Expression Of Kv1 Research Articles

The antipsychotic chlorpromazine reduces neuroinflammation by inhibiting microglial voltage-gated potassium channels.

Neuroinflammation, the result of microglial activation, is associated with the pathogenesis of a wide range of psychiatric and neurological disorders. Recently, chlorpromazine (CPZ), a dopaminergic D2 receptor antagonist and schizophrenia therapy, was proposed to exert antiinflammatory effects in the central nervous system. Here, we report that the expression of Kv1.3 channel, which is abundant in T cells, is upregulated in microglia upon infection, and that CPZ specifically inhibits these channels to reduce neuroinflammation. In the mouse medial prefrontal cortex, we show that CPZ lessens Kv1.3 channel activity and reduces proinflammatory cytokine production. In mice treated with LPS, we found that CPZ was capable of alleviating both neuroinflammation and depression-like behavior. Our findings suggest that CPZ acts as a microglial Kv1.3 channel inhibitor and neuroinflammation modulator, thereby exerting therapeutic effects in neuroinflammatory psychiatric/neurological disorders.

Involvement of KV3.4 Channel in Parkinson's Disease: A Key Player in the Control of Midbrain and Striatum Differential Vulnerability during Disease Progression?

Parkinson's disease (PD), the second most common neurodegenerative disease in the elderly, is characterized by selective loss of dopaminergic neurons and accumulation of α-synuclein (α-syn), mitochondrial dysfunction, Ca2+ dyshomeostasis, and neuroinflammation. Since current treatments for PD merely address symptoms, there is an urgent need to identify the PD pathophysiological mechanisms to develop better therapies. Increasing evidence has identified KV3.4, a ROS-sensitive KV channel carrying fast-inactivating currents, as a potential therapeutic target against neurodegeneration. In fact, it has been hypothesized that KV3.4 channels could play a role in PD etiopathogenesis, controlling astrocytic activation and detrimental pathways in A53T mice, a well-known model of familial PD. Here, we showed that the A53T midbrain, primarily involved in the initial phase of PD pathogenesis, displayed an early upregulation of the KV3.4 channel at 4 months, followed by its reduction at 12 months, compared with age-matched WT. On the other hand, in the A53T striatum, the expression of KV3.4 remained high at 12 months, decreasing thereafter, in 16-month-old mice. The proteomic profile highlighted a different detrimental phenotype in A53T brain areas. In fact, the A53T striatum and midbrain differently expressed neuroprotective/detrimental pathways, with the variation of astrocytic p27kip1, XIAP, and Smac/DIABLO expression. Of note, a switch from protective to detrimental phenotype was characterized by the upregulation of Smac/DIABLO and downregulation of p27kip1 and XIAP. This occurred earlier in the A53T midbrain, at 12 months, compared with the striatum proteomic profile. In accordance, an upregulation of Smac/DIABLO and a downregulation of p27kip1 occurred in the A53T striatum only at 16 months, showing the slowest involvement of this brain area. Of interest, HIF-1α overexpression was associated with the detrimental profile in midbrain and its major vulnerability. At the cellular level, patch-clamp recordings revealed that primary A53T striatum astrocytes showed hyperpolarized resting membrane potentials and lower firing frequency associated with KV3.4 ROS-dependent hyperactivity, whereas primary A53T midbrain astrocytes displayed a depolarized resting membrane potential accompanied by a slight increase of KV3.4 currents. Accordingly, intracellular Ca2+ homeostasis was significantly altered in A53T midbrain astrocytes, in which the ER Ca2+ level was lower than in A53T striatum astrocytes and the respective littermate controls. Collectively, these results suggest that the early KV3.4 overexpression and ROS-dependent hyperactivation in astrocytes could take part in the different vulnerabilities of midbrain and striatum, highlighting astrocytic KV3.4 as a possible new therapeutic target in PD.

Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10μM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5μM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Ubiquitination is involved in PKC-mediated degradation of cell surface Kv1.5 channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K+ current (IKur) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

Oxidative stress-related cellular aging causes dysfunction of the Kv3.1/KCNC1 channel reverted by melatonin.

