Eag Potassium Channel Research Articles

Eag1 potassium channel as cancer therapy target

Eag1 potassium channel as cancer therapy target

Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and eag potassium channels.

Unintended block of HERG K+ channels is a side effect of many common medications and is the most common cause of acquired long QT syndrome associated with increased risk of life-threatening arrhythmias. The molecular mechanism of high-affinity HERG block by structurally diverse compounds has been attributed to pi-stacking and cation-pi interactions of a drug (e.g., cisapride) with specific aromatic amino acid residues (Tyr-652 and Phe-656) in the S6 alpha-helical domain that face the central cavity of the channel. It also has been proposed that strong C-type inactivation of HERG facilitates or is the primary determinant of high-affinity drug binding. The structurally related, but noninactivating eag channel is insensitive to HERG blockers unless inactivation is induced by specific amino acid mutations [Ficker, E., Jarolimek, W. & Brown, A. M. (2001) Mol. Pharmacol. 60, 1343-1348]. Here we examine the relative importance of inactivation vs. positioning of S6 aromatic residues in determining sensitivity of HERG and eag channels to block by cisapride. The repositioning of Tyr-652 or Phe-656 along the S6 alpha-helical domain of HERG reduced sensitivity of channels to block by cisapride. Moreover, independent of inactivation, repositioning of the equivalent aromatic residues in Drosophila eag channels induced sensitivity to block by cisapride. These findings suggest that positioning of S6 aromatic residues relative to the central cavity of the channel, not inactivation per se determines drug block of HERG or eag channels.

Functional distinction of human EAG1 and EAG2 potassium channels

Human ether à go-go potassium channel 2 (hEAG2) was cloned and its properties were compared with the previously characterized isoform hEAG1. In the Xenopus oocyte expression system the time course of activation was about four times slower and the voltage required for half-maximal subunit activation was about 10 mV greater for hEAG2 channels. However, its voltage dependence was smaller and, therefore, hEAG2 channels start to open at more negative voltages than hEAG1. Coexpression of both isoforms and kinetic analysis of the resulting currents indicated that they can form heteromeric channel complexes in which the slow activation phenotype of hEAG2 is dominant. Upon expression in mammalian cells, quinidine blocked hEAG1 channels (IC 50 1.4 μM) more potently than hEAG2 channels (IC 50 152 μM), thus providing a useful tool for the functional distinction between hEAG1 and hEAG2 potassium channels.

Potassium channel ether à go-go mRNA expression in the spiral ligament of the rat

Identification of the K + transporters located in the lateral wall of the cochlea is essential for a better understanding of the mechanisms by which a positive endocochlear potential and a high K + concentration are achieved in endolymph. In this study, we have determined the distribution of the K + channel rat ether à go-go (eag) mRNA in the cochlea. After reverse transcription of adult rat cochlear tissues, cDNA was amplified with primers specific to eag channel. The eag mRNA was localized in cochlear tissues by in situ hybridization using specific oligonucleotide probes tailed with digoxigenin conjugated UTP. Eag mRNA was detected in the organ of Corti but mainly in the fibrocytes of the spiral ligament but not in spiral prominence or in stria vascularis. The expression pattern of rat eag transcript in spiral ligament is complementary to the Na +,K +-ATPase distribution in the cochlear lateral wall. The localization of eag mRNA suggests that eag potassium channel may be produced in the corresponding cells. Considering the importance of the K + gradient in the cochlea, the result reported here suggests that eag channel may play a role in the control of K + fluxes in the spiral ligament.

Individual Subunits Contribute Independently to Slow Gating of Bovine EAG Potassium Channels

The bovine ether à go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability.

Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia.

1. Three new members of the EAG potassium channel gene family were identified in rat and the complete coding sequence of one of these genes (elk1) was determined by cDNA cloning. 2. The elk1 gene, when expressed in Xenopus oocytes, encodes a slowly activating and slowly deactivating potassium channel. 3. The elk1 gene is expressed in sympathetic ganglia and is also expressed in sciatic nerve. 4. Six of the seven known EAG genes were found to be expressed in rat sympathetic ganglia, suggesting an important functional role for these channels in the sympathetic nervous system.

Slob, a Novel Protein that Interacts with the Slowpoke Calcium-Dependent Potassium Channel

Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat KV1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins.

Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment.

1. Rat eag potassium channels (r-eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole-Moore effect. 2. Deletions in the N-terminal structure of r-eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (delta 2-190, delta 2-12 and delta 7-12) or resulted in a shift to more positive potentials and faster activation kinetics (delta 150-162). 3. The impact of the deletion delta 7-12 was investigated in more detail: it almost abolished the Cole-Moore effect and markedly slowed down channel deactivation. 4. Unlike wild-type channels, the deletion mutants delta 7-12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG. 5. Both the slowing of deactivation and the inactivation induced by the deletion delta 7-12 were compensated by a single histidine-to-arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full-length r-eag channel. 6. These results demonstrate a functional role of the N-terminus in the voltage-dependent gating of potassium channels which is presumably mediated by an interaction of the N-terminal protein structure with the S4 motif during the gating process.

Extracellular Mg2+ regulates activation of rat eag potassium channel

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg2+ on the activation of rat eag. Extracellular Mg2+ at physiological concentrations dramatically slowed the activation in a dose- and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg2+ as the effect of Mg2+ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg2+-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg2+ concentration constitutes a system for fine-tuning K channel availability in neuronal cells.