Drug-resistant epileptic encephalopathies of infancy have been associated with KCNT1 gain-of-function mutations, which increase the activity of KNa1.1 sodium-activated potassium channels. Pharmacological inhibition of hyperactive KNa1.1 channels by quinidine has been proposed as a stratified treatment, but mostly this has not been successful, being linked to the low potency and lack of specificity of the drug. Here we describe the use of a previously determined cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify how quinidine binds to the channel pore and, using computational methods, screened for compounds predicated to bind to this site. We describe six compounds that inhibited KNa1.1 channels with low- and sub-micromolar potencies, likely also through binding in the intracellular pore vestibule. In hERG inhibition and cytotoxicity assays, two compounds were ineffective. These may provide starting points for the development of new pharmacophores and could become tool compounds to study this channel further.

Canonical transient receptor potential 3 (TRPC3) channel is a non-selective cation permeable channel that plays an essential role in calcium signalling. TRPC3 is highly expressed in the brain and also found in endocrine tissues and smooth muscle cells. The channel is activated directly by binding of diacylglycerol downstream of G-protein coupled receptor activation. In addition, TRPC3 is regulated by endogenous factors including Ca²⁺ ions, other endogenous lipids, and interacting proteins. The molecular and structural mechanisms underlying activation and regulation of TRPC3 are incompletely understood. Recently, several high-resolution cryogenic electron microscopy structures of TRPC3 and the closely related channel TRPC6 have been resolved in different functional states and in the presence of modulators, coupled with mutagenesis studies and electrophysiological characterisation. Here, we review the recent literature which has advanced our understanding of the complex mechanisms underlying modulation of TRPC3 by both endogenous and exogenous factors. TRPC3 plays an important role in Ca²⁺ homeostasis and entry into cells throughout the body, and both pathological variants and downstream dysregulation of TRPC3 channels have been associated with a number of diseases. As such, TRPC3 may be a valuable therapeutic target, and understanding its regulatory mechanisms will aid future development of pharmacological modulators of the channel.

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase K _Na 1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric K _Na 1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant K _Na 1.1 assemblies were more hyperpolarised than WT K _Na 1.1 and, unlike WT K _Na 1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant K _Na 1.1 had activation kinetics intermediate of homomeric WT and variant K _Na 1.1 channels, with residual sodium-independent activity. In general, WT and variant K _Na 1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the K _Na 1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that K _Na 1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated K _Na 1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

The Na⁺-activated K⁺ channel K_Na1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na⁺ ions bind and increase channel activity is not well understood. Analysis of K_Na1.1 channel structures indicate that there is a large twisting of the βN-αQ loop in the intracellular RCK2 domain between the inactive and Na⁺-activated conformations, with a lysine (K885, human subunit numbering) close enough to potentially form a salt bridge with an aspartate (D839) in βL in the Na⁺-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within a pocket formed by the βO strand. In carrying out mutagenesis and electrophysiology with human K_Na1.1, we found that alanine substitution of selected residues in these regions resulted in almost negligible currents in the presence of up to 40 mM intracellular Na⁺. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na⁺. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active K_Na1.1, and D884I had Na⁺ dependence similar to wild-type K_Na1.1, while increasing the side-chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na⁺. We conclude that Na⁺ binding results in a conformational change that accommodates D884 in the βO pocket, which triggers further conformational changes in the RCK domains and channel activation.

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

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.