The voltage-gated Kv3.1/KCNC1 channel is abundantly expressed in fast-spiking principal neurons and GABAergic inhibitory interneurons throughout the ascending auditory pathway and in various brain regions. Inactivating mutations in the KCNC1 gene lead to forms of epilepsy and a decline in the expression of the Kv3.1 channel is involved in age-related hearing loss. As oxidative stress plays a fundamental role in the pathogenesis of epilepsy and age-related hearing loss, we hypothesized that an oxidative insult might affect the function of this channel. To verify this hypothesis, the activity and expression of endogenous and ectopic Kv3.1 were measured in models of oxidative stress-related aging represented by cell lines exposed to 100 mM d-galactose. In these models, intracellular reactive oxygen species, thiobarbituric acid reactive substances, sulfhydryl groups of cellular proteins, and the activity of catalase and superoxide dismutase were dysregulated, while the current density of Kv3.1 was significantly reduced. Importantly, the antioxidant melatonin reverted all these effects. The reduction of function of Kv3.1 was not determined by direct oxidation of amino acid side chains of the protein channel or reduction of transcript or total protein levels but was linked to reduced trafficking to the cell surface associated with Src phosphorylation as well as metabolic and endoplasmic reticulum stress. The data presented here specify Kv3.1 as a novel target of oxidative stress and suggest that Kv3.1 dysfunction might contribute to age-related hearing loss and increased prevalence of epilepsy during aging. The pharmacological use of the antioxidant melatonin can be protective in this setting.

MiR-21-5p alleviates trigeminal neuralgia in rats through down-regulation of voltage-gated potassium channel Kv1.1.

Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN. A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.

Kv1.3 Blockade Alleviates White Matter Injury through Reshaping M1/M2 Phenotypes via the NF-κB Signaling Pathway after Intracerebral Hemorrhage.

White matter injury (WMI) in basal ganglia usually induces long-term disability post intracerebral hemorrhage (ICH). Kv1.3 is an ion channel expressed in microglia and induces neuroinflammation after ICH. Here, we investigated the functions and roles of Kv1.3 activation-induced inflammatory response in WMI and the Kv1.3 blockade effect on microglia polarization after ICH. Mice ICH model was constructed by autologous blood injection. The expression of Kv1.3 was measured using immunoblot, real-time quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays. Then, the effect of administration of 5-(4-Phenoxybutoxy) psoralen (PAP-1), a selectively pharmacological Kv1.3 blocker, was investigated using open field test (OFT) and basso mouse score (BMS). RT-qPCR, immunoblot, and enzyme-linked immunosorbent assay (ELISA) were taken to elucidate the expression of pro-inflammatory or anti-inflammatory factors around hematoma. PAP-1's function in regulating microglia polarization was investigated using immunoblot, RT-qPCR, and immunostaining assays. The downstream PAP-1 signaling pathway was determined by RT-qPCR and immunoblot. Kv1.3 expression was increased in microglia around the hematoma significantly after ICH. PAP-1 markedly improved neurological outcomes and the WMI by reducing pro-inflammatory cytokine accumulation and upregulating anti-inflammatory factors. Mechanistically, PAP-1 reduces NF-κB p65 and p50 activation, thus facilitating microglia polarization into M2-like microglia, which exerts this beneficial effect. PAP-1 reduced pro-inflammatory cytokines accumulation and increased anti-inflammatory factors by facilitating M2-like microglia polarization via the NF-κB signaling pathway. Thus, the current study shows that the Kv1.3 blockade is capable of ameliorating WMI by facilitating M2-like phenotype microglia polarization after ICH.

Environmental Enrichment Improves the Recognition Memory in Adult Mice Following Social Isolation via Downregulation of Kv4.2 Potassium Channels.

