E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.

Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.

Inhibitory Effects of 2-Aminoethoxydiphenyl Borate (2-APB) on Three KV1 Channel Currents.

2-Aminoethoxydiphenyl borate (2-APB), a boron-containing compound, is a multitarget compound with potential as a drug precursor and exerts various effects in systems of the human body. Ion channels are among the reported targets of 2-APB. The effects of 2-APB on voltage-gated potassium channels (KV) have been reported, but the types of KV channels that 2-APB inhibits and the inhibitory mechanism remain unknown. In this paper, we discovered that 2-APB acted as an inhibitor of three representative human KV1 channels. 2-APB significantly blocked A-type Kv channel KV1.4 in a concentration-dependent manner, with an IC50 of 67.3 µM, while it inhibited the delayed outward rectifier channels KV1.2 and KV1.3, with IC50s of 310.4 µM and 454.9 µM, respectively. Further studies on KV1.4 showed that V549, T551, A553, and L554 at the cavity region and N-terminal played significant roles in 2-APB's effects on the KV1.4 channel. The results also indicated the importance of fast inactivation gating in determining the different effects of 2-APB on three channels. Interestingly, a current facilitation phenomenon by a short prepulse after 2-APB application was discovered for the first time. The docked modeling revealed that 2-APB could form hydrogen bonds with different sites in the cavity region of three channels, and the inhibition constants showed a similar trend to the experimental results. These findings revealed new molecular targets of 2-APB and demonstrated that 2-APB's effects on KV1 channels might be part of the reason for the diverse bioactivities of 2-APB in the human body and in animal models of human disease.

Pharmacology of A-Type K+ Channels.

Transient outward potassium currents were first described nearly 60 years ago, since then major strides have been made in understanding their molecular basis and physiological roles. From the large family of voltage-gated potassium channels members of 3 subfamilies can produce such fast-inactivating A-type potassium currents. Each subfamily gives rise to currents with distinct biophysical properties and pharmacological profiles and a simple workflow is provided to aid the identification of channels mediating A-type currents in native cells. Their unique properties and regulation enable A-type K+ channels to perform varied roles in excitable cells including repolarisation of the cardiac action potential, controlling spike and synaptic timing, regulating dendritic integration and long-term potentiation as well as being a locus of neural plasticity.

Differential localization of voltage-gated potassium channels during Drosophila metamorphosis.

Neuronal excitability is determined by the combination of different ion channels and their sub-neuronal localization. This study utilizes protein trap fly strains with endogenously tagged channels to analyze the spatial expression patterns of the four Shaker-related voltage-gated potassium channels, Kv1-4, in the larval, pupal, and adult Drosophila ventral nerve cord. We find that all four channels (Shaker, Kv1; Shab, Kv2; Shaw, Kv3; and Shal, Kv4) each show different spatial expression patterns in the Drosophila ventral nerve cord and are predominantly targeted to different sub-neuronal compartments. Shaker is abundantly expressed in axons, Shab also localizes to axons but mostly in commissures, Shaw expression is restricted to distinct parts of neuropils, and Shal is found somatodendritically, but also in axons of identified motoneurons. During early pupal life expression of all four Shaker-related channels is markedly decreased with an almost complete shutdown of expression at early pupal stage 5 (∼30% through metamorphosis). Re-expression of Kv1-4 channels at pupal stage 6 starts with abundant channel localization in neuronal somata, followed by channel targeting to the respective sub-neuronal compartments until late pupal life. The developmental time course of tagged Kv1-4 channel expression corresponds with previously published data on developmental changes in single neuron physiology, thus indicating that protein trap fly strains are a useful tool to analyze developmental regulation of potassium channel expression. Finally, we take advantage of the large diameter of the giant fiber (GF) interneuron to map channel expression onto the axon and axon terminals of an identified interneuron. Shaker, Shaw, and Shal but not Shab channels localize to the non-myelinated GF axonal membrane and axon terminals. This study constitutes a first step toward systematically analyzing sub-neuronal potassium channel localization in Drosophila. Functional implications as well as similarities and differences to Kv1-4 channel localization in mammalian neurons are discussed.

Open channel block of Kv1.4 potassium channels by aripiprazole.

