In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go-related genes (ERG1-3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole-cell patch-clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1 μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2-20 μM) augmented spontaneous contractions to a greater extent than BKCa channel blockers, enhanced carbachol-driven activity, increased nerve stimulation-mediated contractions, and impaired β-adrenoceptor-mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle.

HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance.

In ether-à-go-go (eag) K(+) channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are approximately 4 A apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg(2+). In I287D+F324D, Mg(2+) significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg(2+) binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg(2+) shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg(2+) binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.

Drugs that inhibit the cardiac rapid delayed rectifier potassium ion current (IKr) channel can be proarrhythmic and their clinical use has been associated with sudden unexpected death (SUD). Since SUD is about 20 times more common among people with epilepsy than in the general population, and some data indicate that drug treatment may contribute, we tested the hypothesis that the classic antiepileptic drugs phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB) have a potential to block IKr. The whole cell patch-clamp recording technique was used to study the effects on IKr channels expressed by the human ether-a-go-go related gene (HERG) stably expressed in Human Embryo Kidney (HEK) 293 cells. Tail currents, which are purely related to HERG, were blocked with an IC50 (the concentration when 50% inhibition was obtained compared to control values) of 240 microM for PHT and 3 mM for PB. A 20% inhibition of tail currents was obtained at CBZ concentrations of 250 and 500 microM. Collective data show that drugs with the same margins (ratio HERG IC50/unbound therapeutic concentration), as PHT and PB, may have arrhythmogenic potential, especially when used in predisposed patients and in the case of drug-drug interactions. SUD in epilepsy is generally a seizure-related phenomenon. However, our data suggest that PHT and PB may play a contributing role, perhaps by making some patients more vulnerable to the cardiovascular depression induced by seizures.

The prevalence of concurrent use of two or more drugs that block human ether-a-go-go-related gene product (hERG) K(+) channels is not uncommon, but is not well characterized. This study defined the effects of concurrent exposure of two hERG-blocking drugs on hERG current amplitude. Experiments were conducted to determine if concomitant exposure to two potent pore hERG blockers, thioridazine and terfenadine and a weak hERG blocker, erythromycin, would result in an additive, synergistic or inhibitory effect.

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, IK1, a Kir current mediated by Kir2.1 channel and IKACh, a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC50 value of 1.62μM and 1.15μM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit IKACh current with an IC50 value of 3.32μM but has no significant effects on IK1. Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks.

Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.

The human ether-a-go-go related gene (HERG) potassium channel has elicited intense scientific interest due to its role in cardiac repolarization and its association with arrhythmia and sudden cardiac death. Increasing evidence indicates the involvement of HERG channels in the pathophysiology of cancer. In the present study we investigated the expression of HERG protein in MDA-MB-435S melanoma cells, and its importance in regulating cell proliferation and migration. Our results showed that HERG was expressed on protein and mRNA levels in MDA-MB-435S melanoma cells. In these cells blockade of HERG channels with the HERG blockers E 4301 or cisapride attenuated both proliferation and migration of the cells. Activation of HERG with PD118057 stimulated cell migration. Furthermore, HERG small interfering (si) RNA attenuated the proliferation and migration of the cells. Incubation of MDA-MB-435S cells with E 4301 decreased the phosphorylation of mitogen-activated protein (MAP) kinase and the expression of the c-fos transcription factor. In control experiments, overexpression of HERG channels in HEK-293 cells dramatically increased the proliferation and migration of the cells and blocking HERG in these cells attenuated both proliferation and migration. Our results indicate that MDA-MB-435S cells express HERG channels and blockade of HERG results in the attenuation of both proliferation and migration by a mechanism dependent, at least in part, on an inhibition of the MAP kinase/c-fos pathway.

Potassium channels vary in their function and regulation, yet they maintain a number of important features - they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities (Curr. Opin. Neurobiol. 5 (1995) 268; Annu. Rev. Physiol. 59 (1997) 171). While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as ether-a-go-go and shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, we initiated studies on Dir.

The human ether-a-go-go-related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K+ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K+ channel in Caenorhabditis elegans Loss-of-function mutations of cnx-1 decreased the protein level and current density of the UNC-103 K+ channel and suppressed the behavioral defects caused by a gain-of-function mutation in unc-103 Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of calnexin reduced the expression and current densities of endogenous hERG K+ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K+ channels.

Drugs that inhibit the cardiac rapid delayed rectifier potassium ion current (I(Kr)) can be proarrhythmic and their clinical use has been associated with sudden unexpected death (SUD) due to cardiac arrhythmia. SUD is 20-40 times more common among people with epilepsy than in the general population and case-control studies have identified polytherapy with antiepileptic drugs (AEDs) as a risk factor. In a previous study, it was described that the old AEDs phenytoin and phenobarbital had the potential to inhibit the I(Kr) channel and it was suggested that this could contribute to the increased risk for SUD in patients with epilepsy. In this study, we have investigated the I(Kr) blocking potential of some more recently introduced AEDs, lamotrigine (LTG), topiramate (TPM) and gapapentin (GBP). The whole cell patch-clamp recording technique was used to study the effects on I(Kr) channels expressed by the human ether-a-go-go related gene (hERG) stably expressed in human embryo kidney (HEK) 293 cells. Tail currents, which are purely related to hERG currents, were blocked with IC50 and IC20 (the concentrations when 50% and 20% inhibition was obtained compared to control values) of 229 and 21 microM, respectively, for LTG. A 40% inhibition of tail currents was obtained at GBP concentrations of 100 mM and a 20% inhibition at 54 mM. A 35% inhibition of tail currents was obtained at TPM concentrations of 1000 microM and a 20% inhibition at 87 microM, respectively. Collective data show that drugs with the same margins (ratio hERG IC50/unbound therapeutic concentration) as LTG, may have arrhythmogenic potential. The risk for arrhythmia may be clinically significant in the presence of predisposing factors such as seizure-induced acidosis and in the case of concurrent treatment with other I(Kr) blocking drugs, or in case of pharmacokinetic drug-drug interactions resulting in excessively high concentrations of LTG.