Recognition memory is a cognitive process that enables us to distinguish familiar objects and situations from new items, which is essential for mammalian survival and adaptation to a changing environment. Social isolation (SI) has been implicated as a detrimental factor for recognition memory. The medial prefrontal cortex (mPFC) has been shown to carry information concerning the relative familiarity of individual stimuli, and modulating neuronal function in this region may contribute to recognition memory. The present study aimed to investigate the neuronal mechanisms in the mPFC of environmental enrichment (EE) on recognition memory in adult mice following SI. Mice were assigned into three groups: control, SI, and SI + EE groups. Novel location recognition (NLR) and novel object recognition (NOR) tests were performed to evaluate the recognition memory. The levels of Kv4 channels were assessed by qRT-PCR and western blotting. The effects of SI and SI + EE on the excitability of pyramidal neurons in the mPFC were measured using whole-cell recording. We found that SI led to a reduction in the excitability of pyramidal neurons. Specifically, we have identified that the reduction in the firing activity of pyramidal neurons resulted from alterations in the function and expression of Kv4.2 channels. Furthermore, EE regulated Kv4.2 channels, normalized the activity of pyramidal neurons, and restored the behavioral deficits following SI. Thus, the roles of Kv4.2 channels in excitability of pyramidal neurons suggest that the Kv4.2 channels present a promising therapeutic target for recognition memory impairment.

Altered placental ion channel gene expression in preeclamptic high-altitude pregnancies.

High-altitude (>2,500 m) residence increases the risk of pregnancy vascular disorders such as fetal growth restriction and preeclampsia, each characterized by impaired placental function. Genetic attributes of highland ancestry confer relative protection against vascular disorders of pregnancy at high altitudes. Although ion channels have been implicated in placental function regulation, neither their expression in high-altitude placentas nor their relationship to high-altitude preeclampsia has been determined. Here, we measured the expression of 26 ion-channel genes in placentas from preeclampsia cases and normotensive controls in La Paz, Bolivia (3,850 m). In addition, we correlated gene transcription to maternal and infant ancestry proportions. Gene expression was assessed by PCR, genetic ancestry evaluated by ADMIXTURE, and ion channel proteins localized by immunofluorescence. In preeclamptic placentas, 11 genes were downregulated (ABCC9, ATP2A2, CACNA1C, KCNE1, KCNJ8, KCNK3, KCNMA1, KCNQ1, KCNQ4, PKD2, and TRPV6) and two were upregulated (KCNQ3 and SCNN1G). KCNE1 expression was positively correlated with high-altitude Amerindian ancestry and negatively correlated with non-high altitude. SCNN1G was negatively correlated with African ancestry, despite minimal African admixture. Most ion channels were localized in syncytiotrophoblasts (Cav1.2, TRPP2, TRPV6, and Kv7.1), whereas expression of Kv7.4 was primarily in microvillous membranes, Kir6.1 in chorionic plate and fetal vessels, and MinK in stromal cells. Our findings suggest a role for differential placental ion channel expression in the development of preeclampsia. Functional studies are needed to determine processes affected by these ion channels in the placenta and whether therapies directed at modulating their activity could influence the onset or severity of preeclampsia.

Effect of Zhenwu Decoction on electrical remodeling of cardiomyocytes in heart failure via I_(to)/Kv channels