Aripiprazole is a quinolinone derivative approved as an atypical antipsychotic drug for the treatment of schizophrenia and bipolar disorder. It acts as with partial agonist activities at the dopamine D2 receptors. Although it is known to be relatively safe for patients with cardiac ailments, less is known about the effect of aripiprazole on voltage-gated ion channels such as transient A-type K+ channels, which are important for the repolarization of cardiac and neuronal action potentials. Here, we investigated the effects of aripiprazole on Kv1.4 currents expressed in HEK293 cells using a whole-cell patch-clamp technique. Aripiprazole blocked Kv1.4 channels in a concentration-dependent manner with an IC50 value of 4.4 µM and a Hill coefficient of 2.5. Aripiprazole also accelerated the activation (time-to-peak) and inactivation kinetics. Aripiprazole induced a voltage-dependent (δ = 0.17) inhibition, which was use-dependent with successive pulses on Kv1.4 currents without altering the time course of recovery from inactivation. Dehydroaripiprazole, an active metabolite of aripiprazole, inhibited Kv1.4 with an IC50 value of 6.3 µM (p < 0.05 compared with aripiprazole) with a Hill coefficient of 2.0. Furthermore, aripiprazole inhibited Kv4.3 currents to a similar extent in a concentration-dependent manner with an IC50 value of 4.9 µM and a Hill coefficient of 2.3. Thus, our results indicate that aripiprazole blocked Kv1.4 by preferentially binding to the open state of the channels.

The Effects of 4-Hydroxybenzoic Acid Identified from Bamboo (Dendrocalamus asper) Shoots on Kv1.4 Channel.

BACKGROUND: Bamboo shoot has been used as a treatment for epilepsy in traditional Chinese medicine for generations to treat neuronal disorders such as convulsive, dizziness and headaches. 4-hydroxybenzoic acid (4-hba) is a non-flavonoid phenol found abundantly in Dendrocalamus asper shoots (bamboo), fruits (strawberries and apples) and flowers. Kv1.4 is a rapidly inactivating Shaker-related member of the voltage-gated potassium channels with two inactivation mechanisms; the fast N-type and slow C-type. It plays vital roles in repolarisation, hyperpolarisation and signaling the restoration of resting membrane potential through the regulation of the movement of K+ across the cellular membrane. METHODS: Chemical compounds from Dendrocalamus asper bamboo shoots were purified and identified as major palmitic acids mixed with other minor fatty acids, palmitic acid, 4-hydroxybenzaldehyde, lauric acid, 4-hydroxybenzoic acid and cholest-4-ene-3-one. The response of synthetic 4-hydroxybenzoic acid was tested on Kv1.4 potassium channel which was injected into viable oocytes that was extracted from Xenopus laevis. The current were detected by the two-microelectrode voltage clamp, holding potential starting from -80 mV with 20 mV step-up until +80 mV. Readings of treatments with 0.1% DMSO, 4-hba concentrations and K channel blockers were taken at +60 mV. The ratio of tail/peak amplitude is the index of the activity of the Kv1.4 channels with n ≥ 6 (number of oocytes tested). The decreases of the ratios of five different concentrations (1 µM, 10 µM, 100 µM, 1 mM and 2.5 mM) were compared with 0.1% DMSO as the control. RESULTS: All concentration showed statistically significant results with P < 0.05 except for 100 µM. The normalised current of the 4-hba concentrations were compared with potassium channel blockers (TEA and 4-AP) and all groups showed statistically significant results. This study also showed that time taken for each concentration to affect Kv1.4 does not play any significant roles. CONCLUSION: 4-hydroxybenzoic acid was found to be able to enhance the inactivation of Kv1.4 by lowering the membrane potential so that the abnormal neuronal firing can be inhibited. With IC50 slightly higher than 10 µM, increasing concentrations (100 µM, 1 mM and 2.5 mM) had shown to exhibit toxicity effects. The best concentration from this study is 10 µM with Hill slope of 0.1799.

Properties of Slo1 K+ channels with and without the gating ring.

High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.

Cardiac involvements in myasthenia gravis associated with anti-Kv1.4 antibodies.

