Erg Currents Research Articles

Ether-à-go-go K+ channels: effective modulators of neuronal excitability.

Mammalian ether-à-go-go (EAG) channels are voltage-gated K+ channels. They are encoded by the KCNH gene family and divided into three subfamilies, eag (Kv10), erg (eag-related gene; Kv11) and elk (eag-like; Kv12). All EAG channel subtypes are expressed in the brain where they effectively modulate neuronal excitability. This Topical Review describes the biophysical properties of each of the EAG channel subtypes, their function in neurons and the neurological diseases induced by EAG channel mutations. In contrast to the function of erg currents in the heart, where they contribute to repolarization of the cardiac action potential, erg currents in neurons are involved in the maintenance of the resting potential, setting of action potential threshold and frequency accommodation. They can even support high frequency firing by preventing a depolarization-induced Na+ channel block. EAG channels are modulated differentially, e.g. eag channels by intracellular Ca2+ , erg channels by extracellular K+ and GPCRs, and elk channels by changes in pH. So far, only currents mediated by erg channels have been recorded in neurons with the help of selective blockers. Neuronal eag and elk currents have not been isolated due to the lack of suitable channel blockers. However, findings in KO mice indicate a physiological role of eag1 currents in synaptic transmission and an involvement of elk2 currents in cognitive performance. Human eag1 and eag2 gain-of-function mutations underlie syndromes associated with epileptic seizures.

Erg Potassium Currents of Neonatal Mouse Purkinje Cells Exhibit Fast Gating Kinetics and Are Inhibited by mGluR1 Activation

We investigated the subthreshold properties of an erg (ether-à-go-go-related gene) K(+) current in Purkinje cells of neonatal mice. Action potentials recorded from Purkinje cells in cerebellar slices exhibited a decreased threshold potential and increased frequency of spontaneous and repetitive activity following application of the specific erg channel blocker E-4031. Accommodation was absent before and after drug application. The erg current of these Purkinje cells activated at membrane potentials near -60 mV and exhibited fast gating kinetics. The functional importance of fast gating subthreshold erg channels in Purkinje cells was corroborated by comparing the results of action potential clamp experiments with erg1a, erg1b, erg2, and erg3 currents heterologously expressed in HEK cells. Computer simulations based on a NEURON model of Purkinje cells only reproduced the effects of the native erg current when an erg channel conductance like that of erg3 was included. Experiments with subunit-sensitive toxins (BeKm-1, APETx1) indicated that erg channels in Purkinje cells are presumably mediated by heteromeric erg1/erg3 or modified erg1 channels. Following mGluR1 activation, the native erg current was reduced by ∼70%, brought about by reduction of the maximal erg current and a shift of the activation curve to more positive potentials. The Purkinje cell erg current contributed to the sustained current component of the biphasic mGluR1 response. Activation of mGluR1 by the agonist 3,4-dihydroxyphenylglycol increased Purkinje cell excitability, similar to that induced by E-4031. The results indicated that erg currents can be modulated and may contribute to the mGluR1-induced plasticity changes in Purkinje cells.

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K+ channels

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca(2+) levels has been shown to rely on G(q/11) and PLC activation, the molecular mechanisms involved in the regulation of ERG K(+) channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH(3) cells β-ARK/CT, Gα(t), and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V (1/2). Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH(3) cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH(3) cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca(2+) and ERG responses in GH(3) cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH(3) and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

Gonadotropin-Releasing Hormone Inhibits Ether-à-Go-Go-Related Gene K+ Currents in Mouse Gonadotropes