This study aimed to investigate the underlying mechanism of Zhenwu Decoction in the treatment of heart failure by regulating electrical remodeling through the transient outward potassium current(I_(to))/voltage-gated potassium(Kv) channels. Five normal SD rats were intragastrically administered with Zhenwu Decoction granules to prepare drug-containing serum, and another seven normal SD rats received an equal amount of distilled water to prepare blank serum. H9c2 cardiomyocytes underwent conventional passage and were treated with angiotensin Ⅱ(AngⅡ) for 24 h. Subsequently, 2%, 4%, and 8% drug-containing serum, simvastatin(SIM), and BaCl_2 were used to interfere in H9c2 cardiomyocytes for 24 h. The cells were divided into a control group [N, 10% blank serum + 90% high-glucose DMEM(DMEM-H)], a model group(M, AngⅡ + 10% blank serum + 90% DMEM-H), a low-dose Zhenwu Decoction-containing serum group(Z1, AngⅡ + 2% drug-containing serum of Zhenwu Decoction + 8% blank serum + 90% DMEM-H), a medium-dose Zhenwu Decoction-containing serum group(Z2, AngⅡ + 4% drug-containing serum of Zhenwu Decoc-tion + 6% blank serum + 90% DMEM-H), a high-dose Zhenwu Decoction-containing serum group(Z3, AngⅡ + 8% drug-containing serum of Zhenwu Decoction + 2% blank serum + 90% DMEM-H), an inducer group(YD, AngⅡ + SIM + 10% blank serum + 90% DMEM-H), and an inhibitor group(YZ, AngⅡ + BaCl_2 + 10% blank serum + 90% DMEM-H). The content of ANP in cell extracts of each group was detected by ELISA. The relative mRNA expression levels of ANP, Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 were detected by real-time quantitative PCR. The protein expression of Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 was detected by Western blot. I_(to) was detected by the whole cell patch-clamp technique. The results showed that Zhenwu Decoction at low, medium, and high doses could effectively reduce the surface area of cardiomyocytes. Compared with the M group, the Z1, Z2, Z3, and YD groups showed decreased ANP content and mRNA level, increased protein and mRNA expression of Kv4.2, Kv4.3, DPP6, and KChIP2, and decreased protein and mRNA expression of Kv1.4, and the aforementioned changes were the most notable in the Z3 group. Compared with the N group, the Z1, Z2, and Z3 groups showed significantly increased peak current and current density of I_(to). The results indicate that Zhenwu Decoction can regulate myocardial remodeling and electrical remodeling by improving the expression trend of Kv1.4, Kv4.2, Kv4.3, KChIP2, and DPP6 proteins and inducing I_(to) to regulate Kv channels, which may be one of the mechanisms of Zhenwu Decoction in treating heart failure and related arrhythmias.

Human leukocyte antigen F-associated transcript 10 regulates the IKs potassium channel by competing for Kv7.1 ubiquitination.

The protein expression and function changes from the slow-delayed rectifying K+ current, IKs, are tightly associated with ventricular cardiac arrhythmias. Human leukocyte antigen F-associated transcript 10 (FAT10), a member of the ubiquitin-like-modifier family, exerts a protective effect against myocardial ischaemia. However, whether or how FAT10 influences the function of IKs remains unclear. Here, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Fat10 knockout HEK293 (Fat10-/-) cells through CRISPR-Cas9 technology were used to evaluate the novel modulation of FAT10 in IKs function. Patch-clamp studies showed that the overexpression of FAT10 significantly enhanced the current density of IKs both in hiPSC-CMs and HEK293-Fat10-/- cells. In addition, a shortened action potential duration (APD) was seen from hiPSC-CMs transfected with the ad-Fat10 virus. Then, a series of molecular approaches from neonatal rat cardiomyocytes, H9C2 cells and HEK293 cells were used to determine the regulatory mechanism of FAT10 in IKs. First, western blot assays indicated that the expression of Kv7.1, the alpha-subunit of IKs, was increased when FAT10 was overexpressed. Furthermore, immunofluorescence and co-immunoprecipitation assays demonstrated that FAT10 could interact with Kv7.1. Notably, FAT10 impedes Kv7.1 ubiquitination and degradation, thereby stabilizing its expression. Finally, a hypoxia model of hiPSC-CMs was established, and the overexpression of FAT10 showed a protective effect against hypoxia-induced decreases in the current density of IKs. Taken together, these findings revealed a novel role of FAT10 in the regulation of the IKs potassium channel by competing for Kv7.1 ubiquitination, which provides a new electrophysiological insight that FAT10 could modulate Kv7.1. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.

