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

Abstract

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.

Keywords: H310Y; K+ channel; MED12L; channelopathy; developmental delay; fluorometry; genetics; seizure.