Secretion of LH from gonadotropes is initiated by a GnRH-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). This increase in [Ca(2+)](i) is the result of Ca(2+) release from intracellular stores and Ca(2+) influx through voltage-dependent Ca(2+) channels. Here we describe an ether-à-go-go-related gene (erg) K(+) current in primary mouse gonadotropes and its possible function in the control of Ca(2+) influx. To detect gonadotropes, we used a knock-in mouse strain, in which GnRH receptor-expressing cells are fluorescently labeled. Erg K(+) currents were recorded in 80-90% of gonadotropes. Blockage of erg currents by E-4031 depolarized the resting potential by 5-8 mV and led to an increase in [Ca(2+)](i), which was abolished by nifedipine. GnRH inhibited erg currents by a reduction of the maximal erg current and in some cells additionally by a shift of the activation curve to more positive potentials. In conclusion, the erg current contributes to the maintenance of the resting potential in gonadotropes, thereby securing a low [Ca(2+)](i) by restricting Ca(2+) influx. In addition, the erg channels are modulated by GnRH by an as-yet unknown signal cascade.

Erg K+ currents modulate excitability in mouse mitral/tufted neurons

Different erg (ether-à-go-go-related gene; Kv11) K+ channel subunits are expressed throughout the brain. Especially mitral cells of the olfactory bulb are stained intensely by erg1a, erg1b, erg2, and erg3 antibodies. This led us to study the erg current in mitral/tufted (M/T) neurons from mouse olfactory bulb in primary culture. M/T neurons were identified by their morphology and presence of mGluR1 receptors, and RT-PCR demonstrated the expression of all erg subunits in cultured M/T neurons. Using an elevated external K+ concentration, a relatively uniform erg current was recorded in the majority of M/T cells and isolated with the erg channel blocker E-4031. With 4-s depolarizations, the erg current started to activate at -65 mV and exhibited half maximal activation at -51 mV. An increase in the external K+ concentration resulted in an increase in erg whole-cell conductance. The specific group 1 mGluR agonist, DHPG, which depolarizes mitral cells, reduced erg channel availability. DHPG accelerated erg current deactivation, reduced the maximum current amplitude, and shifted availability and activation curves to more depolarized potentials. A pharmacological block of erg channels depolarized the resting potential of M/T cells and clearly demonstrated the involvement of erg channels in the control of mitral cell excitability.

Ether‐à‐go‐go‐related gene K+ channels contribute to threshold excitability of mouse auditory brainstem neurons

The ionic basis of excitability requires identification and characterisation of expressed channels and their specific roles in native neurons. We have exploited principal neurons of the medial nucleus of the trapezoid body (MNTB) as a model system for examining voltage-gated K(+) channels, because of their known function and simple morphology. Here we show that channels of the ether-à-go-go-related gene family (ERG, Kv11; encoded by kcnh) complement Kv1 channels in regulating neuronal excitability around threshold voltages. Using whole-cell patch clamp from brainstem slices, the selective ERG antagonist E-4031 reduced action potential (AP) threshold and increased firing on depolarisation. In P12 mice, under voltage-clamp with elevated [K(+)](o) (20 mm), a slowly deactivating current was blocked by E-4031 or terfenadine (V(0.5,act) = -58.4 +/- 0.9 mV, V(0.5,inact) = -76.1 +/- 3.6 mV). Deactivation followed a double exponential time course (tau(slow) = 113.8 +/- 6.9 ms, tau(fast) = 33.2 +/- 3.8 ms at -110 mV, tau(fast) 46% peak amplitude). In P25 mice, deactivation was best fitted by a single exponential (tau(fast) = 46.8 +/- 5.8 ms at -110 mV). Quantitative RT-PCR showed that ERG1 and ERG3 were the predominant mRNAs and immunohistochemistry showed expression as somatic plasma membrane puncta on principal neurons. We conclude that ERG currents complement Kv1 currents in limiting AP firing at around threshold; ERG may have a particular role during periods of high activity when [K(+)](o) is elevated. These ERG currents suggest a potential link between auditory hyperexcitability and acoustic startle triggering of cardiac events in familial LQT2.