Expression of Kv4.2 and Kv4.3 potassium channels in human umbilical veins from normal, diabetic and hypertensive pregnancies

Background/Aim. A substantial line of evidence indicates that Kv4.2 and Kv4.3 channels are the major components of rapid transient-outward potassium currents (A-type cur-rents). It is speculated that those currents may be involved in the maintenance of the membrane potential, as well as in the regulation of propagation and frequency of action potentials. However, very little is known about the presence and function of A-type currents in human vascular smooth muscles such as the human umbilical vein (HUV). Bearing in mind its crucial role in the proper fetal oxygenation, the aim of the study was to determine whether Kv4.2 and Kv4.3 potassium channels are present in HUV smooth muscle and to investigate potential alterations of their expression during maternal pathological conditions such as gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). Methods. Healthy, diabetic, and hypertensive pregnancies were subjects of this investigation. Each group consisted of 6 HUV samples obtained from 6 normal pregnancies, 6 pregnancies with GDM, and 6 with PIH. After pharmacology analysis, immunohistochemistry (IH) and Western blot were performed. Results. IH revealed similar expression patterns of both, Kv4.2 and Kv4.3 subunits in HUV smooth muscle in all groups of patients. Results obtained by Western blot were in agreement with IH staining. The expression of Kv4.2 and Kv4.3 subunits were not significantly different between the groups. Conclusion. Collective-ly, this is the first study that demonstrated the presence of Kv4.2 and Kv4.3 potassium channels in the HUV smooth muscle and their preservation during GDM and PIH pregnancies. These channels are most likely major components of rapid A-type currents that may be relevant for maternal-fetus blood flow and hence fetal development. In addition, they may represent sensors for detecting hemodynamic and/or metabolic changes in the local environment.

Cardiac-specific knockdown of Bhlhe40 attenuates angiotensin II (Ang II)-Induced atrial fibrillation in mice.

Atrial fibrosis and atrial inflammation are associated with the pathogenesis of atrial fibrillation (AF). Basic helix–loop–helix family member E40 (Bhlhe40) is an important transcription factor, which is involved in tumors, inflammation, apoptosis, viral infection, and hypoxia. However, its role and molecular mechanism in AF remain unclear. In this study, a mouse model of AF was induced by Ang II infusion. The atrial diameter was evaluated using echocardiography. Induction and duration of AF were measured by programmed electrical stimulation. Atrial structural remodeling was detected using routine histologic examinations. Our results showed that Bhlhe40 was significantly upregulated in angiotensin II (Ang II)-stimulated atrial cardiomyocytes and atrial tissues and in tissues from patients with AF. Cardiac-specific knockdown of Bhlhe40 in mice by a type 9 recombinant adeno-associated virus (rAAV9)-shBhlhe40 significantly ameliorated Ang II-induced atrial dilatation, atrial fibrosis, and atrial inflammation, as well as the inducibility and duration of AF. Mechanistically, cardiac-specific knockdown of Bhlhe40 attenuated Ang II-induced activation of NF-κB/NLRP3, TGF-1β/Smad2 signals, the increased expression of CX43, and the decreased expression of Kv4.3 in the atria. This is the first study to suggest that Bhlhe40 is a novel regulator of AF progression, and identifying Bhlhe40 may be a new therapeutic target for hypertrophic remodeling and heart failure.

Crim1 inhibits angiotensin II-induced hypertrophy and preserves Kv4.2 expression in cardiomyocytes.

Objective(s):Angiotensin II (Ang II) plays a key role in the regulation of myocardial hypertrophy via downstream cysteine-rich transmembrane bone morphogenetic protein regulator 1 (Crim1). However, it is still unclear whether Crim1 is involved in ionic channel remodeling. The study aimed to explore the effects of Crim1 on transient outward potassium current (Ito) and Kv4.2 (the main subunit of Ito channel) expression in hypertrophic ventricular cardiomyocytes. Materials and Methods:The ventricular cardiomyocytes were isolated from the neonatal rats. Hypertrophy was induced by Ang II. Crim1 expression was modulated by using adenovirus transfection. The expression of myosin heavy chain beta (β-MHC), Crim1, and Kv4.2 was determined by RT-qPCR and western blot. The cellular surface area was assessed using Image J software. Ito was recorded by the whole-cell patch clamp technique. Results:Ang II-induced hypertrophy in cardiomyocytes was identified by their larger cellular surface area and higher mRNA expression of β-MHC. Ang II significantly decreased the expression of Crim1 and Kv4.2 and reduced Ito current density. However, Crim1 overexpression abolished the Ang II-induced hypertrophy and preserved the expression of Kv4.2 and Ito current density. Conclusion:Crim1 overexpression inhibits Ang II-induced hypertrophy and preserves Ito current density via up-regulating Kv4.2 in ventricular cardiomyocytes from neonatal rats. Crim1 could have a role in the development of ventricular arrhythmia in hypertrophic hearts.

