Function and Pharmacological Modulation of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel

Gain-of-function missense pathogenic variants of KCNT1, the gene encoding the
sodium-activated potassium channel subunit KNa1.1, are associated with intractable
early-onset epilepsies. Variants in diverse regions of the channel are hypothesised to
interfere with channel gating, although the mechanisms of gating are poorly defined.
Patients are heterozygous and there is a lack of information about how pathogenic
variants increase channel activity and also the behaviour of heterotetrametric channels comprised of wildtype and variant subunits. Inhibition of hyperactive KNa1.1 channels with class I antiarrhythmic quinidine has had variable success in patients due to low potency, non-selectivity and poor blood-brain-barrier penetration.

Seven disease-causing variants were selected from across the spectrum of disorders
and involving different protein domains. Whole cell electrophysiology was used to
characterise homomeric and heteromeric KNa1.1 channel assemblies carrying epilepsy-causing variants expressed in CHO cells. We found that all disease-causing KNa1.1
variants lowered the energetic barrier associated with the sodium-dependent activation
gate by destabilising the inactivated channel conformation. Some variants also had
effects on selectivity filter gating. Using the previously determined cryo-EM structure of
KNa1.1 and mutational analysis, we identified how quinidine binds within the intracellular
pore vestibule. Using in silico methods and whole cell electrophysiology, six small
molecule compounds and four FDA-approved drugs were identified that inhibit KNa1.1
channels with low- and sub-micromolar potency. Finally, using mutagenesis and whole
cell electrophysiology, six residues involved in Na+-activation were identified, located
on the cytoplasmic RCK (regulators of conductance of K+) domains.

The results provide a common mechanism for how disease-causing variants cause
gain-of-function. In addition to providing more information about the gating of KNa1.1,
identification of the Na+-activation mechanisms will aid development of alternative drug
modalities in future. We have identified potential therapeutic drugs that could be
repurposed for treating KCNT1-related epilepsies, or possible pharmacophores that
could be starting points for developing potent and selective inhibitors.