IL-6 signaling pathway contributes to exercise pressor reflex in rats with femoral artery occlusion in association with Kv4 activity in muscle afferent nerves.

Interleukin-6 (IL-6) via trans-signaling pathway plays a role in modifying muscle sensory nerve-exaggerated exercise pressor reflex in rats with ligated femoral arteries, but the underlying mechanisms are poorly understood. It is known that voltage-gated potassium channel subfamily member Kv4 channels contribute to the excitabilities of sensory neurons and neuronal signaling transduction. Thus, in this study, we determined that 1) IL-6 regulates the exaggerated exercise pressor reflex in rats with peripheral artery disease (PAD) induced by femoral artery ligation and 2) Kv4 channels in muscle dorsal root ganglion (DRG) neurons are engaged in the role played by IL-6 trans-signaling pathway. We found that the protein levels of IL-6 and its receptor IL-6R expression were increased in the DRGs of PAD rats with 3-day of femoral artery occlusion. Inhibition of muscle afferents' IL-6 trans-signaling pathway (gp130) by intra-arterial administration of SC144, a gp130 inhibitor, into the hindlimb muscles of PAD rats alleviated blood pressure response to static muscle contraction. On the other hand, we found that 3-day femoral occlusion decreased amplitude of Kv4 currents in rat muscle DRG neurons. The homo IL-6/IL-6Rα fusion protein (H. IL-6/6Rα), but not IL-6 alone significantly inhibited Kv4 currents in muscle DRG neurons; and the effect of H. IL-6/6Rα was largely reverted by SC144. In conclusion, our data suggest that via trans-signaling pathway upregulated IL-6 in muscle afferent nerves by ischemic hindlimb muscles inhibits the activity of Kv4 channels and thus likely leads to adjustments of the exercise pressor reflex in PAD.

The p.P888L SAP97 polymorphism increases the transient outward current (Ito,f) and abbreviates the action potential duration and the QT interval.

Synapse-Associated Protein 97 (SAP97) is an anchoring protein that in cardiomyocytes targets to the membrane and regulates Na+ and K+ channels. Here we compared the electrophysiological effects of native (WT) and p.P888L SAP97, a common polymorphism. Currents were recorded in cardiomyocytes from mice trans-expressing human WT or p.P888L SAP97 and in Chinese hamster ovary (CHO)-transfected cells. The duration of the action potentials and the QT interval were significantly shorter in p.P888L-SAP97 than in WT-SAP97 mice. Compared to WT, p.P888L SAP97 significantly increased the charge of the Ca-independent transient outward (Ito,f) current in cardiomyocytes and the charge crossing Kv4.3 channels in CHO cells by slowing Kv4.3 inactivation kinetics. Silencing or inhibiting Ca/calmodulin kinase II (CaMKII) abolished the p.P888L-induced Kv4.3 charge increase, which was also precluded in channels (p.S550A Kv4.3) in which the CaMKII-phosphorylation is prevented. Computational protein-protein docking predicted that p.P888L SAP97 is more likely to form a complex with CaMKII than WT. The Na+ current and the current generated by Kv1.5 channels increased similarly in WT-SAP97 and p.P888L-SAP97 cardiomyocytes, while the inward rectifier current increased in WT-SAP97 but not in p.P888L-SAP97 cardiomyocytes. The p.P888L SAP97 polymorphism increases the Ito,f, a CaMKII-dependent effect that may increase the risk of arrhythmias.

Inhibition of cardiac Kv4.3 (Ito) channel isoforms by class I antiarrhythmic drugs lidocaine and mexiletine.