BACKGROUND AND PURPOSE: There is no general consensus as to whether autoimmune myasthenia gravis (MG) is associated with heart diseases, despite the fact that myocarditis, a serious cardiac involvement treatable by immunotherapy, is a complication of MG. It has been observed previously that MG patients with clinically suspected myocarditis had anti-Kv1.4 antibodies. The purpose of this study was to disclose the association between anti-Kv1.4 antibodies and cardiac involvements in MG patients. METHODS: Anti-Kv1.4 antibody was detected by an immunoprecipitation assay using (35) S-labeled rhabdomyosarcome cellular extract as the antigen source. Cardiac findings including electrocardiography (ECG) and clinical features of clinically suspected myocarditis in MG patients with anti-Kv1.4 antibodies were investigated. Ultrasound echocardiography (UCG) of ex vivo chick embryos was performed to determine the suppressive effects of sera with or without anti-Kv1.4 antibodies on heart muscle functions. RESULTS: Seventy (10.8%) of 650 MG patients had anti-Kv1.4 antibodies and 60% of them had abnormal ECG findings with high frequencies of T-wave abnormality and QT prolongation. Clinically suspected myocarditis was found in eight MG patients with anti-Kv1.4 antibodies but in none of the MG patients without anti-Kv1.4 antibodies. Most patients showed rapid deterioration with lethal arrhythmias such as ventricular tachycardia, sick sinus syndrome, or complete atrial ventricular block and severe heart failure. It was concluded using UCG of ex vivo chick embryos that MG serum with anti-Kv1.4 antibodies suppressed heart muscle functions. CONCLUSION: It has been demonstrated that anti-Kv1.4 antibodies are possible markers for cardiac involvements in MG patients.

Amyloid-ß Protein Precursor Regulates Electrophysiological Properties in the Hippocampus via Altered Kv1.4 Expression and Function in Mice.

BACKGROUND: Amyloid-ß protein precursor (AßPP) is enriched in neurons. However, the mechanism underlying AßPP regulation of neuronal activity is poorly understood. Potassium channels are critically involved in neuronal excitability. In hippocampus, A-type potassium channels are highly expressed and involved in determining neuronal spiking. OBJECTIVE: We explored hippocampal local field potential (LFP) and spiking in the presence and absence of AßPP, and the potential involvement of an A-type potassium channel. METHODS: We used in vivo extracellular recording and whole-cell patch-clamp recording to determine neuronal activity, current density of A-type potassium currents, and western blot to detect changes in related protein levels. RESULTS: Abnormal LFP was observed in AßPP-/- mice, including reduced beta and gamma power, and increased epsilon and ripple power. The firing rate of glutamatergic neurons reduced significantly, in line with an increased action potential rheobase. Given that A-type potassium channels regulate neuronal firing, we measured the protein levels and function of two major A-type potassium channels and found that the post-transcriptional level of Kv1.4, but not Kv4.2, was significantly increased in the AßPP-/- mice. This resulted in a marked increase in the peak time of A-type transient outward potassium currents in both glutamatergic and gamma-aminobutyric acid-ergic (GABAergic) neurons. Furthermore, a mechanistic experiment using human embryonic kidney 293 (HEK293) cells revealed that the AßPP deficiency-induced increase in Kv1.4 may not involve protein-protein interaction between AßPP and Kv1.4. CONCLUSION: This study suggests that AßPP modulates neuronal firing and oscillatory activity in the hippocampus, and Kv1.4 may be involved in mediating the modulation.

[A case of anti-acetylcholine receptor antibody-positive ocular myasthenia gravis with anti-titin antibody and anti-Kv1.4 antibody positive inflammatory myopathy].

An 84-year-old man was diagnosed with anti-acetylcholine receptor (AChR) antibody-positive ocular myasthenia gravis (OMG) at the age of 77 and received treatment. The patient was referred to our department with swelling and pain in his right upper arm, which had spread to other limbs. His serum anti-AChR antibody and creatine kinase levels were elevated, and MRI of the limbs displayed signal changes suggesting inflammation in the several muscles. Despite showing no sign of thymoma, he was positive for serum anti-titin and anti-Kv1.4 antibodies. We performed a muscle biopsy, which led to a diagnosis of inflammatory myopathy (IM). IM associated with OMG is relatively mild. Age-related immune dysregulation may cause both OMG and IM. Evaluation of disease activity with serum anti-AChR antibody levels, and assessment of prognosis with examining anti-striational antibodies are necessary for appropriate management of IM associated with MG.

