuron (right). (E) Simulated traces from a model PV neuron in response to 500-ms current injections with control levels of KCNT1 (black traces) and KCNT1 GOF (Na⁺ EC₅₀ = 35 mM) with a 2-fold increase in I_NaP (orange traces). Representative traces are shown at increasing current steps from bottom to top for each level. (F) F-I plot shows the increase in AP firing (left), and a bar graph with overlaid scatter plot of individual neuron values shows the decrease in rheobase associated with modeling KCNT1 GOF (Na⁺ EC₅₀ = 35 mM) together with the increase in I_NaP. For modeling data, statistical significance was tested using a Repeated Measures ANOVA. Asterisks indicate where p < 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Figure 8.. YH-HET SST neurons show increased chemical and electrical coupling and receive increased excitatory input.

(A and B) Evoked postsynaptic currents (PSCs) were recorded from SST neuron pairs by stimulating each neuron at 0.1 Hz and recording the response onto the partner. (A) A bar graph shows the connection probability (mean + SEM) between SST neurons (WT, gray; YH-HET, blue; numbers on bars represent connected pair number/recorded pair number). (B) On the left, example traces of evoked IPSCs [individual IPSCs (light) overlaid by averaged IPSCs (dark)], and on the right, a graph of connection strength between SST neurons (WT, gray; YH-HET, blue; individual measurements and summary violin plots). (C₁) A schematic illustrates representative responses (presynaptic, black; postsynaptic, red) between chemically coupled (upper two panels) and electrically coupled (lower two panels) SST neurons following 100-pA (left two panels) and AP (right two panels) stimulation. (C₂) Summary data of four possible connection motifs (not coupled, chemical: one-way, chemical: two-way, and electrical) tested among WT (gray box) and YH-HET (blue box) SST neuron pairs (20 pairs/group). (D) On the left, example traces of spontaneous EPSCs (sEPSCs) recorded onto SST neurons (WT, black; YH-HET, blue). On the right, a graph shows individual neuron measurements and summary violin plots of the sEPSC frequency onto SST neurons (WT, gray; YH-HET, blue). All significant p-values are displayed at the top of each graph.

Figure 9.. Acute cortical slice recordings confirm the reduced intrinsic excitability of YH-HET SST neurons.

(A) An inverted monochrome image illustrates the recording scheme from GFP-expressing neurons in acute slices from P21–P30 GIN mice that express GFP in a subset of SST-positive neurons. (B) Representative responses to current steps are shown for WT (black) and YH-HET (blue) to three, equivalent 1 s current steps. (C) Plot of the action potential frequency (mean ± SEM) per current injection step shows that YH-HET GFP-expressing neurons (blue) fire fewer action potentials than those of WT (grey) across a range of current inputs. (D) Bar graphs show quantification of the membrane properties and AP parameters for WT (grey) and YH-HET (colors) GFP-expressing neuron groups, with individual neuron measurements overlaid in scatter plots.