Level Of Kv1 Research Articles

Age-related changes in Kv4/Shal and Kv1/Shaker expression in Drosophila and a role for reactive oxygen species.

Age-related changes in ion channel expression are likely to affect neuronal signaling. Here, we examine how age affects Kv4/Shal and Kv1/Shaker K+ channel protein levels in Drosophila. We show that Kv4/Shal protein levels decline sharply from 3 days to 10 days, then more gradually from 10 to 40 days after eclosion. In contrast, Kv1/Shaker protein exhibits a transient increase at 10 days that then stabilizes and eventually declines at 40 days. We present data that begin to show a relationship between reactive oxygen species (ROS), Kv4/Shal, and locomotor performance. We show that Kv4/Shal levels are negatively affected by ROS, and that over-expression of Catalase or RNAi knock-down of the ROS-generating enzyme, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase (NOX), can attenuate the loss of Kv4/Shal protein. Finally, we compare levels of Kv4.2 and Kv4.3 in the hippocampus, olfactory bulb, cerebellum, and motor cortex of mice aged 6 weeks and 1 year. While there was no global decline in Kv4.2/4.3 that parallels what we report in Drosophila, we did find that Kv4.2/4.3 are differentially affected in various brain regions; this survey of changes may help inform mammalian studies that examine neuronal function with age.

Expression, Cellular and Subcellular Localisation of Kv4.2 and Kv4.3 Channels in the Rodent Hippocampus.

The Kv4 family of voltage-gated K+ channels underlie the fast transient (A-type) outward K+ current. Although A-type currents are critical to determine somato-dendritic integration in central neurons, relatively little is known about the precise subcellular localisation of the underlying channels in hippocampal circuits. Using histoblot and immunoelectron microscopic techniques, we investigated the expression, regional distribution and subcellular localisation of Kv4.2 and Kv4.3 in the adult brain, as well as the ontogeny of their expression during postnatal development. Histoblot demonstrated that Kv4.2 and Kv4.3 proteins were widely expressed in the brain, with mostly non-overlapping patterns. During development, levels of Kv4.2 and Kv4.3 increased with age but showed marked region- and developmental stage-specific differences. Immunoelectron microscopy showed that labelling for Kv4.2 and Kv4.3 was differentially present in somato-dendritic domains of hippocampal principal cells and interneurons, including the synaptic specialisation. Quantitative analyses indicated that most immunoparticles for Kv4.2 and Kv4.3 were associated with the plasma membrane in dendritic spines and shafts, and that the two channels showed very similar distribution patterns in spines of principal cells and along the surface of granule cells. Our data shed new light on the subcellular localisation of Kv4 channels and provide evidence for their non-uniform distribution over the plasma membrane of hippocampal neurons.

Long noncoding RNA MALAT1 downregulates cardiac transient outward potassium current by regulating miR-200c/HMGB1 pathway.

The dysregulation of long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) participates in the remodeling of electrophysiological/ion channel in cardiomyocytes during arrhythmia. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is reported to be highly expressed in myocardial ischemia-reperfusion injury and offsets cardioprotective effects of fentanyl. However, the roles of MALAT1 and its related miRNAs during arrhythmia are poorly understood. In this study, the overexpression of MALAT1 was firstly indicated in cardiomyocytes from arrhythmic model rats. After downregulation of MALAT1 by RNA interference, transient outward potassium current (Ito), peak current density, and the levels of Kv4.2 and Kv4.3 channel proteins were increased in rat cardiomyocytes. Then, miR-200c was predicted and convinced to be a direct target of MALAT1, and high-mobility group box 1 (HMGB1) was verified to be a target of miR-200c during arrhythmia. HMGB1 expression reduced by the knockdown of MALAT1 was further decreased by miR-200c overexpression. In addition, cardiac Ito, peak current density, and the levels of Kv4.2 and Kv4.3 in arrhythmic model rats were detected to be negatively correlated with the expression of HMGB1, and to be positively with miR-200c expression. Taken together, these results suggested that MALAT1 may act as a competing endogenous RNA for miR-200c to upregulate the expression of HMGB1 and downregulate cardiac Ito.

Abstract 285: Endoplasmic Reticulum Stress Induces Mitochondrial ROS Overproduction and Contributes to Electrical Remodeling in Heart Failure

