Potassium current inactivation as a novel pathomechanism for KCNQ2 developmental and epileptic encephalopathy

Abstract

De novo variants in KCNQ2 cause neonatal onset developmental and epileptic encephalopathy (KCNQ2-DEE; Online Mendelian Inheritance in Man #613720), most often by loss-of-function in vitro effects. In this study, we describe a neonatal onset DEE proband carrying a recurrent de novo KCNQ2 variant (c.794C>T; p.A265V) affecting the pore domain of KCNQ2-encoded Kv7.2 subunits. Whole-cell patch-clamp measurement in a mammalian heterologous expression system revealed that, when compared to wild-type Kv7.2 channels, channels containing Kv7.2 A265V subunits displayed (1) reduced maximal current density; (2) decreased voltage-dependence of activation; and (3) an unusual inactivation process, with a 50% current reduction during 1-2-s depolarizing pulses at voltages > 0 mV. These effects were proportional to the number of mutant subunits incorporated in heteromeric channels, being overall less dramatic upon coexpression with Kv7.2 or Kv7.2 + Kv7.3 subunits. These results reveal current inactivation as a novel pathogenetic mechanism for KCNQ2-DEE caused by a recurrent variant affecting a critical pore residue, further highlighting the importance of in vitro functional assessment for a better understanding of disease molecular pathophysiology.

Keywords: KCNQ2; developmental encephalopathies; epilepsy; loss‐of‐function variant; potassium channels; potassium current inactivation.

FIGURE 1

Localization and functional characterization of the Kv7.2 A265V variant. (A) Alignment of the amino acid sequence within the S₅–S₆ region of the indicated channels. The position of the A265 residue in  a Kv7.2 subunit is indicated by an arrow. The regions in yellow correspond to the pore helix and the beginning of S6. The boxed area corresponds to the selectivity filter (SF). (B) Kv7.2 structure (Protein Data Bank code: 8J05) of S₅, S₆, and the intervening linker, including the pore domain from two opposite subunits; the other two subunits present in functional tetrameric channels have been removed for clarity. The A265 side‐chain atoms are shown as spheres. (C) Macroscopic currents from Kv7.2 and Kv7.2 A265V channels, in response to the indicated voltage protocol (scale: 200 pA, 200 ms). (D) Superimposed and normalized current traces of Kv7.2 (black) and Kv7.2 A265V (dark green) channels at +20 mV (time scale: 200 ms). (E) G/V curves for the indicated channels; conductance (G) values at each potential (V) were calculated by dividing the peak current values by the K⁺ equilibrium potential. (F) Maximal current density at 0 mV recorded from cells transfected with the indicated cDNAs. (G) Percent of inactivation at +20 mV in the current carried by the indicated channels (same as panel E). *p < .05 versus respective control.