Deletion of Kv10.2 Causes Abnormal Dendritic Arborization and Epilepsy Susceptibility.

The abnormal function of the voltage-gated potassium channel Kv10.2 can induce epilepsy. However, the physiological function of Kv10.2 in the central nervous system remains unclear. In this study, we found that Kv10.2 knockout (KO) increased the complexity of neurons in the CA3 subarea of hippocampus. Kv10.2 KO led to enlarged somata, elongated dendritic length, and increased the number of dendritic tips in cultured rat hippocampus neurons. Kv10.2 KO also increased Synapsin I and PSD95 protein density in cultured rat hippocampal neurons. Whole cell patch-clamp recordings of brain slices in the CA3 subarea of hippocampus revealed that Kv10.2 KO increased the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC), depolarized the resting membrane potential and increased the action potential firing, reduced the rheobase and increased the input resistance, which results in enhanced neuronal excitability. Furthermore, we made electroencephalogram (EEG) recordings of brain activity in freely moving rats before and after inducing seizures by pentylenetetrazole (PTZ) injection. Kv10.2 KO rats dramatically increased the EEG amplitude during epilepsy. Behavioral observation after seizure induction revealed that Kv10.2 KO rats demonstrated shortened onset latency, prolonged duration, and increased seizure severity when compared with wild type rats. Therefore, this study provides a new link between Kv10.2 and neuronal morphology and higher intrinsic excitability.

Up-regulation of miR-21 and miR-23a Contributes to As2 O3 -induced hERG Channel Deficiency.

Arsenic trioxide (As2O3) is used to treat acute pro-myelocytic leukaemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the hERG current via disturbing its trafficking to cellular membrane. Consistent with these findings, in this study, we reported that As2O3 inhibited hERG channel at both protein and mRNA levels and damaged hERG current but did not affect channel kinetics. Further, we demonstrated that As2O3 up-regulated miR-21 and miR-23a expression in hERG-HEK293 cells and neonatal cardiomyocytes. In addition, knock-down of miR-21 by its specific antisense molecules AMO-21 was able to rescue Sp1 and hERG inhibition caused by As2O3. Consistently, phosphorylation of NF-κB, the upstream regulatory factor of miR-21, was significantly up-regulated by As2O3 . This finding revealed that regulation of the NF-κB-miR-21-Sp1 signalling pathway is a novel mechanism for As2O3-induced hERG inhibition. Meanwhile, the expression of Hsp90 and hERG was rescued by transfection with AMO-23a. And the hERG channel inhibition induced by As2O3 was rescued after being transfected with AMO-23a, which may be a molecular mechanism for the role of As2O3 in hERG trafficking deficiency. In brief, our study revealed that miR-21 and miR-23a are involved in As2O3-induced hERG deficiency at transcriptional and transportational levels. This discovery may provide a novel mechanism of As2O3-induced hERG channel deficiency, and these miRNAs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

The rescuable function and mechanism of resveratrol on As2O3-induced hERG K⁺ channel deficiency.

Arsenic trioxide (As2O3) is used to treat acute promyelocytic leukemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the human ether-a-go-go-related gene (hERG) current via disturbing its trafficking to cellular membrane. This study aimed to investigate whether the As2O3-insulted hERG channel can be rescued by resveratrol, a recognized cardioprotective agent. The whole-cell patch clamp technique was used to record the hERG current and action potential duration. Co-immunoprecipitation and Western blot assay were applied to determine the function of hERG-Hsp70/Hsp90 chaperone complexes and the expression alteration of protein-folding-related proteins, respectively. Compared with treatment of As2O3 alone, co-treatment with resveratrol successfully restored the current and surface expression of hERG and obviously shortened action potential duration in guinea pig ventricular myocytes. Further experiments demonstrate that resveratrol relieved As2O3-caused endoplasmic reticulum (ER) stress by restoring the function of hERG-Hsp70/Hsp90 chaperone complexes and downregulating the protein expression of ER chaperone proteins (calnexin and calreticulin) and activating transcription factor 6. In conclusion, resveratrol was able to rescue the trafficking deficiency and relieve the ER stress (ERS). Our findings suggest that resveratrol has a potential effect to alleviate the adverse effect of As2O3 on cardiotoxicity.

Berberine induces hERG channel deficiency through trafficking inhibition.

AIMS: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the IKr, which plays an essential role in repolarization of action potentials. hERG channels are targeted by various pro-arrhythmic drugs. Berberine (BBR) was previously found to acutely inhibit hERG currents and prolong action potential duration. The present study aimed to determine long-term effects of BBR on the expression of 135kDa/155kDa hERG and the mechanism. METHODS AND RESULTS: hERG expression was assessed by western blot. Mature hERG (155 kDa) was reduced, whereas ER-located hERG (135 kDa) was increased by BBR. This indicated that hERG was restricted to the ER and that BBR disrupted channel trafficking. To determine the mechanism of trafficking inhibition, we performed western blot and immunoprecipitation to test folding of hERG by assessing interaction between hERG and Hsp90/Hsp70. Both the expression of Hsp90 and its interaction with hERG were strongly decreased by BBR. These data suggest that BBR reduces channel folding to induce trafficking inhibition. Western blot and confocal imaging were used to further detect whether the unfolded protein response (UPR) was activated. Active ATF6, a marker of the UPR, was activated by BBR. Calnexin and calreticulin, chaperones that are activated by ATF6 to assist channel folding, were also elevated and increasingly colocalized with hERG. These data also demonstrate that the UPR was activated. Immunoprecipitation and western blot assays were performed after BBR treatment to examine ubiquitination and degradation, common endpoints of the UPR. We found that the ER-restricted hERG was ubiquitinized and degraded in the lysosomes and proteasomes. CONCLUSION: Our study demonstrates that BBR induces hERG channel deficiency by inhibiting channel trafficking after incubation for 24h. Trafficking inhibition activated the UPR, and the ER-restricted hERG was ubiquitinized and degraded in lysosomes and proteasomes.