Introduction: Heart failure (HF) is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), which has three branches (PERK, IRE1 and ATF6α). UPR inhibits protein translation, and we hypothesized UPR contributes to electrical remodeling in HF. Methods: HF was induced in C57BL/6 mice 6-7 weeks after unilateral nephrectomy, deoxycorticosterone acetate pellet implantation, and salt water substitution. Sham operated mice were used as controls. Isolated ventricular myocytes were utilized for whole-cell patch clamp recording and ROS measurement by DCFDA with confocal imaging. Heart tissue was used for mRNA and protein measurements. GSK2606414, a specific inhibitor of PERK, was applied to myocytes at 4-30 nM for 6-20 h. Results: The action potential duration was prolonged in HF myocytes (204±26 vs. 100±16 ms of sham, P<0.05). The mRNA and protein levels of the UPR effectors indicated that the PERK and IRE1 branches were activated in HF heart tissue. The peak I Na , I to , I K1 , and I Kslow were decreased significantly in HF group, while I CaL and other K + currents were not affected. A PERK inhibitor, GSK2606414, restored I Na and I K,slow . Current changes were similar to those observed in myocytes under tunicamycin-induced ER stress. Significant reduction of channel protein levels were observed for K v 4.3, Kir2.1, K v 1.5, and Ca v 1.2, but not Na v 1.5, suggesting that I Na reduction was mediated by PERK but not by translational downregulation. Significant ROS production was observed in mouse cardiomyocytes under ER stress, which was suppressed by GSK2606414. Confocal imaging of ROS indicates the source to be mitochondria. Conclusions: TM-treated cardiomyocyte changes indicated that reductions of I Na , I to , I K1 and I K,slow were mediated by ER stress. Proportionally decreased protein and current levels of K v 4.3, Kir2.1, and K v 1.5 suggested direct effects of ER stress. The downregulation of Na v 1.5 and K v 1.5 could be prevented by PERK inhibition. Nevertheless, the effect of PERK inhibition on I Na was indirect and mediated by a PERK-dependent elevation in mitochondrial ROS. In conclusion, the UPR contributes to electrical remodeling in HF by direct and indirect means. Targeting the UPR may be a novel antiarrhythmic strategy.

Disruption of Myelin Leads to Ectopic Expression of KV1.1 Channels with Abnormal Conductivity of Optic Nerve Axons in a Cuprizone-Induced Model of Demyelination

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K+ channels (KV1.1 and 1.2 α subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of KV1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or KV1.1-subunit selective (dendrotoxin K) blockers of K+ currents suggest enhanced KV1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K+ currents mediated by recombinant channels comprised of different KV1.1 and 1.2 stoichiometries revealed that the enrichment of KV1 channels KV1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the patho-physiology of MS and related disorders.

Inhibition of a Mitochondrial Potassium Channel as a New Therapeutic Strategy for Chronic Lymphocytic Leukemia

Ion channels are involved in the regulation of proliferation and apoptosis and are emerging as promising oncological targets. We have recently identified three membrane-permeant inhibitors of the Kv1.3 potassium channel as inducers of apoptosis. These inhibitors induce mitochondrial membrane potential changes, increase of ROS release and opening of the permeability transition pore finally leading to cytochrome c release and cells death. Death occurs only when Kv1.3 is active in the inner mitochondrial membrane (mtKv1.3) and also in the absence of Bax and Bak. Efficiency of one of these inhibitors to reduce tumor volume in vivo was demonstrated in a melanoma orthotopic mouse model (Leanza, et al., 2012 EMBO Molecular Medicine). The same signaling pathway and a selective action of the Kv1.3 inhibitors was observed in B lymphocytes of patients suffering of Chronic lymphocytic leukemia (B-CLL) (Leanza, et al., 2013 Leukemia). Regarding the mechanism of selectivity, we defined that synergy between the level of Kv1.3 expression and an altered redox state in B-CLL cells determines the susceptibility of these cells. Since Kv1.3 inhibitors kill B-CLL by direct interference with mitochondrial functions, they act on these malignant cells independent of classic prognostic factors and Bcl-2 overexpression. To generalize our findings, in addition to melanoma and B-CLL cells, the effect of these inhibitors on cell survival was measured on different cancer cell lines expressing Kv1.3. A correlation between Kv1.3 expression and susceptibility to mtKv1.3 inhibitors was found.

Deficiency of NOX1/Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Oxidase Leads to Pulmonary Vascular Remodeling

Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.

Kv4 potassium channels modulate hippocampal EPSP-spike potentiation and spatial memory in rats

Kv4 channels regulate the backpropagation of action potentials (b-AP) and have been implicated in the modulation of long-term potentiation (LTP). Here we showed that blockade of Kv4 channels by the scorpion toxin AmmTX3 impaired reference memory in a radial maze task. In vivo, AmmTX3 intracerebroventricular (i.c.v.) infusion increased and stabilized the EPSP-spike (E-S) component of LTP in the dentate gyrus (DG), with no effect on basal transmission or short-term plasticity. This increase in E-S potentiation duration could result from the combination of an increase in excitability of DG granular cells with a reduction of GABAergic inhibition, leading to a strong reduction of input specificity. Radioactive in situ hybridization (ISH) was used to evaluate the amounts of Kv4.2 and Kv4.3 mRNA in brain structures at different stages of a spatial learning task in naive, pseudoconditioned, and conditioned rats. Significant differences in Kv4.2 and Kv4.3 mRNA levels were observed between conditioned and pseudoconditioned rats. Kv4.2 and Kv4.3 mRNA levels were transiently up-regulated in the striatum, nucleus accumbens, retrosplenial, and cingulate cortices during early stages of learning, suggesting an involvement in the switch from egocentric to allocentric strategies. Spatial learning performance was positively correlated with the levels of Kv4.2 and Kv4.3 mRNAs in several of these brain structures. Altogether our findings suggest that Kv4 channels could increase the signal-to-noise ratio during information acquisition, thereby allowing a better encoding of the memory trace.

