ERG Channels Research Articles

Mapping the receptor site for ergtoxin, a specific blocker of ERG channels

We show here that ergtoxin (ErgTx) is a bona fide, specific blocker of the human ether-a-go-go-related gene (HERG) channels. It does not affect the function of either M-eag or M-elk channels. A chimeric construction containing a segment of the P-region of M-eag channel inserted into the HERG channel drastically diminished or completely abolished the inhibitory effect of ErgTx, whereas chimeras of the P-region of HERG channel into M-eag channels recovered the inhibitory effect. From the P-region point mutants of HERG channel assays, only the mutant N598Q shows about 25% decrement of the ErgTx inhibitory effect. ErgTx recognizes the P-region of HERG channels, blocking the channel function with a K d in the order of 12 nM.

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.

Both linopirdine- and WAY123,398-sensitive components of I K(M,ng) are modulated by cyclic ADP ribose in NG108-15 cells

The "M-like" current in NG108-15 cells has two components carried by different K+ channels: a fast-deactivating component, analogous to IK(M) in sympathetic neurones and carried by KCNQ2/3 channels, and a more slowly deactivating component carried by murine erg1 (merg1) channels. The former is selectively blocked by linopirdine (< or =10 microM), the latter by WAY123,398 (< or =10 microM). Bradykinin (100 nM) inhibited 76% of the KCNQ component of current compared with 12% of the merg component. Cyclic ADP ribose (cADPR, 2 microM), introduced via the patch pipette, caused a rundown of both current components. Acetylcholine (100 microM) inhibited 89% of the KCNQ component of current compared to 34% of the merg component. After 15 min of intracellular dialysis with the cADPR antagonist 8-amino-cADP ribose (100 microM), the inhibition reduced to 40% and 19% and after 30 min it was further reduced to 8% and 5% for the KCNQ currents and merg currents respectively. These data show that both KCNQ and merg currents in NG108-15 cells can be modulated by either bradykinin or M1 muscarinic receptors. The inhibition of the KCNQ current component is more pronounced than that of the merg component. These results suggest that cADPR might be involved in M1-muscarinic inhibition of both KCNQ2/3 and merg1 channels.

The ether-à-go-go-Related Gene K(+) Current: Functions of a Strange Inward Rectifier.

The erg channels mediate an inward-rectifying K(+) current because of their peculiar gating kinetics. They are involved in repolarization of the cardiac action potential, frequency adaptation, and maintenance of the resting potential. Reduction of erg currents via an intracellular signal cascade underlies the thyrotropin-releasing hormone-induced increase in prolactin secretion.

A toxin to nervous, cardiac, and endocrine ERG K+channels isolated fromCentruroides noxiusscorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila 'seizure' mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16+/-1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure-function relationships and novel pharmacological tools with potential therapeutic efficacy.

Functional Analysis of a Mouse Brain Elk-Type K+Channel

Members of the Ether à go-go (Eag) K+ channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functions in vivo remain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene, Melk2, that encodes a predicted polypeptide with 48% amino acid identity to Drosophila Elk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively. Melk2 RNA appears to be expressed at high levels only in brain tissue. Functional expression of Melk2 in Xenopus oocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole-Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-à-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system.

The erg inwardly rectifying K+ current and its modulation by thyrotrophin-releasing hormone in giant clonal rat anterior pituitary cells.

1. The voltage-dependent inwardly rectifying K+ current (IK,IR) of clonal rat anterior pituitary cells (GH3/B6) was investigated in solutions with physiological K+ gradient using giant polynuclear cells. 2. IK,IR was isolated by the use of the selective erg (ether-à-go-go-related gene) channel blocker E-4031. In external 5 mM K+ solution, IK,IR carried steady-state outward current in the potential range between -60 and 0 mV, with a maximum current amplitude at -40 mV. Negative to the K+ equilibrium potential, EK, large transient inward currents occurred. 3. A selective pharmacological block of IK,IR induced a sustained depolarization of the membrane potential when Ca2+ action potentials were blocked, confirming the contribution of IK,IR to the resting membrane potential of GH3/B6 cells. 4. Thyrotrophin-releasing hormone (TRH) reduced effectively the sustained outward and the transient inward IK,IR. The magnitude of a TRH-induced depolarization of the membrane potential was consistent with an almost complete reduction of IK,IR. 5. The results demonstrate that the TRH-induced reduction of IK,IR is able to mediate the resting potential depolarization, suggesting that the increase in the frequency of action potentials occurring during the second phase of the TRH response in GH cells should be sustained by IK,IR inhibition. Moreover, this is the first evidence of a ligand-induced physiological modulation of an erg-mediated current.

Time, voltage and ionic concentration dependence of rectification of h- erg expressed in Xenopus oocytes

The rapid delayed rectifier, I Kr is believed to have h- erg ( h uman e ther-à-go-go r elated g ene) as its molecular basis. A recent study has shown that rectification of h- erg involves a rapid inactivation process that involves rapid closure of the external mouth of the pore or C-type inactivation. We measured the instantaneous current to voltage relationship for h- erg channels using the saponin permeabilized variation of the cut-open oocyte clamp technique. In contrast to C-type inactivation in other voltage-gated K + channels, the rate of inactivation was strongly voltage dependent at depolarized potentials. This voltage dependence could be modulated independently of activation by increasing [K +] o from 2 to 98 mM. These results suggest that inactivation of h- erg has its own intrinsic voltage sensor.