Abstracts

Rationale: Heterozygous KCNQ2 de novo variants (DNVs) are a cause of neonatal-onset developmental and epileptic encephalopathy (Q2-DEE). Most Q2-DEE variants are missense, and are clustered in gene regions encoding domains for key protein functions, namely voltage sensing, ion pore, and sites regulating PIP2 and calmodulin binding. Under voltage-clamp, many Q2-DEE DNVs exert dominant negative effects when co-expressed with wild type (wt) subunits to mimic heterozygosity. Ezogabine (EZO) increases currents through channels containing mutated subunits in vitro and has been used in Q2-DEE patients. Novel small molecules acting as potent neuronal KCNQ channel openers are candidate precision medicines for Q2-DEE. Methods: The highly recurrent Q2-DEE missense variants T274M (pore helix), A294V (pore loop), and R581Q/L (C-helix) were selected for analysis and introduced into KCNQ2 cDNA by QuickChange site-directed mutagenesis. Using lipid mediated transfection, plasmids including the pathogenic variants were co-expressed with wt KCNQ2 or KCNQ2 and KCNQ3 subunits in Chinese hamster ovary (CHO) cells. The KCNQ2 S5 helix point mutation W236L, which abolishes EZO modulation, was used to characterize KB-3061 binding. Whole cell voltage-clamp protocols were used to measure gating and conductance density of wt channels and mutated subunit containing channels, before, during and after KB-3061 application via focal pipette perfusion. Results: Wt KCNQ2 homomer and KCNQ2/KCNQ3 heteromer channels were activated by KB-3061 (0.3 - 3.0 µM). The half-activating voltage (V1/2), determined by Boltzmann fits of the conductance-voltage (G/V) curves, were left-shifted by 26.02 mV (homomers, 1.0 M KB-3061, n=5), 19.79 mV (heteromers, 1.0 µM KB-3061, n=3) and 24.44 mV (heteromers, 3.0 µM, n=3). Homomeric KCNQ2 W236L current density at -40 mV was not modulated by 3 µM KB-3061 (control: 5.45 ± 0.99 pA/pF, KB-3061: 5.80 ± 1.07 pA/pF, n=10) or 10 µM EZO (control: 5.45 ± 0.99 pA/pF, EZO: 5.74 ± 0.97 pA/pF n=10). Compared to currents at -40 mV in cells expressing wt KCNQ2 only (4.85 ± 0.45 pA/pF, n=8), currents in cells coexpressing wt with T274M, A294V, or R581L were strongly suppressed (T274M: 1.46 ± 0.21 pA/pF, n=4; A294V: 1.28 ± 0.31 pA, n=7; R581L: 0.63 ± 0.09 pA/pF, n=6). Treatment with 0.3 µM KB-3061 increased currents strongly for each variant: (T274M: 7.20 ± 0.60 pA/pF, n=4; A294V: 8.17 ± 1.21, n=7 pA/pF; R581L: 4.90 ± 0.59 pA/pF, n=6). Conclusions: KB-3061 activates KCNQ2-containing homomers and heteromers by shifting activation and increasing current. Such KCNQ2 modulation requires the pore domain residue W236. Variants from three different Q2-DEE mutational hotspots exhibited strong dominant-negative current suppression under conditions mimicking heterozygosity. KB-3061 (0.3 µM) augmented currents in cells expressing each of these variants, increasing currents at -40 mV to wt levels. These results suggest potential for utility in Q2-DEE. Funding: Supported by a grant from Knopp Biosciences, Inc. to Baylor College of Medicine. SID, KEP are employees of Knopp Biosciences, Inc.