The paradoxical effects of K⁺channel gain-of-function are mediated by GABAergic neuron hypoexcitability and hyperconnectivity

Abstract

Gain-of-function (GOF) variants in K + channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K + currents lead to network hyperexcitability. Here, we introduced a human Na + -activated K + (KNa) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, found motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increased KNa currents in cortical excitatory and inhibitory neurons, there was a selective increase in the KNa current across subthreshold voltages in inhibitory neurons, particularly in those with non-fast spiking properties, resulting in impaired excitability and AP generation. We further observed evidence of synaptic rewiring associated with hyperexcitable networks, including increases in homotypic synaptic connectivity and the ratio of excitatory-to-inhibitory synaptic input. These findings support inhibitory neuron-specific mechanisms in mediating the epileptogenic effects of K + channel GOF, offering cell-type-specific currents and effects as promising targets for therapeutic intervention. discharges were detected using previously employed frequency and duration features (Gelinas et al., 2016). Multi-electrode arrays (MEA) Primary neuron cultureOne to seven days before dissection, 48-well MEA plates (Axion Biosystems #M768-KAP-48) were coated with 50 µg/mL poly-D-lysine (Sigma-Aldrich #P0899-50MG) in borate buffer, then washed three times with phosphate buffered saline (PBS) and stored in PBS at 4°C until use. Prior to use, PBS was aspirated and plates were allowed to dry in a sterilized hood. Cortical or hippocampal neurons were dissociated from the brains of postnatal day 0 (P0) C57BL/6NJ WT or Kcnt1 m/m mice. Pups were decapitated, weighed and genotyped. The entire cerebral cortex was rapidly dissected and cut into small pieces under sterile conditions in cold Hibernate A solution (Gibco, #A1247501). Cortices from two WT or Kcnt1 m/m pups were pooled together. The dissected cortices were then enzymatically digested in 20 U mL -1 Papain plus DNAse (Worthington Biochemical Corporation, #LK003178 and #LK003172) diluted in Hibernate A for 20 min at 37°C. Cells were pelleted by centrifugation at 300RCF for 5 min, then the digestion was neutralized by aspirating off the supernatant and adding warm Hibernate A media. Cells were mechanically dissociated by trituration, and counted using a hemocytometer with Trypan blue counterstain. Cells were pelleted by centrifugation at 300RCF for 5 min and re-suspended at a density of 6,000 cells/µl in warm Neurobasal-A (Gibco #10888022) + 1X B27 supplement (Gibco #17504044) + 1X GlutaMax (Gibco #35050061) + 1% HEPES (Gibco #15630080) + 1% Penicillin/Streptinomycin (Gibco # 15140122) + 1% fetal bovine serum (Gibco #26140079) + 5 ug/mL Laminin (Sigma-Aldrich #L2020). 50,000 cells were plated on a pre-coated 48-well MEA plate in a 40 uL drop. The day after plating (DIV1), 100% of the media was removed and replaced with warm Neu...

VU0606170, a Selective Slack Channels Inhibitor, Decreases Calcium Oscillations in Cultured Cortical Neurons

“…Recently, data supporting the mechanism that gain-of-function Slack mutations, such as A934T, lead to epilepsy through inhibitory neuron-mediated mechanisms by prolonging afterhyperpolari-zation and increasing currents at subthreshold voltages. 24 Indeed, we found that our synchronously firing neuronal cultures contained a mixture of inhibitory and excitatory neurons as well as astrocytes (Figure S5, Supporting Information), and it is tempting to speculate that the decrease in spike rate observed when neurons were treated with VU0606170 resulted from increasing inhibitory tone. Additionally, we were encouraged by the face that application of VU0606170 decreased the spike rate but did not completely suppress neuronal activity.…”

“…One proposed mechanism by which mutant Slack channels cause seizures is by enhancing K + currents in GABAergic neurons . This increase in current, especially in nonfast spiking GABAergic neurons, leads to prolonged hyperpolarization, resulting in a decrease in inhibitory interneuron activity and an overall decrease in inhibitory tone. ,, Second, this reduced excitability has been accompanied by an increase in excitatory–excitatory and inhibitory–inhibitory synaptic connectivity . It is thought that this increase in synaptic connectivity early in development predisposes the nervous system to develop circuits that generate epileptic discharges. , The resulting overall imbalance between neuronal excitation and inhibition leads to the development of seizures. ,,, …”

“…20,24,25 Second, this reduced excitability has been accompanied by an increase in excitatory−excitatory and inhibitory−inhibitory synaptic connectivity. 24 It is thought that this increase in synaptic connectivity early in development predisposes the nervous system to develop circuits that generate epileptic discharges. 5,24 The resulting overall imbalance between neuronal excitation and inhibition leads to the development of seizures.…”

“…24 It is thought that this increase in synaptic connectivity early in development predisposes the nervous system to develop circuits that generate epileptic discharges. 5,24 The resulting overall imbalance between neuronal excitation and inhibition leads to the development of seizures. 1,5,24,26 Mutations within the Slack channel have also been described in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) 11,27,28 and other epilepsies, including Ohtahara syndrome, 29 multifocal epilepsy, 27,29 and West syndromes.…”

“…5,24 The resulting overall imbalance between neuronal excitation and inhibition leads to the development of seizures. 1,5,24,26 Mutations within the Slack channel have also been described in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) 11,27,28 and other epilepsies, including Ohtahara syndrome, 29 multifocal epilepsy, 27,29 and West syndromes. 10,18,27 Several of the Slack mutations implicated in seizure disorders all share a common association with severe intellectual debility and other developmental delays and together give rise to an emerging role of Slack channels in intellectual disability.…”