Connexin 37 Regulates the Kv1.3 Pathway and Promotes the Development of Atherosclerosis.

Objective To investigate the mechanism of Connexin 37 (Cx37) and Kv1.3 pathways in atherosclerosis (AS). Methods ApoE−/− mice were given a high-fat diet to establish atherosclerosis (AS) model, and macrophages in mice were isolated and extracted to transfect Cx37 vectors with silencing or overexpressing, and Kv1.3 pathway blockers were used to inhibit the pathway activity. The indexes of body weight, blood glucose, and blood lipid of mice were collected. The protein and mRNA expression levels of Cx37 and Kv1.3 were detected by reverse transcription-PCR (RT-PCR), Western blot, and immunofluorescence technique. Oil red O staining was used to observe plaque area. Masson staining was used to detect collagen content. The concentrations of chemokine CCL7 were quantified using the ELISA kits. CCK8 was used to detect cell proliferation. Results Cx37 and Kv1.3 were highly expressed in macrophages of AS mice, and the expression of Kv1.3 and CCL7 decreased after Cx37 was silenced, and the proliferation of macrophages was also decreased. Wild-type mice and AS model mice were treated with Cx37 overexpression vectors and Kv1.3 pathway blocking, and it was found that Cx37 overexpression could improve the blood lipid and blood glucose levels and increase the area of AS in AS mice. However, blocking the activity of Kv1.3 pathway can reduce the levels of blood lipid and blood glucose, increase the body weight of mice, and reduce the area of AS mice. Blocking the activity of Kv1.3 pathway can slow down the plaque development of AS mice and make its indexes close to wild-type mice. And the use of Kv1.3 pathway blockers on the basis of overexpression of Cx37 indicated that inhibition of Kv1.3 pathway activity did not affect the expression of Cx37, but could inhibit the collagen content in the plaque area of AS mice, inhibit the expression of chemokine CCL7, and reverse the effect of Cx37 overexpression. Conclusion Cx37 can improve the activity of macrophages by regulating the expression of chemokines and the activity of Kv1.3 pathway in AS mice, and enrich macrophages in inflammatory tissues and expand the area of plaque formation.

K+ channel Kv4.1 is expressed in the nociceptors/secondary nociceptive neurons and participates in pain regulation.

Kv4 channels are key components controlling neuronal excitability at membrane potentials below action potential thresholds. It remains elusive whether Kv4.1 participates in pain regulation. We raised a Kv4.1 antibody to map Kv4.1+ neurons in the superficial dorsal horn of the spinal cord and dorsal root ganglion (DRG) of rats. Behavioural, biochemical and immunohistochemical methods were used to examine whether the activity of Kv4.1+ neurons or Kv4.1 expression level is altered after peripheral nerve injury. In lamina I of the spinal cord, Kv4.1 immunoreactivity (IR) was detected in neurokinin-1 receptor positive (NK1R)+ projection neurons (the secondary nociceptive neurons) and NK1R+ excitatory interneurons. Kv4.1, KChIP2 and DPP10 were co-expressed in these neurons. Peripheral nerve injury evoked by lumbar spinal nerve ligation (SNL) immediately induced phosphorylated extracellular regulated protein kinase (pERK, an indicator of enhanced neuronal activity) in lamina I Kv4.1+ neurons and lamina II Kv4.2/Kv4.3+ neurons of the spinal cord. Furthermore, Kv4.1 appeared in 59.9% of DRG neurons with variable sizes. Kv4.1 mRNA and protein levels in DRG neurons were gradually decreased after SNL. Following intrathecal injection of Kv4.1 antisense oligodeoxynucleotide (ASO) into naive rats, Kv4.1 protein level was reduced in the DRG, and mechanical but not thermal hypersensitivity was induced. Kv4.1 appears in the secondary nociceptive neurons, and peripheral nerve injury increases the activity of these neurons. Kv4.1 expression in DRG neurons (including half of the nociceptors) is gradually reduced after peripheral nerve injury, and knockdown of Kv4.1 in DRG neurons induces pain. Thus, Kv4.1 participates in pain regulation. Based on the expression of Kv4.1 and Kv4.3 in the nociceptors, Kv4.1 in the secondary nociceptive neurons, Kv4.1 in spinal lamina I excitatory interneurons that regulate the activity of the secondary nociceptive neurons, as well as Kv4.2 and Kv4.3 in spinal lamina II excitatory interneurons that also regulate the activity of the secondary nociceptive neurons, developing Kv4 activators or genetic manipulation to increase Kv4 channel activity in these pain-related Kv4+ neurons will be useful in future pain therapeutics.