Transient outward K+ current, Ito, contributes to cardiac action potential generation and is primarily carried by Kv4.3 (KCND3) channels. Two Kv4.3 isoforms are expressed in human ventricle and show differential remodeling in heart failure (HF). Lidocaine and mexiletine may be applied in selected patients to suppress ventricular arrhythmias, without effects on sudden cardiac death or mortality. Isoform-dependent effects of antiarrhythmic drugs on Kv4.3 channels and potential implications for remodeling-based antiarrhythmic management have not been assessed to date. We sought to test the hypotheses that Kv4.3 channels are targeted by lidocaine and mexiletine, and that drug sensitivity is determined in isoform-specific manner. Expression of KCND3 isoforms was quantified using qRT-PCR in left ventricular samples of patients with HF due to either ischemic or dilated cardiomyopathies (ICM or DCM). Long (Kv4.3-L) and short (Kv4.3-S) isoforms were heterologously expressed in Xenopus laevis oocytes to study drug sensitivity and effects on biophysical characteristics activation, deactivation, inactivation, and recovery from inactivation. In the present HF patient cohort KCND3 isoform expression did not differ between ICM and DCM. In vitro, lidocaine (IC50-Kv4.3-L: 0.8 mM; IC50-Kv4.3-S: 1.2 mM) and mexiletine (IC50-Kv4.3-L: 146 µM; IC50-Kv4.3-S: 160 µM) inhibited Kv4.3 with different sensitivity. Biophysical analyses identified accelerated and enhanced inactivation combined with delayed recovery from inactivation as primary biophysical mechanisms underlying Kv4.3 current reduction. In conclusion, differential effects on Kv4.3 isoforms extend the electropharmacological profile of lidocaine and mexiletine. Patient-specific remodeling of Kv4.3 isoforms may determine individual drug responses and requires consideration during clinical application of compounds targeting Kv4.3.

Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation.

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.SIGNIFICANCE STATEMENT The sparse activity of dentate granule cells (GCs), which is essential for pattern separation, is supported by high inhibitory inputs and low intrinsic excitability of GCs. Low excitability of GCs is thought to be attributable to a high K+ conductance at resting membrane potentials, but this study identifies Kv4.1, a depolarization-activated K+ channel, as a key ion channel that regulates firing of GCs without affecting resting membrane potentials. Kv4.1 expression is developmentally regulated and Kv4.1 currents are detected only in mature GCs that show low-frequency firing, but not in less mature high-frequency firing GCs. Furthermore, mice depleted of Kv4.1 transcripts in the dentate gyrus show impaired pattern separation, suggesting that Kv4.1 is crucial for sparse coding and pattern separation.

Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability.

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning-like and evening-like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.

Modulator-Gated, SUMOylation-Mediated, Activity-Dependent Regulation of Ionic Current Densities Contributes to Short-Term Activity Homeostasis.

Neurons operate within defined activity limits, and feedback control mechanisms dynamically tune ionic currents to maintain this optimal range. This study describes a novel, rapid feedback mechanism that uses SUMOylation to continuously adjust ionic current densities according to changes in activity. Small ubiquitin-like modifier (SUMO) is a peptide that can be post-translationally conjugated to ion channels to influence their surface expression and biophysical properties. Neuronal activity can regulate the extent of protein SUMOylation. This study on the single, unambiguously identifiable lateral pyloric neuron (LP), a component of the pyloric network in the stomatogastric nervous system of male and female spiny lobsters (Panulirus interruptus), focused on dynamic SUMOylation in the context of activity homeostasis. There were four major findings: First, neuronal activity adjusted the balance between SUMO conjugation and deconjugation to continuously and bidirectionally fine-tune the densities of two opposing conductances: the hyperpolarization activated current (Ih) and the transient potassium current (IA). Second, tonic 5 nm dopamine (DA) gated activity-dependent SUMOylation to permit and prevent activity-dependent regulation of Ih and IA, respectively. Third, DA-gated, activity-dependent SUMOylation contributed to a feedback mechanism that restored the timing and duration of LP activity during prolonged modulation by 5 µm DA, which initially altered these and other activity features. Fourth, DA modulatory and metamoduatory (gating) effects were tailored to simultaneously alter and stabilize neuronal output. Our findings suggest that modulatory tone may select a subset of rapid activity-dependent mechanisms from a larger menu to achieve homeostasis under varying conditions.SIGNIFICANCE STATEMENT Post-translational SUMOylation of ion channel subunits controls their interactions. When subunit SUMOylation is dysregulated, conductance densities mediated by the channels are distorted, leading to nervous system disorders, such as seizures and chronic pain. Regulation of ion channel SUMOylation is poorly understood. This study demonstrated that neuronal activity can regulate SUMOylation to reconfigure ionic current densities over minutes, and this regulation was gated by tonic nanomolar dopamine. Dynamic SUMOylation was necessary to maintain specific aspects of neuronal output while the neuron was being modulated by high (5 µm) concentrations of dopamine, suggesting that the gating function may ensure neuronal homeostasis during extrinsic modulation of a circuit.

Control of Sleep Onset by Shal/Kv4 Channels in Drosophila Circadian Neurons.

Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. Here, we demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels [Shal; also known as voltage-gated K+ channel 4 (Kv4)] in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk ∼ZT13, when the resting membrane potentials and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. We propose that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. Our results have important implications for the understanding and treatment of sleep disorders such as insomnia.SIGNIFICANCE STATEMENT The mechanisms by which our brains reversibly switch from waking to sleep state remain an unanswered and intriguing question in biological research. In this study, we identified that Shal/Kv4, a well known voltage-gated K+ channel, acts as a controller of wake-sleep transition at dusk in Drosophila circadian neurons. We find that interference of Kv4 function with a dominant-negative form (DNKv4) in subsets of circadian neurons specifically disrupts sleep onset at dusk, although Kv4 itself does not exhibit circadian oscillation. Kv4 preferentially downregulates neuronal firings at ZT9-ZT17, supporting that it plays an essential role in wake-sleep transition at dusk. Our findings may help understand and eventually treat sleep disorders such as insomnia.

A-type K+ channels impede supralinear summation of clustered glutamatergic inputs in layer 3 neocortical pyramidal neurons.

A-type K+ channels restrain the spread of incoming signals in tufted and apical dendrites of pyramidal neurons resulting in strong compartmentalization. However, the exact subunit composition and functional significance of K+ channels expressed in small diameter proximal dendrites remain poorly understood. We focus on A-type K+ channels expressed in basal and oblique dendrites of cortical layer 3 pyramidal neurons, in ex vivo brain slices from young adult mice. Blocking putative Kv4 subunits with phrixotoxin-2 enhances depolarizing potentials elicited by uncaging RuBi-glutamate at single dendritic spines. A concentration of 4-aminopyridine reported to block Kv1 has no effect on such responses. 4-aminopyridine and phrixotoxin-2 increase supralinear summation of glutamatergic potentials evoked by synchronous activation of clustered spines. The effect of 4-aminopyridine on glutamate responses is simulated in a computational model where the dendritic A-type conductance is distributed homogeneously or in a linear density gradient. Thus, putative Kv4-containing channels depress excitatory inputs at single synapses. The additional recruitment of Kv1 subunits might require the synchronous activation of multiple inputs to regulate the gain of signal integration.

A Model of the Block of Voltage-Gated Potassium Kv4.2 Ionic Currents by 4-Aminopyridine.

Voltage clamp recordings of macroscopic currents were made from rat potassium-gated potassium 4.2(Kv4.2) channels expressed in human embryonic kidney (HEK293) cells with the main goals of quantifying the concentration, time, and voltage dependence of the block and to generate a state model that replicates the features of the block. When applied either externally or internally, the block of Kv4.2 currents by 4-aminopyridine (4AP) occurs at the holding potential (-80 mV), is affected by the stimulus frequency, and is relieved by membrane depolarization. The Kd for the tonic block at -80 mV was 0.9 ± 0.07 mM and was consistent with 1:1 binding. Relief of block during a step to 50 mV was well fitted by a single exponential with a time constant of ∼40 milliseconds. At -80 mV, the association rate constant was 0.08 mM-1 s-1, and the off-rate was 0.08 s-1 The state model replicates the features of the experimental data reasonably well by assuming that 4AP binds only to closed states, that 4AP binding and inactivation are mutually exclusive processes, and that the activation of closed-bound channels is the same as for closed channels. Since the open channel has a very low or no affinity for 4AP, channel opening promotes the unbinding of 4AP, which accounts for the reverse use dependence of the block.

MiR-223-3p as a Novel MicroRNA Regulator of Expression of Voltage-Gated K+ Channel Kv4.2 in Acute Myocardial Infarction.

BACKGROUND/AIMS: Acute myocardial infarction (AMI) is a devastating cardiovascular disease with a high rate of morbidity and mortality, partly due to enhanced arrhythmogenicity. MicroRNAs (miRNAs) have been shown to participate in the regulation of cardiac ion channels and the associated arrhythmias. The purpose of this study was to test our hypothesis that miR-223-3p contributes to the electrical disorders in AMI via modulating KCND2, the gene encoding voltage-gated channel Kv4.2 that carries transient outward K+ current Ito. METHODS: AMI model was established in male Sprague-Dawley (SD) rats by left anterior descending artery (LAD) ligation. Evans blue and TTC staining was used to measure infarct area. Ito was recorded in isolated ventricular cardiomyocytes or cultured neonatal rat ventricular cells (NRVCs) by whole-cell patch-clamp techniques. Western blot analysis was employed to detect the protein level of Kv4.2 and real-time RT-PCR to determine the transcript level of miR-223-3p. Luciferase assay was used to examine the interaction between miR-223-3p and KCND2 in cultured NRVCs. RESULTS: Expression of miR-223-3p was remarkably upregulated in AMI relative to sham control rats. On the contrary, the protein level of Kv4.2 and Ito density were significantly decreased in AMI. Consistently, transfection of miR-223-3p mimic markedly reduced Kv4.2 protein level and Ito current in cultured NRVCs. Co-transfection of AMO-223-3p (an antisense inhibitor of miR-223-3p) reversed the repressive effect of miR-223-3p. Luciferase assay showed that miR-223-3p, but not the negative control, substantially suppressed the luciferase activity, confirming the direct binding of miR-223-3p to the seed site within the KCND2 sequence. Finally, direct intramuscular injection of AMO-223-3p into the ischemic myocardium to knockdown endogenous miR-223-3p decreased the propensity of ischemic arrhythmias. CONCLUSIONS: Upregulation of miR-223-3p in AMI repressed the expression of KCND2/Kv4.2 resulting in reduction of Ito density that can cause APD prolongation and promote arrhythmias in AMI, and therefore knockdown of endogenous miR-223-3p might be considered a new approach for antiarrhythmic therapy of ischemic arrhythmias.

A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons.

Tuft dendrites of layer 5 pyramidal neurons form a separate biophysical and processing compartment. Presently, little is known about plasticity mechanisms in this isolated compartment. Here, we describe a novel form of plasticity in which unpaired low-frequency (0.1 Hz) stimulation of tuft inputs resulted in prolonged transient (86.3 ± 7.3 min) potentiation of EPSPs (286.1% ± 30.5%) and enhanced local excitability that enabled more-efficient back-propagation of axo-somatic action potentials and dendritic calcium spikes selectively into the activated dendritic segments. This plasticity was exclusive to tuft dendrites and did not occur in basal dendrites. Induction of this plasticity depended on activation of Kv4.2 potassium and NMDAR channels, internalization of membrane proteins, and insertion of AMPAR. This unique form of tuft plasticity increases proximal-distal electrical coupling of activated tuft dendrites and opens a prolonged time window for binding and storing feedforward and feedback information in a branch-specific manner.

IA Channels Encoded by Kv1.4 and Kv4.2 Regulate Circadian Period of PER2 Expression in the Suprachiasmatic Nucleus.

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 (Kv1.4(-/-)) or Kcnd2 (Kv4.2(-/-); but not Kcnd3, Kv4.3(-/-)), voltage-gated K(+) (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K(+) currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse (Per2(Luc)) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4(-/-)Per2(Luc) and Kv4.2(-/-) Per2(Luc), but not Kv4.3(-/-)Per2(Luc), mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2(Luc) mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4(-/-)/Kv4.2(-/-)Per2(Luc) SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within and/or outside of the SCN to regulate circadian phase of daily activity.

Kv4.3-Encoded Fast Transient Outward Current Is Presented in Kv4.2 Knockout Mouse Cardiomyocytes.

Gradients of the fast transient outward K+ current (Ito,f) contribute to heterogeneity of ventricular repolarization in a number of species. Cardiac Ito,f levels and gradients change notably with heart disease. Human cardiac Ito,f appears to be encoded by the Kv4.3 pore-forming α-subunit plus the auxiliary KChIP2 ß-subunit while mouse cardiac Ito,f requires Kv4.2 and Kv4.3 α-subunits plus KChIP2. Regional differences in cardiac Ito,f are associated with expression differences in Kv4.2 and KChIP2. Although Ito,f was reported to be absent in mouse ventricular cardiomyocytes lacking the Kv4.2 gene (Kv4.2-/-) when short depolarizing voltage pulses were used to activate voltage-gated K+ currents, in the present study, we showed that the use of long depolarization steps revealed a heteropodatoxin-sensitive Ito,f (at ~40% of the wild-type levels). Immunohistological studies further demonstrated membrane expression of Kv4.3 in Kv4.2-/- cardiomyocytes. Transmural Ito,f gradients across the left ventricular wall were reduced by ~3.5-fold in Kv4.2-/- heart, compared to wild-type. The Ito,f gradient in Kv4.2-/- hearts was associated with gradients in KChIP2 mRNA expression while in wild-type there was also a gradient in Kv4.2 expression. In conclusion, we found that Kv4.3-based Ito,f exists in the absence of Kv4.2, although with a reduced transmural gradient. Kv4.2-/- mice may be a useful animal model for studying Kv4.3-based Ito,f as observed in humans.

Effect of tyrphostin AG879 on Kv 4.2 and Kv 4.3 potassium channels.

BACKGROUND AND PURPOSE: A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels. EXPERIMENTAL APPROACH: To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique. KEY RESULTS: Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. CONCLUSION AND IMPLICATIONS: AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.

Neuroplasticity of A-type potassium channel complexes induced by chronic alcohol exposure enhances dendritic calcium transients in hippocampus.

RATIONALE: Chronic alcohol-induced cognitive impairments and maladaptive plasticity of glutamatergic synapses are well-documented. However, it is unknown if prolonged alcohol exposure affects dendritic signaling that may underlie hippocampal dysfunction in alcoholics. Back-propagation of action potentials (bAPs) into apical dendrites of hippocampal neurons provides distance-dependent signals that modulate dendritic and synaptic plasticity. The amplitude of bAPs decreases with distance from the soma that is thought to reflect an increase in the density of Kv4.2 channels toward distal dendrites. OBJECTIVE: The aim of this study was to quantify changes in hippocampal Kv4.2 channel function and expression using electrophysiology, Ca(2+) imaging, and western blot analyses in a well-characterized in vitro model of chronic alcohol exposure. RESULTS: Chronic alcohol exposure significantly decreased expression of Kv4.2 channels and KChIP3 in hippocampus. This reduction was associated with an attenuation of macroscopic A-type K(+) currents in CA1 neurons. Chronic alcohol exposure increased bAP-evoked Ca(2+) transients in the distal apical dendrites of CA1 pyramidal neurons. The enhanced bAP-evoked Ca(2+) transients induced by chronic alcohol exposure were not related to synaptic targeting of N-methyl-D-aspartate (NMDA) receptors or morphological adaptations in apical dendritic arborization. CONCLUSIONS: These data suggest that chronic alcohol-induced decreases in Kv4.2 channel function possibly mediated by a downregulation of KChIP3 drive the elevated bAP-associated Ca(2+) transients in distal apical dendrites. Alcohol-induced enhancement of bAPs may affect metaplasticity and signal integration in apical dendrites of hippocampal neurons leading to alterations in hippocampal function.

Mutant α-synuclein enhances firing frequencies in dopamine substantia nigra neurons by oxidative impairment of A-type potassium channels.

Parkinson disease (PD) is an α-synucleinopathy resulting in the preferential loss of highly vulnerable dopamine (DA) substantia nigra (SN) neurons. Mutations (e.g., A53T) in the α-synuclein gene (SNCA) are sufficient to cause PD, but the mechanism of their selective action on vulnerable DA SN neurons is unknown. In a mouse model overexpressing mutant α-synuclein (A53T-SNCA), we identified a SN-selective increase of in vivo firing frequencies in DA midbrain neurons, which was not observed in DA neurons in the ventral tegmental area. The selective and age-dependent gain-of-function phenotype of A53T-SCNA overexpressing DA SN neurons was in part mediated by an increase of their intrinsic pacemaker frequency caused by a redox-dependent impairment of A-type Kv4.3 potassium channels. This selective enhancement of "stressful pacemaking" of DA SN neurons in vivo defines a functional response to mutant α-synuclein that might be useful as a novel biomarker for the "DA system at risk" before the onset of neurodegeneration in PD.

Inhibition of A-type potassium current by the peptide toxin SNX-482.