Emerging roles for ether‐á‐go‐go‐related gene potassium channels in the brain

Emerging roles for ether‐á‐go‐go‐related gene potassium channels in the brain

Effects of TRH on heteromeric rat erg1a/1b K+ channels are dominated by the rerg1b subunit

The erg1a (HERG) K+ channel subunit and its N-terminal splice variant erg1b are coexpressed in several tissues and both isoforms have been shown to form heteromultimeric erg channels in heart and brain. The reduction of erg1a current by thyrotropin-releasing hormone (TRH) is well studied, but no comparable data exist for erg1b. Since TRH and TRH receptors are widely expressed in the brain, we have now studied the different TRH effects on the biophysical properties of homomeric rat erg1b as well as heteromeric rat erg1a/1b channels. The erg channels were overexpressed in the clonal somatomammotroph pituitary cell line GH3/B6, which contains TRH receptors and endogenous erg channels. Compared to rerg1a, homomeric rerg1b channels exhibited not only faster deactivation kinetics, but also considerably less steady-state inactivation, and half-maximal activation occurred at about 10 mV more positive potentials. Coexpression of both isoforms resulted in erg currents with intermediate properties concerning the deactivation kinetics, whereas rerg1a dominated the voltage dependence of activation and rerg1b strongly influenced steady-state inactivation. Application of TRH induced a reduction of maximal erg conductance for all tested erg1 currents without effects on the voltage dependence of steady-state inactivation. Nevertheless, homomeric rerg1b channels significantly differed in their response to TRH from rerg1a channels. The TRH-induced shift in the activation curve to more positive potentials, the dramatic slowing of activation and the acceleration of deactivation typical for rerg1a modulation were absent in rerg1b channels. Surprisingly, most effects of TRH on heteromeric rerg1 channels were dominated by the rerg1b subunit.

Specificity of TRH receptor coupling to G‐proteins for regulation of ERG K+ channels in GH3 rat anterior pituitary cells

The identity of the G-protein coupling thyrotropin-releasing hormone (TRH) receptors to rat ether-à-go-go related gene (r-ERG) K+ channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH(3) cells and dominant-negative variants (Galpha-QL/DN) of G-protein alpha subunits. Expression of dominant-negative Galpha(q/11) that minimizes the TRH-induced Ca2+ signal had no effect on r-ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant-negative variants of Galpha13 and the small G-protein Rho caused a significant loss of the inhibitory effect of TRH on r-ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant-negative Galpha(s) or transducin alpha subunits, an agent known to sequester free G-protein betagamma dimers. As a further indication of specificity of the dominant-negative effects, only the dominant-negative variants of Galpha13 and Rho (but not Galpha(s)-QL/DN or Galpha(t)) were able to reduce the TRH-induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a G(q/11) protein for transducing the Ca2+ signal during the initial response to TRH, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in pituitary cells. They also identify G(s) (or a G(s)-like protein) and G13 as important contributors to the hormonal effect in these cells and suggest that betagamma dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.

Fast erg K+ currents in rat embryonic serotonergic neurones

Ether-á-go-go-related gene (erg) channels form one subfamily of the ether-á-go-go (EAG) K(+) channels and all three erg channels (erg1-3) are expressed in the brain. In the present study we characterize a fast erg current in neurones in primary culture derived from the median part of rat embryonic rhombencephala (E15-16). The relatively uniform erg current was regularly found in large multipolar serotonergic neurones, and occurred also in other less well characterized neurones. The erg current was blocked by the antiarrhythmic substance E-4031. Single-cell RT-PCR revealed the expression of erg1a, erg1b, erg2 and erg3 mRNA in different combinations in large multipolar neurones. These cells also contained neuronal tryptophan hydroxylase, a key enzyme for serotonin production. To characterize the molecular properties of the channels mediating the native erg current, we compared the voltage and time dependence of activation and deactivation of the neuronal erg current to erg1a, erg1b, erg2 and erg3 currents heterologously expressed in CHO cells. The biophysical properties of the neuronal erg current were well within the range displayed by the different heterologously expressed erg currents. Activation and deactivation kinetics of the neuronal erg current were fast and resembled those of erg3 currents. Our data suggest that the erg channels in rat embryonic rhombencephalon neurones are heteromultimers formed by different erg channel subunits.

