Spontaneous seizure and memory loss in mice expressing an epileptic encephalopathy variant in the calmodulin-binding domain of Kv7.2

Epileptic encephalopathy (EE) is characterized by seizures that respond poorly to antiseizure drugs, psychomotor delay, and cognitive and behavioral impairments. One of the frequently mutated genes in EE is KCNQ2, which encodes the K_v7.2 subunit of voltage-gated K_v7 potassium channels. K_v7 channels composed of K_v7.2 and K_v7.3 are enriched at the axonal surface, where they potently suppress neuronal excitability. Previously, we reported that the de novo dominant EE mutation M546V in human K_v7.2 blocks calmodulin binding to K_v7.2 and axonal surface expression of K_v7 channels via their intracellular retention. However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral comorbidities remains unknown. Here, we report conditional transgenic cKcnq2^+/M547V mice, in which expression of mouse K_v7.2-M547V (equivalent to human K_v7.2-M546V) is induced in forebrain excitatory pyramidal neurons and astrocytes. These mice display early mortality, spontaneous seizures, enhanced seizure susceptibility, memory impairment, and repetitive behaviors. Furthermore, hippocampal pathology shows widespread neurodegeneration and reactive astrocytes. This study demonstrates that the impairment in axonal surface expression of K_v7 channels is associated with epileptic seizures, cognitive and behavioral deficits, and neuronal loss in KCNQ2-related EE.

Epileptic encephalopathies (EEs) are a collection of heterogeneous disorders in which early-onset severe seizures contribute to developmental delay and progressive cognitive and behavioral impairments (1). Current treatments for EEs have limited efficacy in alleviating seizures and comorbidities (2), posing an urgent need to understand the etiology of EEs and find new therapeutic targets. Recent discoveries of epilepsy-related genes in multiple laboratories and through the large Epi4K, EpiPM, and EuroEPINOMICS-RES consortia have identified a diverse array of proteins that may contribute to epileptogenesis (3–5). Among them, dominant variants associated with benign familial neonatal epilepsy (BFNE) and EE have been found in KCNQ2 and KCNQ3 genes, which encode the K_v7.2 and K_v7.3 subunits of voltage-gated potassium (K⁺) channel subfamily Q (K_v7) (https://www.rikee.org; ClinVar Database, National Center for Biotechnology Information [NCBI]).Neuronal K_v7 channels are mostly heterotetrameric channels of K_v7.2 and K_v7.3 subunits (6), which have overlapping distribution in the central nervous system including the cerebral cortex and hippocampus (7). In neurons, they are preferentially localized to the axonal plasma membrane with the highest concentration at the axonal initial segment (AIS) (7, 8), where the action potential (AP) initiates (9). They give rise to the slowly activating and noninactivating outward K⁺ current termed M current (I_M) (6). Because they open at subthreshold potentials (6), I_M potently suppresses AP firing (6, 10, 11), underscoring their critical roles in reducing neuronal excitability. By contrast, activation of G_q-coupled receptors, including muscarinic acetylcholine receptors, inhibits I_M by depleting the lipid cofactor PIP₂, resulting in enhanced AP firing (12).To date, 193 dominant variants in KCNQ2 and 2 variants in KCNQ3 have been identified in patients with EE (https://www.rikee.org; ClinVar Database, NCBI). EE variants are clustered at the functional domains of K_v7.2 important for voltage-dependent opening of K_v7 channels (13) and typically decrease the function of heterotetrameric channels by 20 to 75% (13–17). EE variants are also enriched at helices A and B in the intracellular C-terminal tail of K_v7.2 (13, 14, 16), which mediate calmodulin (CaM) binding critical for axonal enrichment of K_v7 channels (18). Among these variants, a mutation of methionine at amino acid position 546 to valine (M546V) was found in a male patient who displayed drug-resistant neonatal tonic-clonic seizures and later developed profound intellectual and language disability, spasticity, and autistic behavior (15). While this mutation in helix B abolishes current expression of homomeric but not heteromeric channels in heterologous cells (14, 17), it severely reduces CaM binding and axonal surface expression of heteromeric channels in cultured hippocampal neurons (14). This mutation also induces ubiquitination and proteasomal degradation of K_v7.2, whereas the presence of K_v7.3 blocks this degradation and accumulates ubiquitinated K_v7.2 (14). However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral deficits in EE remains unknown.In this study, we investigated the contribution of the EE variant M546V by generating conditional transgenic mice in which heterozygous expression of mouse K_v7.2-M547V was induced in forebrain excitatory pyramidal neurons. M546 in the human K_v7.2 is conserved in the mouse K_v7.2 at amino acid position 547. These mice showed widespread neurodegeneration and reactive astrogliosis in the hippocampus and cortex, and displayed spontaneous seizures and cognitive deficit, providing a causal link between M546V-mediated disruption of axonal surface expression of K_v7 channels and KCNQ2-associated EE.