A molecular switch in RCK2 triggers sodium-dependent activation of KNa.1 (KCNT1) potassium channels

The Na⁺-activated K⁺ channel KNa1.1, 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 KNa1.1 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 KNa1.1, we found 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 KNa1.1, D884I had Na⁺-dependence similar to wild-type KNa1.1, whilst 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

Drosophila expressing mutant human KCNT1 transgenes make an effective tool for targeted drug screening in a whole animal model of KCNT1-epilepsy

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with 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 responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy