Ether-a-go-go-related Gene Channels Research Articles

Locus Coeruleus Neurons’ Firing Pattern Is Regulated by ERG Voltage-Gated K+ Channels

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG (ether-à-go-go-related gene) K+ channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

ERG Channels Regulate Excitability in Stellate and Bushy Cells of Mice Ventral Cochlear Nucleus.

ERG (ether-a-go-go-related gene) channels are the members of the voltage-dependent potassium channel family, which have three subtypes, as ERG1 (Kv 11.1), ERG2 (Kv 11.2), and ERG3 (Kv11.3). There is no information on ERG channels in the cochlear nucleus (CN) neurons, which is the first relay station of the auditory pathway. As occur in some of congenital long QT Syndromes (LQTS), mutation of the KCNQ11 genes for ERG channel has been reported to be accompanied by hearing loss. For that reason, we aimed to study biophysical properties and physiological importance, and contribution of ERG K+ currents to the formation of action potentials in the stellate and bushy neurons of the ventral cochlear nucleus (VCN). A total of 70 mice at 14-17days old were used for this study. Electrophysiological characterization of ERG channels was performed using patch-clamp technique in the CN slices. In current clamp, ERG channel blockers, terfenadine (10µM) and E-4031 (10µM), were applied in both cell types. The activation, inactivation, and deactivation kinetics of the ERG channels were determined by voltage clamp. In conclusion, the findings obtained in the present study suggest that stellate and bushy neurons express ERG channels and ERG channels appear to contribute to setting action potential (AP) frequency, threshold for AP induction, and, possibly, resting membrane potentials in this cells.

Modulation of Ether-à-Go-Go Related Gene (ERG) Current Governs Intrinsic Persistent Activity in Rodent Neocortical Pyramidal Cells.

While cholinergic receptor activation has long been known to dramatically enhance the excitability of cortical neurons, the cellular mechanisms responsible for this effect are not well understood. We used intracellular recordings in rat (both sexes) neocortical brain slices to assess the ionic mechanisms supporting persistent firing modes triggered by depolarizing stimuli following cholinergic receptor activation. We found multiple lines of evidence suggesting that a component of the underlying hyperexcitability associated with persistent firing reflects a reduction in the standing (leak) K+ current mediated by Ether-a-go-go-Related Gene (ERG) channels. Three chemically diverse ERG channel blockers (terfenadine, ErgToxin-1, and E-4031) abolished persistent firing and the underlying increase in input resistance in deep pyramidal cells in temporal and prefrontal association neocortex. Calcium accumulation during triggering stimuli appears to attenuate ERG currents, leading to membrane potential depolarization and increased input resistance, two critical elements generating persistent firing. Our results also suggest that ERG current normally governs cortical neuron responses to depolarizing stimuli by opposing prolonged discharges and by enhancing the poststimulus repolarization. The broad expression of ERG channels and the ability of ERG blocks to abolish persistent firing evoked by both synaptic and intracellular step stimuli suggest that modulation of ERG channels may underlie many forms of persistent activity observed in vivoSIGNIFICANCE STATEMENT Persistent activity, where spiking continues beyond the triggering stimulus, is a common phenomenon observed in many types of neurons. Identifying the mechanism underlying this elementary process of memory is a step forward in understanding higher cognitive function including short-term memory. Our results suggest that a reduction in the currents normally mediated by Ether-a-go-go-Related Gene (ERG) K+ channels contributes to persistent firing in neocortical pyramidal cells. ERG currents have been previously studied primarily in the heart; relatively little is known about ERG function in the brain, although mutations in ERG channels have recently been linked to schizophrenia. The present study is among the first to describe its role in neocortex in relation to biophysical correlates of memory function.

