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At least 55 records · Page 3Linked to original sources
PubMed1998

Characterization of an eag-like potassium channel in human neuroblastoma cells.

1. SH-SY5Y human neuroblastoma cells were investigated with whole-cell and perforated patch recording methods. 2. Besides a quickly activating delayed rectifier channel and a HERG-like channel, a slowly activating potassium channel with biophysical properties identical to those of rat eag (r-eag) channels was detected, here referred to as h-eag. 3. h-eag shows a marked Cole-Moore shift, i.e. the activation kinetics become very slow when the depolarization starts from a very negative holding potential. In addition, extracellular Mg2+ and Ni2+ strongly slow down activation. 4. Application of acetylcholine induces a fast block of the current when recorded in the perforated patch mode. This block is presumably mediated by Ca2+, as about 1 microM intracellular Ca2+ completely abolished h-eag outward current. 5. When cells were grown in the presence of 10 microM retinoic acid in order to synchronize the cell line in the G1 phase of the cell cycle, h-eag current was reduced to less than 5 % of the control value, while the delayed rectifier channel was expressed more abundantly. Down-regulation of h-eag by long-term exposure to retinoic acid was paralleled by a right shift in the activation potential of HERG-like channels. 6. Acute application of 10 microM retinoic acid blocked the delayed rectifier channel but enhanced the h-eag current. 7. Thus, our results show that human neuroblastoma cells express in a cell cycle-dependent manner an [Mg2+]o-dependent potassium channel (h-eag) which is blocked by submicromolar concentrations of intracellular Ca2+.

Acetylcholine↗
PubMed2004

Block of wild-type and inactivation-deficient human ether-a-go-go-related gene K+ channels by halofantrine.

Halofantrine is an antimalarial drug developed as a treatment of P. falciparum resistant to chloroquine. However, halofantrine can also induce long QT syndrome (LQTS) and torsades de pointes, a potentially life-threatening ventricular arrhythmia. Drug-induced LQTS is usually caused by block of the human ether-a-go-go-related gene (HERG) channels that conduct the rapid delayed rectifier K(+) current, I(Kr), in the heart. Here we show that halofantrine preferentially blocks open and inactivated HERG channels heterologously expressed in Xenopus laevis oocytes. The half-maximal inhibitory concentration (IC(50)) for block of wild-type (WT) HERG was 1.0 microM. As we reported previously for other HERG channel blockers, the potency of halofantrine was reduced by mutation to Ala of aromatic residues (Y652, F656) located in the S6 domain, or a Val (V625) located in the pore helix. Halofantrine at a concentration 10 microM did not affect the transient outward potassium channel, Kv4.3, the slow delayed rectifier potassium channel, KvLQT1+minK and inward rectifier potassium channel, Kir2.1. An inactivation deficient mutant (G628C/S631C HERG) was only slightly less sensitive (IC(50)=2.0 microM). The rate of block onset by halofantrine at 0 mV was used to estimate the apparent association (k(on)) and dissociation (k(off)) rate constants for drug binding. For WT and G628C/S631C HERG, k(on) was similar (0.0114 and 0.0163 M(-1)/s(-1) respectively). In contrast, k(off) was significantly faster for G628C/S631C (0.357 s(-1)) than WT (0.155 s(-1)), and explains the observed decrease in drug potency for the inactivation-deficient mutant channel. We conclude that halofantrine requires channels to open before it can gain access to its binding site located in the central cavity of the HERG channel.