Interneurons transiently express the ERG K + channels during development of mouse spinal networks in vitro

During spinal cord maturation neuronal excitability gradually differentiates to meet different functional demands. Spontaneous activity, appearing early during spinal development, is regulated by the expression pattern of ion channels in individual neurons. While emerging excitability of embryonic motoneurons has been widely investigated, little is known about that of spinal interneurons. Voltage-dependent K + channels are a heterogeneous class of ion channels that accomplish several functions. Recently voltage-dependent K + channels encoded by erg subfamily genes (ERG channels) were shown to modulate excitability in immature neurons of mouse and quail. We investigated the expression of ERG channels in immature spinal interneurons, using organotypic embryonic cultures of mouse spinal cord after 1 and 2 weeks of development in vitro. We report here that all the genes of the erg family known so far ( erg1a, erg1b, erg2, erg3) are expressed in embryonic spinal cultures. We demonstrate for the first time that three ERG proteins (ERG1A, ERG2 and ERG3) are co-expressed in the same neuronal population, and display a spatio-temporal distribution in the spinal slices. ERG immuno-positive cells, representing mainly GABAergic interneurons, were present in large numbers at early stages of development, while declining later, with a ventral to dorsal gradient. Patch clamp recordings confirmed these data, showing that ventral interneurons expressed functional ERG currents only transiently. Similar expression of the erg genes was observed at comparable ages in vivo. The role of ERG currents in regulating neuronal excitability during the earliest phases of spinal circuitry development will be examined in future studies.

Functions of erg K+ channels in excitable cells.

Ether-à-go-go-related gene (erg) channels are voltage-dependent K+ channels mediating inward-rectifying K+ currents because of their peculiar gating kinetics. These characteristics are essential for repolarization of the cardiac action potential. Inherited and acquired malfunctioning of erg channels may lead to the long QT-syndrome. However, erg currents have also been recorded in many other excitable cells, like smooth muscle fibres of the gastrointestinal tract, neuroblastoma cells or neuroendocrine cells. In these cells erg currents contribute to the maintenance of the resting potential. Changes in the resting potential are related to cell-specific functions like increase in hormone secretion, frequency adaptation or increase in contractility.

Protein kinase C is necessary for recovery from the thyrotropin-releasing hormone-induced r-ERG current reduction in GH3 rat anterior pituitary cells.

The biochemical cascade linking activation of phospholipase C-coupled thyrotropin-releasing hormone (TRH) receptors to rat ERG (r-ERG) channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH3 cells and pharmacological inhibitors. To check the recent suggestion that Rho kinase is involved in the TRH-induced r-ERG current suppression, the hormonal effects were studied in cells pretreated with the Rho kinase inhibitors Y-27632 and HA-1077. The TRH-induced r-ERG inhibition was not significantly modified in the presence of the inhibitors. Surprisingly, the hormonal effects became irreversible in the presence of HA-1077 but not in the presence of the more potent Rho kinase inhibitor Y-27632. Further experiments indicated that the effect of HA-1077 correlated with its ability to inhibit protein kinase C (PKC). The hormonal effects also became irreversible in cells in which PKC activity was selectively impaired with GF109203X, Gö6976 or long-term incubation with phorbol esters. Furthermore, the reversal of the effects of TRH, but not its ability to suppress r-ERG currents, was blocked if diacylglycerol generation was prevented by blocking phospholipase C activity with U-73122. Our results suggest that a pathway involving an as yet unidentified protein kinase is the main cause of r-ERG inhibition in perforated-patch clamped GH3 cells. Furthermore, they demonstrate that although not necessary to trigger the ERG current reductions induced by TRH, an intracellular signal cascade involving phosphatidylinositol-4,5-bisphosphate hydrolysis by phospholipase C, activation of an alpha/betaII conventional PKC and one or more dephosphorylation steps catalysed by protein phosphatase 2A, mediates recovery of ERG currents following TRH withdrawal.