Inhibition of Schistosoma mansoni ether-a-go-go related gene-encoded potassium channels leads to hypermotility and impaired egg production

The purpose of this work was to investigate the effect of ether-a-go-go related gene (ERG) potassium channel inhibition on Schistosoma mansoni. Use of dofetilide to block the schistosome ERGs resulted in a striking ‘corkscrew’ effect. The worms were unable to control their motility; they were hypermotile. The treated worms produced abnormal eggs, some of which consisted of little more than a spine. One of the S. mansoni ERGs (SmERGs), Smp_161140, was chosen for further study by RNAi. The transcript was knocked down to 50% compared to the controls. These RNAi-treated worms demonstrated seizure-like movements. In S. mansoni, as in other organisms, ERG channels seem to play a role in regulating muscle excitability. This work shows that egg production can be greatly reduced by effectively targeting muscle coordination in these important parasites.

ICA-105574 Interacts with a Common Binding Site to Elicit Opposite Effects on Inactivation Gating of EAG and ERG Potassium Channels

Rapid and voltage-dependent inactivation greatly attenuates outward currents in ether-a-go-go-related gene (ERG) K(+) channels. In contrast, inactivation of related ether-a-go-go (EAG) K(+) channels is very slow and minimally reduces outward currents. ICA-105574 (ICA, or 3-nitro-N-[4-phenoxyphenyl]-benzamide) has opposite effects on inactivation of these two channel types. Although ICA greatly attenuates ERG inactivation by shifting its voltage dependence to more positive potentials, it enhances the rate and extent of EAG inactivation without altering its voltage dependence. Here, we investigate whether the inverse functional response to ICA in EAG and ERG channels is related to differences in ICA binding site or to intrinsic mechanisms of inactivation. Molecular modeling coupled with site-directed mutagenesis suggests that ICA binds in a channel-specific orientation to a hydrophobic pocket bounded by the S5/pore helix/S6 of one subunit and S6 of an adjacent subunit. ICA is a mixed agonist of mutant EAG and EAG/ERG chimera channels that inactivate by a combination of slow and fast mechanisms. With the exception of three residues, the specific amino acids that form the putative binding pocket for ICA in ERG are conserved in EAG. Mutations introduced into EAG to replicate the ICA binding site in ERG did not alter the functional response to ICA. Together these findings suggest that ICA binds to the same site in EAG and ERG channels to elicit opposite functional effects. The resultant agonist or antagonist activity is determined solely by channel-specific differences in the mechanisms of inactivation gating.

Functional characterization of ether-à-go-go-related gene potassium channels in midbrain dopamine neurons - implications for a role in depolarization block.

Bursting activity by midbrain dopamine neurons reflects the complex interplay between their intrinsic pacemaker activity and synaptic inputs. Although the precise mechanism responsible for the generation and modulation of bursting in vivo has yet to be established, several ion channels have been implicated in the process. Previous studies with nonselective blockers suggested that ether-à-go-go-related gene (ERG) K(+) channels are functionally significant. Here, electrophysiology with selective chemical and peptide ERG channel blockers (E-4031 and rBeKm-1) and computational methods were used to define the contribution made by ERG channels to the firing properties of midbrain dopamine neurons in vivo and in vitro. Selective ERG channel blockade increased the frequency of spontaneous activity as well as the response to depolarizing current pulses without altering spike frequency adaptation. ERG channel block also accelerated entry into depolarization inactivation during bursts elicited by virtual NMDA receptors generated with the dynamic clamp, and significantly prolonged the duration of the sustained depolarization inactivation that followed pharmacologically evoked bursts. In vivo, somatic ERG blockade was associated with an increase in bursting activity attributed to a reduction in doublet firing. Taken together, these results show that dopamine neuron ERG K(+) channels play a prominent role in limiting excitability and in minimizing depolarization inactivation. As the therapeutic actions of antipsychotic drugs are associated with depolarization inactivation of dopamine neurons and blockade of cardiac ERG channels is a prominent side effect of these drugs, ERG channels in the central nervous system may represent a novel target for antipsychotic drug development.