Anti-Kv1.4 Antibody Without Myasthenia Gravis: A Rare Cause of Autoimmune Myocarditis and Myositis.

Anti-Kv1.4 antibody is often detected in thymoma-associated myasthenia gravis patients with anti-acetylcholine receptor antibody. Herein, we describe 2 patients with concurrent myocarditis and myositis. In both cases, anti-Kv1.4 antibody was positive despite the absence of thymoma and anti-acetylcholine receptor antibody, and immunosuppressants eventually resolved their symptoms and cardiac function. (Level of Difficulty: Advanced.).

Nivolumab-induced Myositis and Myocarditis with Positive Anti-titin Antibody and Anti-voltage-gated Potassium Channel Kv1.4 Antibody.

Immune checkpoint inhibitors (ICIs) are complicated by immune-related adverse events (irAEs), such as myositis, myocarditis, and myasthenia gravis (MG). Anti-titin antibody and anti-voltage-gated potassium channel Kv1.4 antibody are anti-striated antibodies that are frequently detected in MG patients with myositis and/or myocarditis. However, the clinical relationship between positive anti-striated antibodies and irAEs of ICIs remains unknown. We herein report a case of nivolumab-induced myositis and myocarditis with positive anti-titin antibody and anti-voltage-gated potassium channel Kv1.4 antibody in a patient with non-small-cell lung cancer. We also review reported cases of positive anti-striated antibodies related to irAEs of ICIs.

Pre-synaptic and post-synaptic A-type K+ channels regulate glutamatergic transmission and switching of the network into epileptiform oscillations.

BACKGROUND AND PURPOSE: Anticonvulsants targeting K+ channels have not been clinically available, although neuronal hyperexcitability in seizures could be suppressed by activation of K+ channels. Voltage-gated A-type K+ channel (A-channel) inhibitors may be prescribed for diseases of neuromuscular junction but could cause seizures. Consistently, genetic loss of function of A-channels may also cause seizures. It is unclear why inhibition of A-channels, compared with other types of K+ channels, is particularly prone to seizure induction. This hinders the development of relevant therapeutic interventions. EXPERIMENTAL APPROACH: Mechanisms underlying epileptogenesis with A-channel inhibition and antiepileptic actions of A-channel activation were investigated with electrophysiological, pharmacological, optogenetic, and behavioral approaches. KEY RESULTS: Pre-synaptic KV 1.4 and post-synaptic KV 4.3 A-channels act synergistically to gate glutamatergic transmission and control rhythmogenesis in the amygdala. The interconnected neurons set into the oscillatory mode by A-channel inhibition would reverberate with regular paces and the same top frequency, demonstrating a spatio-temporally well-orchestrated system with built-in oscillatory rhythms normally curbed by A-channels. Accordingly, selective over-excitation of glutamatergic neurons or inhibition of A-channels can induce behavioural seizures, which may be ameliorated by A-channel activators (e.g. NS-5806) or AMPA receptor antagonists (e.g. perampanel). CONCLUSION AND IMPLICATIONS: Trans-synaptic voltage-dependent A-channels serve as a biophysical-biochemical transducer responsible for a novel form of synaptic plasticity. Such a network-level switch into and out of the oscillatory mode may underlie a wide scope of telencephalic information processing or, at its extreme, epileptic seizures. A-channels thus constitute a potential target of antiepileptic therapy.

Functional role of endogenous Kv1.4 in experimental demyelination.

During neuroinflammation, the shaker type potassium channel Kv1.4 is re-expressed in oligodendrocytes (Ol), but not immune cells. Here, we analyze the role of endogenous Kv1.4 in two demyelinating animal models of multiple sclerosis. While Kv1.4 deficiency in primary murine Ol led to a decreased proliferation rate in vitro, it did not exert an effect on Ol proliferation or on the extent of de- or remyelination in the cuprizone model in vivo. However, in experimental autoimmune encephalomyelitis, Kv1.4-/- mice exhibited a milder disease course and reduced Th1 responses. These data argue for an indirect effect of Kv1.4 on immune cells, possibly via glial cells.

Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel.

Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.

Altered gating of Kv1.4 in the nucleus accumbens suppresses motivation for reward.

Deficient motivation contributes to numerous psychiatric disorders, including withdrawal from drug use, depression, schizophrenia, and others. Nucleus accumbens (NAc) has been implicated in motivated behavior, but it remains unclear whether motivational drive is linked to discrete neurobiological mechanisms within the NAc. To examine this, we profiled cohorts of Sprague-Dawley rats in a test of motivation to consume sucrose. We found that substantial variability in willingness to exert effort for reward was not associated with operant responding under low-effort conditions or stress levels. Instead, effort-based motivation was mirrored by a divergent NAc shell transcriptome with differential regulation at potassium and dopamine signaling genes. Functionally, motivation was inversely related to excitability of NAc principal neurons. Furthermore, neuronal and behavioral outputs associated with low motivation were linked to faster inactivation of a voltage-gated potassium channel, Kv1.4. These results raise the prospect of targeting Kv1.4 gating in psychiatric conditions associated with motivational dysfunction.

A-Type KV Channels in Dorsal Root Ganglion Neurons: Diversity, Function, and Dysfunction.

A-type voltage-gated potassium (Kv) channels are major regulators of neuronal excitability that have been mainly characterized in the central nervous system. By contrast, there is a paucity of knowledge about the molecular physiology of these Kv channels in the peripheral nervous system, including highly specialized and heterogenous dorsal root ganglion (DRG) neurons. Although all A-type Kv channels display pore-forming subunits with similar structural properties and fast inactivation, their voltage-, and time-dependent properties and modulation are significantly different. These differences ultimately determine distinct physiological roles of diverse A-type Kv channels, and how their dysfunction might contribute to neurological disorders. The importance of A-type Kv channels in DRG neurons is highlighted by recent studies that have linked their dysfunction to persistent pain sensitization. Here, we review the molecular neurophysiology of A-type Kv channels with an emphasis on those that have been identified and investigated in DRG nociceptors (Kv1.4, Kv3.4, and Kv4s). Also, we discuss evidence implicating these Kv channels in neuropathic pain resulting from injury, and present a perspective of outstanding challenges that must be tackled in order to discover novel treatments for intractable pain disorders.

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases.

Over the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.

Differential responses of rabbit ventricular and atrial transient outward current (Ito) to the Ito modulator NS5806.

Transient outward potassium current (Ito) in the heart underlies phase 1 repolarization of cardiac action potentials and thereby affects excitation-contraction coupling. Small molecule activators of Ito may therefore offer novel treatments for cardiac dysfunction, including heart failure and atrial fibrillation. NS5806 has been identified as a prototypic activator of canine Ito This study investigated, for the first time, actions of NS5806 on rabbit atrial and ventricular Ito Whole cell patch-clamp recordings of Ito and action potentials were made at physiological temperature from rabbit ventricular and atrial myocytes. 10 µmol/L NS5806 increased ventricular Ito with a leftward shift in Ito activation and accelerated restitution. At higher concentrations, stimulation of Ito was followed by inhibition. The EC50 for stimulation was 1.6 µmol/L and inhibition had an IC50 of 40.7 µmol/L. NS5806 only inhibited atrial Ito (IC50 of 18 µmol/L) and produced a modest leftward shifts in Ito activation and inactivation, without an effect on restitution. 10 µmol/L NS5806 shortened ventricular action potential duration (APD) at APD20-APD90 but prolonged atrial APD NS5806 also reduced atrial AP upstroke and amplitude, consistent with an additional atrio-selective effect on Na+ channels. In contrast to NS5806, flecainide, which discriminates between Kv1.4 and 4.x channels, produced similar levels of inhibition of ventricular and atrial Ito NS5806 discriminates between rabbit ventricular and atrial Ito, with mixed activator and inhibitor actions on the former and inhibitor actions against the later. NS5806 may be of significant value for pharmacological interrogation of regional differences in native cardiac Ito.