Microglia-derived PDGFB promotes neuronal potassium currents to suppress basal sympathetic tonicity and limit hypertension

Although many studies have addressed the regulatory circuits affecting neuronal activities, local non-synaptic mechanisms that determine neuronal excitability remain unclear. Here, we found that microglia prevented overactivation of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) at steady state. Microglia constitutively released platelet-derived growth factor (PDGF) B, which signaled via PDGFRα on neuronal cells and promoted their expression of Kv4.3, a key subunit that conducts potassium currents. Ablation of microglia, conditional deletion of microglial PDGFB, or suppression of neuronal PDGFRα expression in the PVN elevated the excitability of pre-sympathetic neurons and sympathetic outflow, resulting in a profound autonomic dysfunction. Disruption of the PDGFBMG-Kv4.3Neuron pathway predisposed mice to develop hypertension, whereas central supplementation of exogenous PDGFB suppressed pressor response when mice were under hypertensive insult. Our results point to a non-immune action of resident microglia in maintaining the balance of sympathetic outflow, which is important in preventing cardiovascular diseases.

KV 1/D-type potassium channels inhibit the excitability of bronchopulmonary vagal afferent nerves.

The KV1/D‐type potassium current (I D) is an important determinant of neuronal excitability. This study explored whether and how I D 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, I D channel‐forming KV1.1, KV1.2 and/or KV1.6 α‐subunits, with the expression of KV1.6 being most prevalent. Patch‐clamp recordings showed that I D, 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 I D 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, I D 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 I D 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 (I D) is an important determinant of neuronal excitability.Nearly all bronchopulmonary nodose afferent neurons in the mouse express I D and the transcripts of α‐DTX‐sensitive, I D channel‐forming KV1.1, KV1.2 and/or KV1.6 α‐subunits.Inhibition of I D 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 I D 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.

Effect of allisartan on blood pressure and left ventricular hypertrophy through Kv1.5 channels in hypertensive rats

ABSTRACT Background The objective of the present work was to study the anti-hypertensive effect of allisartan on blood pressure (BP) and in facilitating left ventricular remodeling through voltage-gated potassium channels (Kv) 1.5 channels. Methods A total of 30 SD rats were randomly divided into sham operation group, hypertension control group, and allisartan treatment group. Hypertension was induced by renal artery stenosis. The animals of treatment group were administered with allisartan once a day at a dose of 30 mg/kg body weight through an oral gavage for 4 weeks. The heart function of animals post 4 weeks of treatment was evaluated by echocardiography, and the degree of ventricular hypertrophy and cardiomyocyte hypertrophy were evaluated by histomorphology. The expression of Kv1.5 is detected by real-time quantitative polymerase chain reaction while Western blotting was used to detect the protein expression. Results Four weeks after renal artery stenosis, a significant difference was observed in the whole heart ratio, left heart ratio, and cardiomyocyte area between allisartan treatment group and the hypertension control group (P< .01). A significant decrease in BP of allisartan treatment group compared to hypertension control group (P< .01) was observed. The expression of Kv1.5 mRNA was increased significantly (P< .01) in allisartan treatment group compared to hypertension control group. Western blot analysis also confirmed the increased expression of Kv1.5 channel. Conclusion The results showed that allisartan lowers BP and improves left ventricular remodeling through increased expression of Kv1.5 mRNA.