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na+ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K+ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na+ currents verified here, this venom certainly warrant further investigation.

A team of potassium channels tunes up auditory neurons

Mammals harbour a diverse array of voltage-gated K+ channels. How those channels team up in a neuron to do a job is becoming clear in the principal cells of the medial nucleus of the trapezoid body (MNTB). Ian Forsythe and his colleagues have used the electrically compact nature of MNTB neurons to measure K+ currents and combined those findings with molecular biological studies to identify, characterize and localize diverse K+ channels on MNTB neurons. A series of reports, the latest by Hardman and Forsythe being in this issue of The Journal of Physiology (Hardman & Forsythe, 2009), show that the firing of action potentials in MNTB neurons is finely controlled by a team of K+ conductances with different properties. Neurons in the MNTB are part of a neuronal circuit that assesses differences in intensity at the two ears, cues that are used to determine where in the horizontal plane sounds arise (Fig. 1A). Neurons in the lateral superior olive (LSO) compare interaural intensity by summing excitation from the ipsilateral ear with inhibition from the contralateral ear. Anyone who has participated in a conversation knows that sounds change rapidly in frequency and intensity; for interaural comparisons of intensity to be useful for localizing sounds, the time of arrival of excitation and inhibition at the LSO must be matched to within a few hundred microseconds. Figure 1 The medial nucleus of the trapezoid body (MNTB) feeds information about contralateral sounds to neurons in the lateral superior olive (LSO) that compare interaural intensity by balancing ipsilateral excitation (red) with contralateral inhibition (blue) ... How are the delays in ipsi- and contralateral inputs to the LSO matched in timing when path from the contralateral ear to the LSO is longer and includes an extra synapse? Neurons from the ipsilateral cochlear nucleus (CN) bring excitation to LSO neurons through relatively fine axons. Globular bushy cells from the contralateral CN, whose axons are particularly large and rapidly conducting, innervate MNTB neurons through calyces of Held. A calyx excites an MNTB neuron with a short and constant synaptic delay, generating large, suprathreshold EPSPs. The team of K+ channels allows MNTB neurons to relay the temporal firing pattern of globular bushy cells to contralateral LSO neurons as precisely timed, glycinergic IPSPs. Which are the members of the team of K+ channels? Voltage-gated K+ channels are formed a tetrameric combinations of α subunits from within a class of channels (e.g. Kv1). Figure 1B summarizes what is known about the presence of various α subunits in the MNTB. (1) Channels of the Kv1 family, located in the pre- and postsynaptic axons as well as on somas of MNTB neurons, provide a prominent, rapidly activating, low-voltage-activated K+ current whose activation around rest shapes synaptic potentials and prevents repetitive firing in both the calyceal terminals and in MNTB neurons (Dodson et al. 2003). (2) Channels that contain Kv2.2 are prominent in the initial segments of axons of MNTB neurons. These high-voltage-activated, slowly activating and deactivating channels hyperpolarize neurons between action potentials when they fire rapidly. In promoting repolarization between action potentials, these channels enhance Na+ currents by removing inactivation when MNTB neurons fire rapidly (Johnston et al. 2008b). (3) Channels containing Kv3.1b mediate a prominent high-voltage-activated, rapidly activating and inactivating K+ current that repolarizes action potentials, making them narrow. Kv3.1b channels are in calyceal terminals as well as on the somatic membranes of MNTB neurons. When they are phosphorylated, Kv3.1b subunits have a lower open probability. Rapid firing induced by electrical stimulation or by a noisy acoustic environment causes a decrease in phosphorylation and an increase in this K+ current (Song et al. 2005). (4) Kv4.3 subunits have been found in the MNTB of mice but not rats (Johnston et al. 2008a). Channels of the Kv4 family mediate A-currents. These K+ currents inactivate rapidly at relatively hyperpolarized potentials making them transient and most evident when depolarization is preceded by hyperpolarization. This small current would be expected to shorten and diminish EPSPs early in a train but becomes inactivated with strong stimulation. (5) Now Hardman and Forsythe show that Kv11 channels encoded by an ether-a-go-go-related gene (ERG) are also present in MNTB neurons of mice. These channels inactivate more rapidly than they activate so that they conduct current mainly upon repolarization after inactivation is removed but before they deactivate. The current through ERG channels repolarizes MNTB neurons, its action being most prominent near the resting potential after a depolarization. K+ channels work as a team to sharpen EPSPs, to sharpen action potentials, and to assure that repolarization removes Na+ inactivation even during rapid firing. K+ channels enable MNTB neurons to fire robustly and to signal with temporal precision even when they are driven at high rates.

