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Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy.

ABSTRACT: K_v7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in K_v7.2 and K_v7.3 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_v7.2 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_v7.2 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 (PIP₂), and our molecular dynamics simulation suggests PIP₂ interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in K_v7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of K_v7-associated EE.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7075958 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy.

Zhang Jiaren J Kim Eung Chang EC Chen Congcong C Procko Erik E Pant Shashank S Lam Kin K Patel Jaimin J Choi Rebecca R Hong Mary M Joshi Dhruv D Bolton Eric E Tajkhorshid Emad E Chung Hee Jung HJ

Scientific reports 20200316 1

Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 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 Kv7.2 including voltage sensing S4, the pore loop and S6 in t ...[more]

PMID: 32179837

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Project description:ObjectiveEarly onset epileptic encephalopathy with suppression-burst is one of the most severe epilepsy phenotypes in human patients. A significant proportion of cases have a genetic origin, and the most frequently mutated gene is KCNQ2, encoding Kv7.2, a voltage-dependent potassium channel subunit, leading to so-called KCNQ2-related epileptic encephalopathy (KCNQ2-REE). To study the pathophysiology of KCNQ2-REE in detail and to provide a relevant preclinical model, we generated and described a knock-in mouse model carrying the recurrent p.(Thr274Met) variant.MethodsWe introduced the p.(Thr274Met) variant by homologous recombination in embryonic stem cells, injected into C57Bl/6N blastocysts and implanted in pseudopregnant mice. Mice were then bred with 129Sv Cre-deleter to generate heterozygous mice carrying the p.(Thr274Met), and animals were maintained on the 129Sv genetic background. We studied the development of this new model and performed in vivo electroencephalographic (EEG) recordings, neuroanatomical studies at different time points, and multiple behavioral tests.ResultsThe Kcnq2Thr274Met/+ mice are viable and display generalized spontaneous seizures first observed between postnatal day 20 (P20) and P30. In vivo EEG recordings show that the paroxysmal events observed macroscopically are epileptic seizures. The brain of the Kcnq2Thr274Met/+ animals does not display major structural defects, similar to humans, and their body weight is normal. Kcnq2Thr274Met/+ mice have a reduced life span, with a peak of unexpected death occurring for 25% of the animals by 3 months of age. Epileptic seizures were generally not observed when animals grew older. Behavioral characterization reveals important deficits in spatial learning and memory in adults but no gross abnormality during early neurosensory development.SignificanceTaken together, our results indicate that we have generated a relevant model to study the pathophysiology of KCNQ2-related epileptic encephalopathy and perform preclinical research for that devastating and currently intractable disease.

Project description:The gene kcnma1 encodes the α-subunit of high-conductance calcium- and voltage-dependent K+ (BK) potassium channel. With the development of generation gene sequencing technology, many KCNMA1 mutants have been identified and are more closely related to generalized epilepsy and paroxysmal dyskinesia. Here, we performed a genetic screen of 26 patients with febrile seizures and identified a novel mutation of KCNMA1 (E155Q). Electrophysiological characterization of different KCNMA1 mutants in HEK 293T cells, the previously-reported R458T and E884K variants (not yet determined), as well as the newly-found E155Q variant, revealed that the current density amplitude of all the above variants was significantly smaller than that of the wild-type (WT) channel. All the above variants caused a positive shift of the I-V curve and played a role through the loss-of-function (LOF) mechanism. Moreover, the β4 subunit slowed down the activation of the E155Q mutant. Then, we used kcnma1 knockout (BK KO) mice as the overall animal model of LOF mutants. It was found that BK KO mice had spontaneous epilepsy, motor impairment, autophagic dysfunction, abnormal electroencephalogram (EEG) signals, as well as possible anxiety and cognitive impairment. In addition, we performed transcriptomic analysis on the hippocampus and cortex of BK KO and WT mice. We identified many differentially expressed genes (DEGs). Eight dysregulated genes [i.e., (Gfap and Grm3 associated with astrocyte activation) (Alpl and Nlrp10 associated with neuroinflammation) (Efna5 and Reln associated with epilepsy) (Cdkn1a and Nr4a1 associated with autophagy)] were validated by RT-PCR, which showed a high concordance with transcriptomic analysis. Calcium imaging results suggested that BK might regulate the autophagy pathway from TRPML1. In conclusion, our study indicated that newly-found point E155Q resulted in a novel loss-of-function variant and the dysregulation of gene expression, especially astrocyte activation, neuroinflammation and autophagy, might be the molecular mechanism of BK-LOF meditated epilepsy.

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Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy.

Publications

Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy.

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