Animals↗
PubMed2005

Structure-function studies of the outer mouth and voltage sensor domain of hERG.

hERG has uniquely fast inactivation but slow activation processes. We study the structural basis for these unique gating properties using the following approaches: site-specific mutagenesis, the MTS accessibility test, disulfide bond formation, thermodynamic mutant cycle analysis, peptide toxin 'foot-printing', NMR spectroscopy, and molecular modelling. We propose the following: (1) two structural features in hERG's outer mouth contribute to its fast inactivation rate: a lack of 'open mouth'-stabilizing hydrogen bonds and an unusually long extracellular 'S5-P' linker that contains an alpha-helix. During membrane depolarization, four such 'S5-P helices' from the tetramer channel come near each other to occlude the outer mouth. This occurs rapidly due to the dynamic nature of the S5-P helices. (2) Two structural features in hERG's voltage sensor domain contribute to its slow activation rate: hERG's major voltage-sensor, S4, has three (instead of four as in Shaker) positive charges involved in gating charge transfer, and hERG has six (instead of three as in Shaker) negative charges in the other transmembrane segments (S1-S3) of the voltage sensor domain. Thus a less voltage-sensitive S4, in conjunction with more surrounding negative charges (some of which can form salt-bridges with S4's positive charges in the pre-open state), retards channel activation.

Amino Acid Sequence↗
PubMed2006

Flunarizine is a highly potent inhibitor of cardiac hERG potassium current.

Flunarizine has been widely used for the management of a variety of disorders such as peripheral vascular diseases, migraine, and epilepsy. The majority of its beneficial effects have been attributed to its ability to inhibit voltage-gated Ca2+ channels in the low micromolar range, albeit non-selectively, as flunarizine has been shown to inhibit a variety of ion channels. We examined the effects of flunarizine on potassium currents through cardiac channels encoded by the human ether-a-go-go related gene (hERG) stably expressed in CHO cells. In this study, we have characterized the effect of flunarizine on biophysical properties of hERG potassium currents with standard whole-cell voltage-clamp techniques. Notably, flunarizine is a highly potent inhibitor of hERG current with an IC50 value of 5.7 nM. The effect of flunarizine on hERG potassium current is concentration and time dependent, and displays voltage dependence over the voltage range between -40 and 0 mV. At concentrations near or above the IC50, flunarizine causes a negative shift in the voltage dependence of hERG current activation and accelerates tail current deactivation. Flunarizine preferentially blocks the activated state of the channel and displays weak frequency dependence of inhibition. Flunarizine also inhibits KCNQ1/KCNE1 channel current with an IC50 of 0.76 microM.

Animals↗
PubMed1997

Identification of two nervous system-specific members of the erg potassium channel gene family.

Two new potassium channel genes, erg2 and erg3, that are expressed in the nervous system of the rat were identified. These two genes form a small gene family with the previously described erg1 (HERG) gene. The erg2 and erg3 genes are expressed exclusively in the nervous system, in marked contrast to erg1, which is expressed in both neural and non-neural tissues. All three genes are expressed in peripheral sympathetic ganglia. The erg3 channel produces a current that has a large transient component at positive potentials, whereas the other two channels are slowly activating delayed rectifiers. Expression of the erg1 gene in the sympathetic nervous system has potential implications for the etiology of the LQT2 form of the human genetic disease long QT syndrome.

Animals↗
PubMed1997

The human delta1261 mutation of the HERG potassium channel results in a truncated protein that contains a subunit interaction domain and decreases the channel expression.

HERG (human eag-related gene) encodes an inward-rectifier potassium channel formed by the assembly of four subunits. Since the truncated HERG protein in patients with long QT syndrome induces a dominant phenotype, that is, cardiac sudden death, the assembly of nonfunctional complexes between wild-type and mutated subunits was implicated in causing the disease. To understand HERG-mediated cardiac sudden death at the molecular level, it is important to determine which regions in the HERG protein participate in subunit interaction. We therefore report the identification of a subunit interaction domain, NAB(HERG), that is localized at the hydrophilic cytoplasmic N terminus and can form a tetramer in the absence of the rest of the HERG protein. Truncated HERG proteins containing NAB(HERG), including one that resulted from the delta1261 human mutation, inhibit the functional expression of the HERG channel in transfected cells. Together, these results support the notion that the expression of HERG in the human heart may be decreased in the presence of the truncated subunit. Such a decrease of potassium channel expression can contribute to the longer QT intervals observed in the patients with the HERG mutation.

Cation Transport Proteins↗
PubMed1999

Mechanism of block and identification of the verapamil binding domain to HERG potassium channels.

Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 micromol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.

Anti-Arrhythmia Agents↗
PubMed2001

Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain.

The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses.

Animals↗
PubMed2003

Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241.

1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3 The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6 Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

Animals↗
PubMed2003

Modulation of human ether-à-go-go-related K+ (HERG) channel inactivation by Cs+ and K+.

Unlike many other native and cloned K+ channels, human ether-à-go-go-related K+ (HERG) channels show significant Cs+ permeability with a PCs/PK (the permeability of Cs+ relative to that of K+) of 0.36 +/- 0.03 (n = 10). Here, we find that raising the concentration of external Cs+ (Cs+o) dramatically slows HERG channel inactivation without affecting activation. Replacement of 5 mM K+o by 135 mM Cs+o increased both inactivation and recovery time constants and shifted the mid-point of the steady-state inactivation curve by 25 mV in the depolarized direction (n = 6, P < 0.01). Raising [Cs+]o also modulated the voltage sensitivity of inactivation gating. With 130 mM Cs+i and 135 mM NMDG+o, the inactivation time constant decreased e-fold per 47.5 +/- 1.1 mV (n = 5), and when 20 mM Cs+ was added to the bath solution, the inactivation time constant decreased e-fold per 20.6 +/- 1.3 mV (n = 5, P < 0.01). A quantitative analysis suggests that Cs+o binds to a site in the pore that is influenced by the transmembrane electrical field, so that Cs+o-induced slowing of HERG inactivation is less prominent at strong depolarizations. K+o has effects that are similar to Cs+o and their effects were additive, suggesting Cs+o and K+o may share a common mechanism of action. The strong effects of Cs+ on inactivation but not on activation highlight the importance of ion and channel interactions during the onset of inactivation in the HERG channel.

Cation Transport Proteins↗
PubMed2003

Expression and function of KCNH2 (HERG) in the human jejunum.

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.

Acetylcholine↗
PubMed1998

Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density.

BACKGROUND: Despite advances in medical therapy, congestive heart failure remains a major cause of death in the developed world. A disproportionate number of the deaths of patients with heart failure are sudden and presumed to be arrhythmic. Heart failure in humans and in animal models is associated with prolongation of the action potential duration (APD), the result of downregulation of K+ currents-prominently, the Ca2+-independent transient outward current (Ito). The mechanism for the reduction of Ito in heart failure is unknown. The K+ channel alpha-subunit Kv4.3, a homolog of the Drosophila Shal family, is most likely to encode all or part of the native cardiac Ito in humans. METHODS AND RESULTS: We used ribonuclease protection assays and whole-cell electrophysiological recording to study changes in the level of Kv4.3 mRNA and Ito in human tissues and isolated ventricular myocytes, respectively. We found that the level of Kv4.3 mRNA decreased by 30% in failing hearts compared with nonfailing controls. Furthermore, this reduction correlated with the reduction in peak Ito density measured in ventricular myocytes isolated from adjacent regions of the heart. There was no significant change in the steady-state level of any other mRNA studied (HERG, Kv1.4, Kir2.1, Kvss1.3, and the alpha1C subunit of the Ca2+ channel). mRNAs encoding Kv1.2, Kv1.5, and Kv2.1 were found in low abundance in human ventricle. CONCLUSIONS: These data provide further support for the hypothesis that Kv4.3 encodes all or part of the native cardiac Ito in humans and that part of the downregulation of this current in heart failure may be transcriptionally regulated.

Action Potentials↗
PubMed2006

Mallotoxin is a novel human ether-a-go-go-related gene (hERG) potassium channel activator.

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.

Acetophenones↗
PubMed2000

Effect of low external calcium on the ERG current of NG108-15 cells.