Two Novel Ergtoxins, Blockers of K+-channels, Purified from the Mexican Scorpion Centruroides elegans elegans

Voltage-gated potassium channels of the ether-a-go-go related gene (ERG) family are implicated in many important cellular processes. Three such genes have been cloned (erg1, erg2 and erg3) and shown to be expressed in the central nervous system (CNS) of mammalians. This communication describes the isolation and characterization of two isoforms of scorpion toxin (CeErg4 and CeErg5, systematic nomenclature gamma-KTx1.7 and gamma-KTx1.8, respectively) that can discriminate the various subtypes of ERG channels of human and rat. These peptides were purified from the venom of the Mexican scorpion Centruroides elegans elegans. They contain 42 amino acid residues, tightly folded by four disulfide bridges. Both peptides block in a reversible manner human and rat ERG1 channels, but have no effect on human ERG2. They also block completely and irreversibly the rat ERG2 and the human ERG3 channels hence are excellent tools for the discrimination of the various sub-types of ion-channels studied.

Functional investigations of ERG potassium channels on INS1 cells with the blocker E-4031

The ERG (ether-a-go-go-related gene) channels are voltage controlled potassium channels, that first have been described in a mutant of Drosophila melanogaster in 1991. In heart muscle cells these channels are essential for the repolarisation of action potentials. Mutations in the ERG gene may lead to a prolonged QT-interval (long QT-syndrome). In human pancreatic β-cells, ERG channels are known to influence the insulin secretion (Rosati et al.). In our group, Erg channel mRNA expression was measured by real-time PCR in rat insulinoma cells (INS1), rat pancreatic islets and endocrine pancreatic tissue (Mühlbauer et al.). Functional properties of ERG channels were investigated by electrophysiology and calcium imaging. The channels were selectively blocked by the methanesulfonanilide E-4031 (1µmol/l). Whole cell voltage clamp measurements from INS1 cells showed E-4031 sensitive currents, leading to a total decrease of the membrane current by 12.2±2.4% (n=6). Current clamp measurements of the membrane potential (perforated patch technique) from spontaneously silent INS1 cells led to a depolarisation of (4.4±0.8) mV (n=9). In spontaneously spiking cells the spiking frequency was raised threefold by the blocker E-4031. These effects of E4031 may influence voltage controlled calcium channels. Thus, an increase of the internal calcium concentration during blocking of the ERG channels was measured by use of confocal laser scanning microscopy. The investigations led to the conclusion, that INS1 cells express functional active and E-4031 sensitive ERG channels which might be linked to insulin secretion.

Ether-a-go-go Related Gene Potassium Channels: What's All the Buzz About?

Antipsychotic drugs are thought to exert their therapeutic action by antagonizing dopamine receptors but are also known to produce side effects in the heart by inhibiting cardiac ether-a-go-go-related gene (ERG) K(+) channels. Recently, it has been discovered that the same channels are present in the brain, including midbrain dopamine neurons. ERG channels are most active after the cessation of intense electrical activity, and blockade of these channels prolongs plateau potentials in bursting dopamine neurons. This change in excitability would be expected to alter dopamine release. Therefore, the therapeutic action of antipsychotic drugs may depend on inhibition of both postsynaptic dopamine receptors and presynaptic ERG K(+) channels.

