Two epilepsy-associated variants in KCNA2 (K_V 1.2) at position H310 oppositely affect channel functional expression

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes K_V 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of K_V 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the K_V -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human K_V 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on K_V 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of K_V 1.2 by K_V β1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for K_V -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of K_V 1.2 voltage-activated, K⁺ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in K_V channels. The p.H310Y variant caused 'dual gain of function', increasing both K_V 1.2-channel activity and the number of K_V 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of K_V 1.2 by K_V β1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of K_V 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.