K(+) currents through ERG (ether-à-go-go related gene) channels were recorded in whole-cell voltage clamped NG108-15 neuroblastomaxglioma hybrid cells. The channels were fully activated by low holding potential (V(H)=-20 mV) and long depolarizing prepulses. Hyperpolarizing pulses elicited inward currents which deactivated after reaching a peak. Lowering [Ca(2+)](o) from 5 to 1. 5 or 0.5 mM decreased tau(-1), the rate constant of deactivation. The effect can be explained by a shift of the tau(-1)(V) curve to more negative potentials caused by an increase in surface charge density. Plotting tau(-1) against [Ca(2+)](o) for different potentials yielded straight lines; their slope was independent of potential at -140 to -120 mV and decreased at more positive potentials. The time to peak curve and the maximum of the steady-state inward current were also shifted to more negative potentials. In addition, peak ERG inward current increased. Raising [Ca(2+)](o) from 5 to 10 mM accelerated deactivation and decreased the peak current. 5 mM Ba(2+) affected tau(-1) similarly and inhibited peak current more strongly whereas 5 mM Mg(2+) was less potent. As found by Faravelli et al. (J. Physiol. 496 (1996) 13), bath solutions devoid of divalent cations (0 Ca(2+), 0 Mg(2+), 0.1 or 1.1 mM EGTA) abolished deactivation almost completely. The phenomenon was seen with bath containing either 40 or 6.5 mM K(+). Its occurrence was favored by raising the temperature to 34 degrees C. It suggests a particular requirement of channel closing for Ca(2+).

Animals↗
PubMed1999

Ion channels: structure of a molecular brake.

A combination of crystallographic and mutagenesis studies on the HERG K+ channel, a key determinant of cardiac excitability, has suggested how the protein's extramembraneous amino-terminal domain might act as a 'molecular brake' that slows down channel deactivation.

Animals↗
PubMed2001

ERG proteins and functional cardiac I(Kr) channels in rat, mouse, and human heart.

The voltage-gated K(+) channel (Kv) pore forming alpha subunit, ERG1 (KCNH2), has been identified as the locus of mutations in one type of inherited long QT syndrome, LQT2. Heterologous expression of ERG1 reveals rapidly activating and inactivating K(+) currents, characterized by marked inward rectification at potentials positive to 0 mV, which are similar to the rapid component of cardiac delayed rectification I(Kr). There are, however, marked differences in the properties of expressed ERG1 and endogenous cardiac I(Kr), suggesting that functional I(Kr) channels reflect the coassembly of full-length ERG1 with splice variants and /or accessory subunits. Consistent with these hypotheses, N- and C-terminal variants of ERG1 have been identified, and it has been demonstrated that heterologously expressed ERG1 and minK (or MiRP1) coimmunoprecipitate. Recent biochemical studies, however, suggest that only full-length ERG1 is expressed in adult mouse, rat, or human heart. Clearly, further studies, focused on identifying the subunits that coassemble with ERG1 in vivo, as well as on post-translational processing of the full-length ERG1 protein will be necessary to define the molecular composition of functional cardiac I(Kr) channels.

Animals↗
PubMed2005

Mechanism of action of a novel human ether-a-go-go-related gene channel activator.

1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) is a newly discovered activator of human ether-a-go-go-related gene (hERG) K(+) channels. Here, we characterize the effects of this compound on cloned hERG channels heterologously expressed in Xenopus laevis oocytes. When assessed with 2-s depolarizations, NS1643 enhanced the magnitude of wild-type hERG current in a concentration- and voltage-dependent manner with an EC(50) of 10.4 microM at -10 mV. The fully activated current-voltage relationship revealed that the drug increased outward but not inward currents, consistent with altered inactivation gating. NS1643 shifted the voltage dependence of inactivation by +21 mV at 10 microM and +35 mV at 30 microM, but it did not alter the voltage dependence of activation of hERG channels. The effects of the drug on three inactivation-deficient hERG mutant channels (S620T, S631A, and G628C/S631C) were determined. In the absence of channel inactivation, NS1643 did not enhance hERG current magnitude. The agonist activity of NS1643 was facilitated by mutations (F656 to Val, Met, or Thr) that are known to greatly attenuate channel inhibition by hERG blockers. We conclude that NS1643 is a partial agonist of hERG channels and that the mechanism of activation is reduced channel inactivation.

Animals↗