Characterization of depolarization‐evoked ERG K+ currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) harbour the ether-a-go-go related gene (ERG) channel as shown by its characteristic rapidly deactivating current upon hyperpolarization. This property, however, does not explain the marked increase in cell excitability by ERG channel blockers, namely an increase in slow wave plateau duration and action potential generation. The objective of the present study was to characterize the depolarization-activated, E4031-sensitive ERG currents in murine ICC within a range of physiologically relevant membrane potentials. Whole cell currents were recorded from ICC isolated from murine neonatal jejunum, superfused with a physiological salt solution and with high intracellular Cs(+) to block most other K(+) currents. Upon depolarizing the cell from the resting membrane potential (approximately -60 mV) towards the region of the slow wave plateau (approximately -30 mV), significant sustained (window) current was generated between the potentials of -40 to 0 mV (maximal at -30 mV) and inhibited by the ERG specific blocker E4031. Channel activation followed by rapid inactivation produced a steady state conductance at -30 mV which was 51.6 +/- 11% of the hyperpolarization-evoked peak conductance value at -100 mV. When the cell repolarized from -30 mV, again, significant currents were generated, indicating recovery from inactivation, a typical characteristic of ERG channels. These data provide evidence that the ERG channel is of significance in the regulation of ICC excitability and provide the mechanism by which ERG channel blockade increases the slow wave duration.

Activation of Human ether-a-go-go-Related Gene Potassium Channels by the Diphenylurea 1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643)

The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased repolarization current could be a new antiarrhythmic principle, because it possibly would attenuate afterdepolarizations, ischemic leak currents, and reentry phenomena. Repolarization of the cardiac myocytes is crucially dependent on the late rapid delayed rectifier current (I(Kr)) conducted by ether-a-go-go-related gene (ERG) potassium channels. We have developed the diphenylurea compound 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and tested whether this small organic molecule could increase the activity of human ERG (HERG) channels expressed heterologously. In Xenopus laevis oocytes, NS1643 increased both steady-state and tail current at all voltages tested. The EC(50) value for HERG channel activation was 10.5 microM. These results were reproduced on HERG channels expressed in mammalian human embryonic kidney 293 cells. In guinea pig cardiomyocytes, studied by patch clamp, application of 10 microM NS1643 activated I(Kr) and significantly decreased the action potential duration to 65% of the control values. The effect could be reverted by application of the specific HERG channel inhibitor 4'-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidinyl]carbonyl]-methanesulfonanilide (E-4031) at 100 nM. Application of NS1643 also resulted in a prolonged postrepolarization refractory time. Finally, cardiomyocytes exposed to NS1643 resisted reactivation by small depolarizing currents mimicking early afterdepolarizations. In conclusion, HERG channel activation by small molecules such as NS1643 increases the repolarization reserve and presents an interesting new antiarrhythmic approach.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K+ channels

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K+ channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastoma×glioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12μg/ml.

Slowing of ERG current deactivation in NG108-15 cells by the histidine-specific reagent diethylpyrocarbonate

The aim of this study was to explore and characterize the effect of the histidine-specific reagent diethylpyrocarbonate (DEPC) on the ERG ( ether-à-go-go related gene) channels of whole-cell voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. The channels were fully activated by long depolarizing prepulses. Hyperpolarizing pulses elicited K + inward currents which deactivated after reaching a peak. DEPC (0.26–2.1 mM, externally applied for 5–12 min) irreversibly decreased τ −1, the rate constant of deactivation. At a pulse potential of −120 mV τ −1 decreased on average by 60%. The effect can be described as a −25 mV shift of the τ −1( V) curve. The activation curve and the curve relating steady-state current to pulse potential were shifted by similar amounts. The decrease of τ −1 was the same at 0.26 and at 2.1 mM, but developed faster at the higher concentration. The slowing of deactivation was only seen when the cells were held at a potential of −20 mV. At this potential it developed with a time constant of 47 s. At more negative holding potentials (−40 or −70 mV) only a slight reduction of the peak occurred. The observations suggest preferential binding of DEPC to the open and inactivated channel states. The DEPC effect can possibly be explained by the reaction of DEPC with histidine residues or other amino acids in the external loops of the channel. However, dichloro-(2,2′:6′,2″-terpyridine)-platinum (II) dihydrate (DTPD), another histidine-specific reagent, markedly decreased the peak current without affecting τ −1. Therefore, the possibility that (some of) the effects of DEPC and DTPD are unrelated to their property as histidine-specific reagents cannot be excluded.