The effects of transient starvation persist through direct interactions between CaMKII and ether-a-go-go K+ channels in C. elegans males.

Prolonged nutrient limitation has been extensively studied due to its positive effects on life span. However, less is understood of how brief periods of starvation can have lasting consequences. In this study, we used genetics, biochemistry, pharmacology and behavioral analysis to show that after a limited period of starvation, the synthesis of egl-2-encoded ether-a-go-go (EAG) K+ channels and its C-terminal modifications by unc-43-encoded CaMKII have a perduring effect on C. elegans male sexual behavior. EGL-2 and UNC-43 interactions, induced after food deprivation, maintain reduced excitability in muscles involved in sex. In young adult males, spastic contractions occur in cholinergic-activated sex muscles that lack functional unc-103-encoded ERG-like K+ channels. Promoting EGL-2 and UNC-43 interactions in unc-103 mutant adult males by starving them for a few hours reduce spastic muscle contractions over multiple days. Although transient starvation during early adulthood has a hormetic effect of suppressing mutation-induced muscle contractions, the treatment reduces the ability of young wild-type (WT) males to compete with well-fed cohorts in siring progeny.

Disruption of the ether-a-go-go K+ channel gene BEC1/KCNH3 enhances cognitive function.

The K+ channel, one of the determinants for neuronal excitability, is genetically heterogeneous, and various K+ channel genes are expressed in the CNS. The therapeutic potential of K+ channel blockers for cognitive enhancement has been discussed, but the contribution each K+ channel gene makes to cognitive function remains obscure. BEC1 (KCNH3) is a member of the K+ channel superfamily that shows forebrain-preferential distribution. Here, we show the critical involvement of BEC1 in cognitive function. BEC1 knock-out mice performed behavioral tasks related to working memory, reference memory, and attention better than their wild-type littermates. Enhanced performance was also observed in heterozygous mutants. The knock-out mice had neither the seizures nor the motor dysfunction that are often observed in K+ channel-deficient mice. In contrast to when it is disrupted, overexpression of BEC1 in the forebrain caused the impaired performance of those tasks. It was also found that altering BEC1 expression could change hippocampal neuronal excitability and synaptic plasticity. The results indicate that BEC1 may represent the first K+ channel that contributes preferentially and bidirectionally to cognitive function.

Not all hERG pore domain mutations have a severe phenotype: G584S has an inactivation gating defect with mild phenotype compared to G572S, which has a dominant negative trafficking defect and a severe phenotype.

INTRODUCTION: Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification. METHODS AND RESULTS: We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584S-hERG channels. CONCLUSIONS: hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.

[Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R].

OBJECTIVE: Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R. METHODS: To study the function of the gene eag of Bacillus anthracis vaccine strain A16R, according to the sequence of Bacillus anthracis Ames strain, we designed primers and constructed a recombinant plasmid by the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of eag cloned in tandem in pKSV7. We introduced the recombinant into A16R by electroporation and screened the mutant using the principle of homologous recombination. We checked the mutant using the PCR and proteomics. RESULTS: We constructed the recombinant plasmid successfully and got the eag deletion mutant. PCR results showed the gene eag was deleted; SDS PAGE showed evident differences between prime strain and mutant strain. Two-dimensional gel electrophoresis results displayed three EA1 protein points of prime strain were absent in the mutant strain. CONCLUSION: We constructed eag deletion mutant of Bacillus anthracis vaccine strain A16R. This research will be helpful to study the functions of eag gene and the other important genes of Bacillus anthracis.

Deficient zebrafish ether-à-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants.

BACKGROUND: Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking. METHODS AND RESULTS: In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish. CONCLUSIONS: With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.

Nonsense mutations in hERG cause a decrease in mutant mRNA transcripts by nonsense-mediated mRNA decay in human long-QT syndrome.

BACKGROUND: Long-QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of the LQT2 mutations result in premature termination codons. Degradation of premature termination codon-containing mRNA transcripts by nonsense-mediated mRNA decay is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of nonsense-mediated mRNA decay in LQT2 mutations has not been explored. METHODS AND RESULTS: We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared with that of the wild-type allele. The decrease in mutant mRNA also was observed in the LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNA interference-mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to nonsense-mediated mRNA decay. CONCLUSIONS: These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by nonsense-mediated mRNA decay rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by nonsense-mediated mRNA decay is an important mechanism in LQT2 patients with nonsense or frameshift mutations.