KCNT1 (K⁺ channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the KCNT1 gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.

Aneuploidy, or having a disrupted genome, is an aberration commonly found in tumours but rare in normal tissues. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift, which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, we investigated the changes in transcription in response to ongoing changes to ploidy (chromosomal instability, CIN). We noticed changes in genes affecting one-carbon metabolism, specifically those affecting the production and use of s-adenosyl methionine (SAM). The depletion of several of these genes has led to cell death by apoptosis in CIN cells but not in normal proliferating cells. We found that CIN cells are particularly sensitive to SAM metabolism at least partly because of its role in generating polyamines. Feeding animals spermine was seen to rescue the cell death caused by the loss of SAM synthase in CIN tissues. The loss of polyamines led to decreased rates of autophagy and sensitivity to reactive oxygen species (ROS), which we have shown to contribute significantly to cell death in CIN cells. These findings suggest that a well-tolerated metabolic intervention such as polyamine inhibition has the potential to target CIN tumours via a relatively well-characterised mechanism.

Objective: Hyperactive KCNT1 potassium channels, caused by gain‐of‐function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug‐resistant seizures and, in cases with infantile onset, developmental regression can follow. KCNT1‐related disorders include epilepsy of infancy with migrating focal seizures and sleep‐related hypermotor epilepsy. There are currently no effective treatments for KCNT1 epilepsies, but suppressing overactive channels poses a potential strategy. Methods: Using the KCNT1 channel structure we in silico screened a library of known drugs for those predicted to block the channel pore to inhibit channel activity. Cellular KCNT1 channel inhibition was analyzed using electrophysiology and Drosophila bang‐sensitive assays were used to analyze seizure suppression. Brain penetration of one drug was analyzed using liquid chromatography–mass spectrometry in a mouse. Results: Eight known drugs were investigated in vitro for their effects on patient‐specific mutant KCNT1 channels, with 4 drugs showing significant reduction of K ⁺ current amplitudes. The action of the 4 drugs was then analyzed in vivo and 2 were found to reduce the seizure phenotype in humanized Drosophila KCNT1 epilepsy models. One drug, antrafenine, was shown to cross the blood–brain barrier in mice. Interpretation: This study identified a known drug, antrafenine, that reduces KCNT1 channel activity, reduces seizure activity in Drosophila , and crosses the blood–brain barrier in the mouse, suggesting its potential applicability as a new treatment for KCNT1 epilepsy. The sequential in silico, in vitro, and in vivo mechanism‐based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified. ANN NEUROL 2025;98:1261–1274

Tubulinopathies are associated with malformations of cortical development but not Walker–Warburg Syndrome. Intensive monitoring of a Croatian infant presenting as Walker–Warburg Syndrome in utero began at 21 weeks due to increased growth of cerebral ventricles and foetal biparietal diameter. Monitoring continued until Caesarean delivery at 34 weeks where the infant was eutrophic. Clinical assessment of a progressive neurological disorder of unknown aetiology found a macrocephalic head and markedly hypoplastic genitalia with a micropenis. Neurological examination showed generalized hypotonia with very rare spontaneous movements, hypotonia-induced respiratory insufficiency and ventilator dependence, and generalized myoclonus intensifying during manipulation. With clinical features of hypotonia, lissencephaly, and brain malformations, Walker–Warburg Syndrome was suspected; however, eye anomalies were absent. Genetic trio analysis via whole-exome sequencing only identified a novel de novo mutation in the TUBA1A gene (NM_006009.4:c.848A>G; NP_006000.2:p.His283Arg) in the infant, who died at 2 months of age, as the likely cause. We report a previously unpublished, very rare heterozygous TUBA1A mutation with clinical features of macrocephaly and hypoplastic genitalia which have not previously been associated with the gene. The absence of eye phenotypes or mutations in Walker–Warburg-associated genes confirm this as not a new presentation of Walker–Warburg Syndrome but a novel TUBA1A tubulinopathy for neonatologists to be aware of.

Objective: Hyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. Methods: Using KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. Results: Eight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K ⁺ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. Interpretation: This study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico , in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are resistant to current treatments. In vitro studies have shown that KCNT1- epilepsy mutations are gain of function, significantly increasing K ⁺ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which was sensitive to drugs currently used to treat patients with KCNT1 -epilepsy. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila can be used to model human KCNT1 -epilepsy and potentially used as a tool to assess new treatments for KCNT1 epilepsy.