The pathobiology of schizophrenia is still unclear. Its current treatment mainly depends on antipsychotic drugs. A leading adverse effect of these medications is the acquired long QT syndrome, which results from the blockade of cardiac HERG1 channels (human ether-a-go-go-related gene potassium channels 1) by antipsychotic agents. The HERG1 channel is encoded by HERG1 (KCNH2, Kv11.1) gene and is most highly expressed in heart and brain. Genetic variations in HERG1 predispose to acquired long QT syndrome. We hypothesized that the blockade of HERG1 channels by antipsychotics might also be significant for their therapeutic mode of action, indicating a novel mechanism in the pathogenesis of schizophrenia.

The congenital long QT syndrome (LQTS) is a cardiac disorder characterized by a prolonged QT interval on the electrocardiogram and an increased susceptibility to ventricular arrhythmias and sudden cardiac death. A frequent cause for LQTS is mutations in the KCNH2 gene (also known as the human ether-a-go-go-related gene or hERG), which reduce or modulate the potassium current IKr and hence alter cardiac repolarization. In a patient with a clinically diagnosed LQTS, we identified the mutation L69P in the N-terminal PAS (Per-Arnt-Sim) domain of hERG. Functional expression in HEK293 cells shows that a homotetrameric hERG channel reconstituted with only mutant subunits exhibits a drastically reduced surface expression of the channel protein thus leading to a diminished hERG current. Unlike many other mutations in the hERG-PAS domain the negative impact of the L69P substitution cannot be rescued by facilitated protein folding at a lower incubation temperature. Further, co-expression of wt and mutant monomers does not restore either wt like surface expression or the full hERG current. These results indicate L69P is a dominant negative mutation, with deficits which most likely occurs at the level of protein folding and subsequently inhibits trafficking to the plasma membrane. The functional deficits of the mutant channel support the clinical diagnosis of a LQTS.

The human ether-a-go-go-related gene (hERG) potassium channel conducts rapid delayed rectifier potassium currents (IKr) and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR) has multiple actions, and its hydrogenated derivative dihydroberberine (DHB) is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD). Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652) and phenylalanine (Phe656), and decreased mature (155-kDa) and simultaneously increased immature (135-kDa) hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90) expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6), a biomarker of unfolded protein response (UPR). Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was disrupted by impaired channel folding associated with altered interactions between hERG proteins and chaperones. Finally, trafficking inhibition activated UPR, and mature hERG channel degradation was increased by DHB.

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome type 2 (LQT2). HERG encodes a voltage-gated potassium channel consisting of four subunits. Tetrameric assembly is required for the formation of functional HERG channels. In the present work, we studied the role of assembly in HERG channel dysfunction of LQT2 mutations Q725X and R1014X, both of which cause truncations of the C-terminus of HERG channels. When expressed in HEK293 cells, Q725X did not generate HERG current, while R1014X generated HERG current with markedly reduced amplitude. Western blot analysis showed that both mutations caused defective trafficking of HERG channel proteins. Using sucrose gradient centrifugation we showed that wild type HERG and R1014X formed a tetrameric structure, whereas Q725X was expressed as a monomer. When coexpressed with wild type HERG, R1014X, but not Q725X, caused dominant negative suppression of wild type HERG current. Coimmunoprecipitation experiments showed that the lack of dominant negative effect by Q725X was due to failure of mutant subunits to coassemble with wild type subunits. These results suggest that the Q725X mutation causes HERG channel dysfunction by disruption of tetrameric assembly of HERG channels. In contrast, the R1014X mutation is capable of forming tetrameric structure, and it causes HERG channel dysfunction by defective trafficking of the mutant protein.

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

Dyslipidemia is associated with greater risk of ventricular tachyarrhythmias in patients with cardiovascular diseases. We aimed to examine whether the most electronegative subfraction of low-density lipoprotein (LDL), L5, is correlated with QTc prolongation in patients with coronary artery disease (CAD) and investigate the effects of human L5 on the electrophysiological properties of cardiomyocytes in relation to the lectin-like oxidized LDL receptor (LOX-1). L5 was isolated from the plasma of 40 patients with angiography documented CAD and 13 patients with no CAD to correlate the QTc interval respectively. The mean concentration of L5 was higher and correlated with QTc in patients with CAD compared to controls. To examine the direct effect of L5 on QTc, mice were intravenously injected with L5 or L1. L5-injected wild-type but not LOX-1-/- mice showed longer QTc compared to L1-injected animals in vivo with corresponding longer action potential duration (APD) in cardiomyocytes incubated with L5 in vitro. The APD prolongation was mediated by an increase of L-type calcium current and a decrease of transient outward potassium current. We show that L5 was positively correlated with QTc prolongation in patients with ischemic heart disease. L5 can modulate cardiac repolarization via LOX-1-mediated alteration sarcolemmal ionic currents.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 μM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.