CONSTITUTIVE OPENING OF THE Kv7.2 PORE ACTIVATION GATE CAUSES KCNQ2-DEVELOPMENTAL ENCEPHALOPATHY

Pathogenic variants in KCNQ2 encoding for Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies ( KCNQ2 -encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S6 activation gate undergoing large structural rearrangements during pore opening. Currents through these mutant channels displayed increased density, hyperpolarizing shifts in activation gating, and insensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in single-channel open probability, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atoms Molecular Dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, a PIP2-independent stabilization of the inner pore gate open configuration is a novel molecular pathogenetic mechanism for KCNQ2 -developmental encephalopathies. ### Competing Interest Statement The authors have declared no competing interest. * CHOL : cholesterol CD : cross-distance CHO : Chinese Hamster Ovary Cryo-EM : cryogenic electron microscopy DEE : developmental and epileptic encephalopathy DMPC : 1,2-Dimyristoyl-sn-glycero-3-phosphocholine ESES GoF : gain-of-function HB : hydrogen bond IG : inner gate Kv : voltage-gated potassium channel LoF : loss-of-function MD : molecular dynamics NDD : neurodevelopmental disorder PBS : phosphate-buffered saline PAG : pore activation gate PD : pore domain PIP2 : phosphatidylinositol 4,5-bisphosphate POPC : 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPE : 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine POPG : 1-Palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) RMSD : root mean square deviation SDS-PAGE : sodium dodecylsulfate polyacrylamide gel electrophoresis SF : selectivity filter SLFNE : self-limited familial neonatal epilepsy TM : transmembrane VSD : voltage-sensing domain WT : wild type