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Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy.

ABSTRACT: Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 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 Kv7.2. 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 Kv7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), revealing novel PIP2 binding residues. While these mutations disrupted Kv7 function to suppress excitability, hyperexcitability was observed in neurons expressing Kv7.2-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 Kv7 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 Kv7.2, whereas the presence of Kv7.3 blocked this degradation. Furthermore, expression of Kv7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of Kv7.2 cause abnormal Kv7 expression and function by disrupting Kv7.2 binding to CaM and/or modulation by PIP2. We propose that such multiple Kv7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

SUBMITTER: Kim EC

PROVIDER: S-EPMC6415549 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy.

Kim Eung Chang EC Zhang Jiaren J Pang Weilun W Wang Shuwei S Lee Kwan Young KY Cavaretta John P JP Walters Jennifer J Procko Erik E Tsai Nien-Pei NP Chung Hee Jung HJ

Neurobiology of disease 20180706

Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 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 epile ...[more]

PMID: 30008368

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Project description:The KV 1/D-type potassium current (ID ) is an important determinant of neuronal excitability. This study explored whether and how ID channels regulate the activation of bronchopulmonary vagal afferent nerves. The single-neuron RT-PCR assay revealed that nearly all mouse bronchopulmonary nodose neurons expressed the transcripts of α-dendrotoxin (α-DTX)-sensitive, ID channel-forming KV 1.1, KV 1.2 and/or KV 1.6 α-subunits, with the expression of KV 1.6 being most prevalent. Patch-clamp recordings showed that ID , defined as the α-DTX-sensitive K+ current, activated at voltages slightly more negative than the resting membrane potential in lung-specific nodose neurons and displayed little inactivation at subthreshold voltages. Inhibition of ID channels by α-DTX depolarized the lung-specific nodose neurons and caused an increase in input resistance, decrease in rheobase, as well as increase in action potential number and firing frequency in response to suprathreshold current steps. Application of α-DTX to the lungs via trachea in the mouse ex vivo vagally innervated trachea-lungs preparation led to action potential discharges in nearly half of bronchopulmonary nodose afferent nerve fibres, including nodose C-fibres, as detected by the two-photon microscopic Ca2+ imaging technique and extracellular electrophysiological recordings. In conclusion, ID channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves by stabilizing the membrane potential, counterbalancing the subthreshold depolarization and promoting the adaptation of action potential firings. Down-regulation of ID channels, as occurs in various inflammatory diseases, may contribute to the enhanced C-fibre activity in airway diseases that are associated with excessive coughing, dyspnoea, and reflex bronchospasm and secretions. KEY POINTS: The α-dendrotoxin (α-DTX)-sensitive D-type K+ current (ID ) is an important determinant of neuronal excitability. Nearly all bronchopulmonary nodose afferent neurons in the mouse express ID and the transcripts of α-DTX-sensitive, ID channel-forming KV 1.1, KV 1.2 and/or KV 1.6 α-subunits. Inhibition of ID channels by α-DTX depolarizes the bronchopulmonary nodose neurons, reduces the minimal depolarizing current needed to evoke an action potential (AP) and increases AP number and AP firing frequency in response to suprathreshold stimulations. Application of α-DTX to the lungs ex vivo elicits AP discharges in about half of bronchopulmonary nodose C-fibre terminals. Our novel finding that ID channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves suggests that their down-regulation, as occurs in various inflammatory diseases, may contribute to the enhanced C-fibre activity in airway inflammation associated with excessive respiratory symptoms.

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.

Dataset Information

Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy.

Publications

Reduced axonal surface expression and phosphoinositide sensitivity in K<sub>v</sub>7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy.

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