Abstracts

Rationale: KCNQ2 and KCNQ3 encode potassium channel subunits (Kv7.2, Kv7.3) that co-assemble to generate neuronal M-current that regulates neuronal excitability. KCNQ2 pathogenic variants identified in children with self-limited familial neonatal epilepsy (SeLFE) and developmental and epileptic encephalopathy (DEE) most often exhibit loss-of-function (LoF). Potentiation of M-current with agents such as BHV-7000 (formally KB-3061) is a potential therapy for these conditions. We determined the effects of BHV-7000 on 50 loss-of-function KCNQ2 variants (10 SeLFNE-associated and 40 DEE-associated).


Methods: We expressed Kv7.2 with Kv7.3 in Chinese hamster ovary (CHO) cells and recorded whole-cell currents using automated planar patch clamp first in the absence then presence of vehicle (DMSO) or BHV-7000. CHO-K1 cells stably expressing WT Kv7.3 were transfected with an equal mixture of wild type (WT) and variant KCNQ2 to generate heteromultimeric channel complexes that recapitulated the heterozygous state without detectable expression of Kv7.2 homotetramers. BHV-7000 effects were assessed at 0.3, 1.0, 3.0, 10 and 30 μM. Specific channel activities were determined by applying the M-current blocker XE-991 (10 µM) at the end of experiments; only XE-991-sensitive currents were analyzed.


Results: We measured the effect of BHV-7000 on current density measured at -30 mV, which we assert is within the physiological relevant range of neuronal M-current. At 1 μM exposure, BHV-7000 restored current density for most variants to ≥ 76% of WT channels that was measured in the absence of drug. Current density for two DEE-associated variants (E140Q, D282N) was restored to approximately 65% of WT levels at this concentration, but current density of both variants was boosted 90-150% at higher concentrations (D282N = 90% at 3 μM; E140Q = 150% at 10 μM). At 3 μM exposure, BHV-7000 induced hyperpolarizing shifts in the voltage-dependence of activation for 42 variants to a degree similar to WT channels (delta V½ of averaged WT channels was -17.5 ± 4.0 mV [mean ± stdev, n =12]). Six variants exhibited smaller degrees of hyperpolarization and 2 showed larger hyperpolarization. The averaged EC_50s for BHV-7000 induced shift in V½ and current density increase measured at -30 mV in WT channels were 1.1 ± 0.9 μM and 3.5 ± 0.9 μM, respectively (mean ± stdev, n =12). For the majority of tested variants, the calculated EC_50s fell within 1 stdev of the WT average for current density and shift in activation V½.


Conclusions: BHV-7000, a selective M-current activator restored current density in all tested pathogenic KCNQ2 variants. For most of the tested variants, current density was restored to near WT levels with 1 μM BHV-7000. The calculated BHV-7000 EC₅₀ values were similar to WT for the majority of the variants tested. These findings support the potential therapeutic value of BHV-7000 in KCNQ2-related epilepsy associated with a wide range of variants.


Funding: Supported by NIH grant NS108874 and research funding from Biohaven Pharmaceuticals.