Clustering and Activity Tuning of Kv1 Channels in Myelinated Hippocampal Axons

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.

Voltage-Gated Ion Channels are Involved in the Signaling Pathway of Differentiating Chondrocytes

Chondrocytes, the matrix-producing cells of cartilage, are non-excitable cells, yet in their mature form they are known to express voltage-gated ion channels. We investigated the signaling pathway and the involvement of plasma membrane ion channels during the early steps of chondrogenesis. Our in vitro chondrogenesis model system is a high density mesenchymal cell culture, in which chondroprogenitor cells are isolated from limb buds of chicken embryos. These cells spontaneously differentiate into chondrocytes mostly on the second and third days of culturing and secrete a significant amount of cartilage-specific extracellular matrix by the sixth day. Using whole-cell patch-clamp, RT-PCR and Western blot experiments we detected the presence of voltage-gated Kv4.1 and Kv1.3 K+ channels and Nav1.4 Na+ channels in these cells. The expression levels of Kv4.1 and Nav1.4 peaked at the critical days 2 and 3 of differentiation, whereas the level of Kv1.3 showed a gradual increase during the six days. However, unlike the other two channels Kv1.3 was not present in the plasma membrane. By confocal microscopy we detected high-frequency, short-duration Ca2+ transients in these cells that were sensitive to the external Ca2+ and K+ concentrations and tetraethylammonium (TEA, Kv channel blocker). Application of TEA, but not tetrodotoxin (TTX, Nav channel blocker), significantly reduced the frequency of Ca2+ transients, cartilage matrix production (assessed by metachromatic staining), cell proliferation, and the protein expression of the transcription factor Sox9, while it did not affect cell viability. Patch-clamp measurements showed that TEA also depolarized cells, whereas TTX had no effect on the membrane potential. Our results indicate the operation of a complex signaling pathway during early chondrocyte differentiation, which heavily relies on the function of Kv channels to stabilize the membrane potential for adequate Ca2+ signaling and the progression of chondrogenesis.

Altered Dynamics of Kv1.3 Channel Compartmentalization in the Immunological Synapse in Systemic Lupus Erythematosus

Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca(2+) influx. Although Kv1.3 channels have such an important role in T cell function, their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells, the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells, but the K channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.

Kv1.4 Subunit Expression is Decreased in Neurons of Painful Human Pulp

Kv1.4, a subunit of voltage-gated K(+) channels, plays a large role in regulating neuronal excitability. The level of Kv1.4 expression is unknown in human sensory neurons innervating healthy or painful tissue. Therefore, we examined Kv1.4 immunoreactivity in axons innervating both clinically diagnosed asymptomatic and painful symptomatic human tooth pulp. Antibodies directed against Kv1.4 and PGP9.5, a protein marker for axons, was used to determine the proportion of PGP9.5 immunopositive tissue that was also immunopositive for Kv1.4. We report that on pulpal axons innervating symptomatic teeth Kv1.4 immunoreactivity, a correlate of decreased Kv1.4 expression, is significantly decreased (p < 0.0001), suggestive of a factor responsible for facilitating chronic dental pain and decreases in currents produced, such as I(A), in neurons innervating painful pulp.

Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents.

Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K(+) channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K(+) current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K(+) channels.

Long QT and ventricular arrhythmias in transgenic mice expressing the N terminus and first transmembrane segment of a voltage-gated potassium channel.

Voltage-gated potassium channels control cardiac repolarization, and mutations of K+ channel genes recently have been shown to cause arrhythmias and sudden death in families with the congenital long QT syndrome. The precise mechanism by which the mutations lead to QT prolongation and arrhythmias is uncertain, however. We have shown previously that an N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K+ channel Kv1.1 (Kv1.1N206Tag) coassembles with other K+ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells. Here we report that transgenic mice overexpressing Kv1.1N206Tag in the heart have a prolonged QT interval and ventricular tachycardia. Cardiac myocytes from these mice have action potential prolongation caused by a significant reduction in the density of a rapidly activating, slowly inactivating, 4-aminopyridine sensitive outward K+ current. These changes correlate with a marked decrease in the level of Kv1.5 polypeptide. Thus, overexpression of a truncated K+ channel in the heart alters native K+ channel expression and has profound effects on cardiac excitability.