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 neuroblastoma×glioma hybrid cells. The channels were fully activated by low holding potential (VH=−20 mV) and long depolarizing prepulses. Hyperpolarizing pulses elicited inward currents which deactivated after reaching a peak. Lowering [Ca2+]o from 5 to 1.5 or 0.5 mM decreased τ−1, the rate constant of deactivation. The effect can be explained by a shift of the τ−1(V) curve to more negative potentials caused by an increase in surface charge density. Plotting τ−1 against [Ca2+]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 [Ca2+]o from 5 to 10 mM accelerated deactivation and decreased the peak current. 5 mM Ba2+ affected τ−1 similarly and inhibited peak current more strongly whereas 5 mM Mg2+ was less potent. As found by Faravelli et al. (J. Physiol. 496 (1996) 13), bath solutions devoid of divalent cations (0 Ca2+, 0 Mg2+, 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°C. It suggests a particular requirement of channel closing for Ca2+.

Inactivation of the ERG Current in NG108-15 Cells

Differentiated NG108-15 neuroblastoma × glioma hybrid cells were whole-cell voltage clamped. The rate of inactivation of ERG (ether-à-go-go related gene) potassium channels was measured with a three-pulse protocol. Contamination with delayed rectifier current at positive potentials was avoided by using the selective ERG channel blocker E-4031. The curve relating time constant of inactivation τ to membrane potential V could be fitted by a Gauss curve. In a bath with 40 mM K+, the curve peaked at V = −36 mV. Lowering [K+]o decreased τ. At V = −20 mV, the average τ was 25.4 ms in 40 mM K+, 20.6 ms in 6.5 mM K+, and 15.0 ms in 0 mM K+. This resembles the relation between τ and [K+]o in ERG channels expressed in Xenopus oocytes.

Functional role of the slow activation property of ERG K+ channels.

ERG (ether-à-go-go-related gene) K+ channels are crucial in human heart physiology (h-ERG), but are also found in neuronal cells and are impaired in Drosophila 'seizure' mutants. Their biophysical properties include the relatively fast kinetics of the inactivation gate and much slower kinetics of the activation gate. In order to elucidate how the complex time- and voltage-dependent activation properties of ERG channels underlies distinct roles in excitability, we investigated different types of ERG channels intrinsically present in cells or heterologously expressed in mammalian cells or Xenopus oocytes. Voltage-dependent activation curves were highly dependent on the features of the eliciting protocols. Only very long preconditioning times produced true steady-state relationships, a fact that has been largely neglected in the past, hampering the comparison of published data on ERG channels. Beyond this technical aspect, the slow activation property of ERG can be responsible for unsuspected physiological roles. We found that around the midpoint of the activation curve, the time constant of ERG open-close kinetics is of the order of 10-15 s. During sustained trains of depolarizations, e.g. those produced in neuronal firing, this leads to the use-dependent accumulation of open-state ERG channels. Accumulation is not observed in a mutant with a fast activation gate. In conclusion, it is well established that other K+ channels (i.e. Ca2+-activated and M) control the spike-frequency adaptation, but our results support the notion that the purely voltage-dependent activation property of ERG channels would allow a slow inhibitory physiological role in rapid neuronal signalling.