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether-a-go-go-related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike-frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single-cell level by means of perforated patch-clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N-methyltransferase and ERG channels suggested expression of these channels in epinephrine- but not in norepinephrine-containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long-QT running heart.

Block of erg current by linoleoylamide, a sleep-inducing agent, in pituitary GH 3 cells

Linoleoylamide is physiological constituent of neurons. The effects of this agent, also a sleep-inducing agent, on ion currents in pituitary GH 3 cells were investigated. Hyperpolarization-elicited K + currents in GH 3 cells bathed in a high-K +, Ca 2+-free solution were studied to determine the effects of linoleoylamide and other related compounds on the I K(IR) that was sensitive to inhibition by E-4031 and identified as an erg ( ether-à-go-go-related-gene) current. Linoleoylamide suppressed the amplitude of I K(IR) in a concentration-dependent manner with an IC 50 value of 5 μM. Oleamide (20 μM) inhibited the amplitude of I K(IR), while neither arachidonic acid (20 μM) nor 14,15-epoxyeicosatrienoic acid (20 μM) had an effect on it. In GH 3 cells incubated with anandamide (20 μM) or arachidonic acid (20 μM), the linoleoylamide-induced inhibition of I K(IR) remained unaltered. In inside-out patches, arachidonic acid (20 μM) and 14,15-epoxyeicosatrienoic acid (20 μM) stimulated large-conductance Ca 2+-activated K + channels; however, linoleoylamide (20 μM) had little or no effect on them. Under current-clamp mode, linoleoylamide (20 μM) increased the firing rate. In IMR-32 neuroblastoma cells, linoleoylamide also suppressed I K(IR). This study provides the evidence that linoleoylamide has a depressant effect on the erg current, and suggests that this effect may affect hormonal secretion.

Functional and pharmacological properties of canine ERG potassium channels.

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K(+) channels and examined their biophysical and pharmacological properties with whole cell patch clamp and (35)S-labeled MK-499 ([(35)S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K(+) current (I(Kr)). Channel opening was time- and voltage dependent with threshold near -40 mV. The half-maximum activation voltage was -7.8 +/- 2.4 mV at 23 degrees C, shifting to -31.9 +/- 1.2 mV at 36 degrees C. Channels activated with a time constant of 13 +/- 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K(+) selective (Na(+)-to-K(+) permeability ratio = 0.007), and were potently inhibited by I(Kr) blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC(50) values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [(35)S]MK-499 binding from cERG and hERG with IC(50) values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

Regulation of an ERG K+ Current by Src Tyrosine Kinase

The human "ether-a-go-go"-related gene (HERG) K(+) channel, and its homologues are present in heart, neuronal tissue, some cancer cells, and the MLS-9 rat microglia cell line (Zhou, W., Cayabyab, F. S., Pennefather, P. S., Schlichter, L. C., and DeCoursey, T. E. (1998) J. Gen. Physiol. 111, 781-794). Despite its importance, there are few studies of ERG modulation. In this first report of regulation by tyrosine phosphorylation we show that MLS-9 cells express transcripts for r-erg1 (rat homologue of HERG) and r-erg2, and an immunoreactive doublet was identified using an anti-HERG antibody. The constitutive tyrosine phosphorylation of the ERG1 protein, detected by co-immunoprecipitation, was reduced by the protein-tyrosine kinase inhibitors, lavendustin A, herbimycin A, or genistein (but not daidzein). The whole cell ERG current was reduced by protein-tyrosine kinase inhibitors or the Src-selective inhibitory peptide, src40-58, but not by a scrambled peptide. Conversely, the current was increased by the Src-activating peptide, srcpY, but not by an inactive analogue. Activating endogenous Src or transfecting constitutively active v-Src altered the voltage dependence and deactivation kinetics to produce more current at negative potentials. Co-immunoprecipitation identified an association between the channel protein and Src. Thus, r-ERG1 and Src tyrosine kinase appear to exist in a signaling complex that is well positioned to modulate this K(+) channel and affect its contribution to cellular functions.

Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via the native signal cascade.

The mechanism of thyrotropin-releasing hormone (TRH)-induced ether-a-go-go-related gene (erg) K+ current modulation was investigated with the perforated-patch whole-cell technique in clonal somatomammotroph GH3/B6 cells. These cells express a small endogenous erg current known to be reduced by TRH. GH3/B6 cells were injected with cDNA coding for rat erg1, erg2, erg3 and HERG K+ channels. The corresponding erg currents were isolated with the help of the specific erg channel blockers E-4031 and dofetilide and their biophysical properties were determined. TRH (1 M) was able to significantly reduce the different erg currents. The voltage dependence of activation was shifted by 15 mV (erg1), 10 mV (erg2) and 6 mV (erg3) to more positive potentials without strongly affecting erg inactivation. TRH reduced the maximal available erg current amplitude by 12% (erg1), 13% (erg2) and 39% (erg3) and accelerated the time course of erg1 and erg2 channel deactivation, whereas erg3 deactivation kinetics were not significantly altered. The effects of TRH on HERG currents did not differ from those on its rat homologue erg1. In addition, coinjection of rat MiRP1 with HERG cDNA did not influence the TRH-induced modulation of HERG channels. Rat erg1 currents recorded in the cell-attached configuration were reduced by application of TRH to the extra-patch membrane in the majority of the experiments, confirming the involvement of a diffusible second messenger. Application of the phorbol ester phorbol 12-myristate 13-acetate (PMA; 1 M) shifted the voltage dependence of erg1 activation in the depolarizing direction, but it did not reduce the maximal current amplitude. The voltage shift could not be explained by a selective effect on protein kinase C (PKC) since the PKC inhibitor bisindolylmaleimide I did not block the effects of TRH and PMA on erg1. In addition, cholecystokinin, known to activate the phosphoinositol pathway similarly to TRH, did not significantly affect the erg1 current. Various agents interfering with different known TRH-elicited cellular responses were not able to completely mimic or inhibit the TRH effects on erg1. Tested substances included modulators of the cAMP-protein kinase A pathway, arachidonic acid, inhibitors of tyrosine kinase and mitogen-activated protein kinase, sodium nitroprusside and cytochalasin D. The results demonstrate that all three members of the erg channel subfamily are modulated by TRH in GH3/B6 cells. In agreement with previous studies on the TRH-induced modulation of the endogenous erg current in prolactin-secreting anterior pituitary cells, the TRH effects on overexpressed erg1 channels are not mediated by any of the tested signalling pathways.

Erg1, erg2 and erg3 K channel subunits are able to form heteromultimers.

Clonal somato-mammotroph GH3/B6 cells and lactotroph MMQ cells express two (ergl, erg2) of the three cloned rat ether-à-go-go-related gene (erg) K channel subunits. To study whether the erg subunits form heteromultimers, dominant-negative mutants of erg and erg2 were constructed by point mutation (erg1G630S, erg2G480S). After co-expression of these mutants with the wild-type erg1, erg2, or erg3 in Chinese hamster ovary (CHO) cells no erg currents could be detected. In contrast, in co-expression experiments with members of the other ether-à-go-go (EAG) subfamilies (eagl, elkl) the mutant erg1G630S had no effect. These results strongly suggest that erg channel subunits are able to form heteromultimers within the erg channel subfamily. Suppression of the endogenous E-4031-sensitive currents in GH3/B6 and MMQ cells by erg1G630S confirms that they are mediated by erg channels despite the differences in gating kinetics in these cells. Reduction of the erg current in GH3/B6 cells by erg2G480S indicates that erg heteromultimers can also be formed in these cells.