SNX-482, a peptide toxin isolated from tarantula venom, has become widely used as an inhibitor of Cav2.3 voltage-gated calcium channels. Unexpectedly, we found that SNX-482 dramatically reduced the A-type potassium current in acutely dissociated dopamine neurons from mouse substantia nigra pars compacta. The inhibition persisted when calcium was replaced by cobalt, showing that it was not secondary to a reduction of calcium influx. Currents from cloned Kv4.3 channels expressed in HEK-293 cells were inhibited by SNX-482 with an IC50 of <3 nM, revealing substantially greater potency than for SNX-482 inhibition of Cav2.3 channels (IC50 20-60 nM). At sub-saturating concentrations, SNX-482 produced a depolarizing shift in the voltage dependence of activation of Kv4.3 channels and slowed activation kinetics. Similar effects were seen on gating of cloned Kv4.2 channels, but the inhibition was less pronounced and required higher toxin concentrations. These results reveal SNX-482 as the most potent inhibitor of Kv4.3 channels yet identified. Because of the effects on both Kv4.3 and Kv4.2 channels, caution is needed when interpreting the effects of SNX-482 on cells and circuits where these channels are present.

Effects of haloperidol on Kv4.3 potassium channels.

Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 µM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials.

Inhibition of cardiac Kv1.5 and Kv4.3 potassium channels by the class Ia anti-arrhythmic ajmaline: mode of action.

Ajmaline is a class Ia anti-arrhythmic compound that is widely used for the diagnosis of Brugada syndrome and the acute treatment of atrial or ventricular tachycardia. For ajmaline, inhibitory effects on a variety of cardiac K(+) channels have been observed, including cardiac Kv1 and Kv4 channels. However, the exact pharmacological properties of channel blockade have not yet been addressed adequately. Using two different expression systems, we analysed pharmacological effects of ajmaline on the potassium channels Kv1.5 and Kv4.3 underlying cardiac I Kur and I to current, respectively. When expressed in a mammalian cell line, we find that ajmaline inhibits Kv1.5 and Kv4.3 with an IC50 of 1.70 and 2.66 µM, respectively. Pharmacological properties were further analysed using the Xenopus expression system. We find that ajmaline is an open channel inhibitor of cardiac Kv1.5 and Kv4.3 channels. Whereas ajmaline results in a mild leftward shift of Kv1.5 activation curve, no significant effect on Kv4.3 channel activation could be observed. Ajmaline did not significantly affect channel inactivation kinetics. Onset of block was fast. For Kv4.3 channels, no significant effect on recovery from inactivation or channel deactivation could be observed. Furthermore, there was no use-dependence of block. Taken together, we show that ajmaline inhibits cardiac Kv1.5 and Kv4.3 channels at therapeutic concentrations. These data add to the current understanding of the electrophysiological basis of anti-arrhythmic action of ajmaline.

Bone morphogenetic protein-4 contributes to the down-regulation of Kv4.3 K+ channels in pathological cardiac hypertrophy.

Kv4.3 K(+) channels contributing to Ito are involved in the repolarization of cardiac action potential. Kv4.3 K(+) channels decrease in pathological cardiac hypertrophy, but the mechanism remains unclear. Our previous study found that the expression of bone morphogenetic protein 4 (BMP4) increased in pressure-overload and Ang II constant infusion induced cardiac hypertrophy. Since the downregulation of Kv4.3 K(+) channels and the upregulation of BMP4 simultaneously occur in pathological cardiac hypertrophy, we hypothesize that the up-regulated BMP4 would contribute to the downregulation of Kv4.3 K(+) channels in cardiac hypertrophy. We found that BMP4 treatment reduced Kv4.3 but not Kv4.2 and Kv1.4 K(+) channel protein expression, and BMP4-induced decrease of Kv4.3 K(+) channel protein expression was reversed by BMP4 inhibitor noggin and DMH1 in cultured cardiomyocytes in vitro. BMP4-induced decrease of Kv4.3 K(+) channel protein expression was also reversed by the NADPH oxidase inhibitor apocynin and the radical scavenger tempol. In in vivo transverse aortic constriction (TAC)-induced cardiac hypertrophy, constant infusion of DMH1 completely rescued TAC-induced down-regulation of Kv4.3 K(+) channel protein expression. We conclude that BMP4 contributes to the downregulation of Kv4.3 K(+) channels in pathological cardiac hypertrophy and the underlying mechanism might be through increasing ROS production.