K v 7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in K v 7.2 and K v 7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of K v 7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of K v 7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP 2), and our molecular dynamics simulation suggests PIP 2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in K v 7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of K v 7-associated EE. Epilepsy is the second most prominent neurological disease (www.epilepsy.com), in which excessive electrical activity within networks of neurons in the brain manifests clinically as recurrent unprovoked seizures 1. Recent discoveries of epilepsy-related genes in multiple laboratories and through large consortia have revealed a diverse array of proteins that may contribute to epileptogenesis 1,2. Among these proteins, neuronal KCNQ/K v 7 potassium (K +) channels have been implicated in epilepsy since mutations in the principle subunits, KCNQ2/K v 7.2 and KCNQ3/K v 7.3, cause Benign Familial Neonatal Epilepsy (BFNE [MIM: 121200]) and Epileptic Encephalopathy (EE [MIM: 613720]) (RIKEE database www.rikee.org). Neuronal K v 7 channels are mainly composed of heterotetramers of K v 7.2 and K v 7.3 3 , which show overlapping distribution in the hippocampus and cortex 4. They generate slowly activating and non-inactivating voltage-dependent K + currents that contribute to resting membrane potential, prevent repetitive and burst firing of action potentials (APs), and modulate AP threshold 3,5-7 .They are enriched at the plasma membrane of axonal initial segments (AIS) and distal axons 8,9 , where APs initiate and propagate 10. Membrane phosphatidylinositol-4,5-bisphosphate (PIP 2) is required for K v 7 channels to open 3 , although its exact binding sites in K v 7.2 and K v 7.3

Neuronal K7/KCNQ channels are voltage-gated potassium channels composed of K7.2/KCNQ2 and K7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in K7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of K7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to K7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP), revealing novel PIP binding residues. While these mutations disrupted K7 function to suppress excitability, hyperexcitability was observed in neurons expressing K7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in K7 channels by severely reducing their CaM binding, K currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of K7.2, whereas the presence of K7.3 blocked this degradation. Furthermore, expression of K7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of K7.2 cause abnormal K7 expression and function by disrupting K7.2 binding to CaM and/or modulation by PIP. We propose that such multiple K7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

KCNQ/K v 7 channels conduct voltage-dependent outward potassium currents that potently decrease neuronal excitability. Heterozygous inherited mutations in their principle subunits K v 7.2/KCNQ2 and K v 7.3/KCNQ3 cause benign familial neonatal epilepsy whereas patients with de novo heterozygous K v 7.2 mutations are associated with early-onset epileptic encephalopathy and neurodevelopmental disorders characterized by intellectual disability, developmental delay and autism. However, the role of K v 7.2-containing K v 7 channels in behaviors especially autism-associated behaviors has not been described. Because pathogenic K v 7.2 mutations in patients are typically heterozygous loss-of-function mutations, we investigated the contributions of K v 7.2 to exploratory, social, repetitive and compulsive-like behaviors by behavioral phenotyping of both male and female KCNQ2 +/− mice that were heterozygous null for the KCNQ2 gene. Compared with their wild-type littermates, male and female KCNQ2 +/− mice displayed increased locomotor activity in their home cage during the light phase but not the dark phase and showed no difference in motor coordination, suggesting hyperactivity during the inactive light phase. In the dark phase, KCNQ2 +/− group showed enhanced exploratory behaviors, and repetitive grooming but decreased sociability with sex differences in the degree of these behaviors. While male KCNQ2 +/− mice displayed enhanced compulsive-like behavior and social dominance, female KCNQ2 +/− mice did not. In addition to elevated seizure susceptibility, our findings together indicate that heterozygous loss of K v 7.2 induces behavioral abnormalities including autism-associated behaviors such as reduced sociability and enhanced repetitive behaviors. Therefore, our study is the first to provide a tangible link between loss-of-function K v 7.2 mutations and the behavioral comorbidities of K v 7.2-associated epilepsy.

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 Kv7.2 subunit of voltage-gated Kv7 potassium channels. Kv7 channels composed of Kv7.2 and Kv7.3 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 Kv7.2 blocks calmodulin binding to Kv7.2 and axonal surface expression of Kv7 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 Kv7.2-M547V (equivalent to human Kv7.2-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 Kv7 channels is associated with epileptic seizures, cognitive and behavioral deficits, and neuronal loss in KCNQ2-related EE.

Objective STriatal‐Enriched protein tyrosine Phosphatase (STEP) is a brain‐specific tyrosine phosphatase. Membrane‐bound STEP61 is the only isoform expressed in hippocampus and cortex. Genetic deletion of STEP enhances excitatory synaptic currents and long‐term potentiation in the hippocampus. However, whether STEP61 affects seizure susceptibility is unclear. Here we investigated the effects of STEP inhibitor TC‐2153 on seizure propensity in a murine model displaying kainic acid (KA)–induced status epilepticus and its effect on hippocampal excitability. Methods Adult male and female C57BL/6J mice received intraperitoneal injection of either vehicle (2.8% dimethylsulfoxide [DMSO] in saline) or TC‐2153 (10 mg/kg) and then either saline or KA (30 mg/kg) 3 h later before being monitored for behavioral seizures. A subset of female mice was ovariectomized (OVX). Acute hippocampal slices from Thy1‐GCaMP6s mice were treated with either DMSO or TC‐2153 (10 μM) for 1 h, and then incubated in artificial cerebrospinal fluid (ACSF) and potassium chloride (15 mM) for 2 min prior to live calcium imaging. Pyramidal neurons in dissociated rat hippocampal culture (DIV 8–10) were pre‐treated with DMSO or TC‐2153 (10 µM) for 1 h before whole‐cell patch‐clamp recording. Results TC‐2153 treatment significantly reduced KA‐induced seizure severity, with greater trend seen in female mice. OVX abolished this TC‐2153‐induced decrease in seizure severity in female mice. TC‐2153 application significantly decreased overall excitability of acute hippocampal slices from both sexes. Surprisingly, TC‐2153 treatment hyperpolarized resting membrane potential and decreased firing rate, sag voltage, and hyperpolarization‐induced current (Ih) of cultured hippocampal pyramidal neurons. Significance This study is the first to demonstrate that pharmacological inhibition of STEP with TC‐2153 decreases seizure severity and hippocampal activity in both sexes, and dampens hippocampal neuronal excitability and Ih. We propose that the antiseizure effects of TC‐2153 are mediated by its unexpected action on suppressing neuronal intrinsic excitability.