Eag Family Research Articles

κO-SrVIA Conopeptide, a Novel Inhibitor Peptide for Two Members of the Human EAG Potassium Channel Family.

The first conotoxin affecting the voltage-gated potassium channels of the EAG family was identified and characterized from the venom of the vermivorous species Conus spurius from the Gulf of Mexico. This conopeptide, initially named Cs68 and later designated κO-SrVIA, is extremely hydrophobic and comprises 31 amino acid residues, including six Cysteines in the framework VI/VII, and a free C-terminus. It inhibits the currents mediated by two human EAG subtypes, Kv10.1 (IC50 = 1.88 ± 1.08 µM) and Kv11.1 (IC50 = 2.44 ± 1.06 µM), and also the human subtype Kv1.6 (IC50 = 3.6 ± 1.04 µM). Despite its clear effects on potassium channels, it shares a high sequence identity with δ-like-AtVIA and δ-TsVIA. Also, κO-SrVIA is the third conopeptide from the venom of C. spurius with effects on potassium channels, and the seventh conotoxin that blocks Kv1.6 channels.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

Structural Basis for Effector Transmembrane Domain Recognition by Type VI Secretion System Chaperones

Type VI secretion systems facilitate the delivery of antibacterial effector proteins between neighbouring Gram-negative bacteria. A subset of these effectors harbour N-terminal transmembrane domains (TMDs) implicated in effector translocation across the target cell membrane. However, the abundance and distribution of these TMD-containing effectors has remained unknown. Here, we report the discovery of prePAAR, a conserved motif found in over 6,000 putative TMD-containing effectors. Based on their differing sizes and number of TMDs these effectors fall into two distinct classes that are unified by their requirement for a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain, which is required for effector interaction with the T6SS spike. Taken together, our findings define the mechanism of chaperone-assisted secretion of a widespread family of T6SS membrane protein effectors.

Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones.

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor.

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.

EAG1 enhances hepatocellular carcinoma proliferation by modulating SKP2 and metastasis through pseudopod formation.

Ether-à-go-go-1 (EAG1), one of the potassium channels, is involved in various physiological processes and plays an important role in the tumorigenesis of many kinds of cancer. EAG1 is highly expressed in hepatocarcinoma cells and is closely related to clinical prognosis, but the molecular mechanism remains elusive. In this study, we verified that EAG1 promotes the proliferation of hepatocellular carcinoma (HCC) both in vitro and in vivo. It promotes cell cycle progression by inhibiting the ubiquitination of SKP2. In addition, EAG1 promotes the migration and invasion of HCC by promoting cell pseudopod formation. Furthermore, in a high-pressure plasmid-injected mouse liver orthotopic carcinoma model, astemizole, an EAG family blocker, can significantly inhibit the formation of liver cancer. Meanwhile, liver-specific EAG1 knockout mice show resistance to hepatocarcinogenesis. This research demonstrated that EAG1 plays an important role in the progression of HCC, and could be a potential therapeutic target for HCC.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

The Cytoplasmic Domain of Voltage-dependent Potassium Channels of the Eag Family May Play a Role in the Regulation of Ion Transport

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The environmental toxicant ziram enhances neurotransmitter release and increases neuronal excitability via the EAG family of potassium channels

Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.

Extracellular HEME Modulates Voltage-gating in CNBD Superfamily Cation Channels

The CNBD superfamily of cation channels evolved from a prokaryotic lineage, is widespread in Eukaryotes and includes EAG family K+ channels, HCN channels and CNG channels in animals as well as plant voltage-gated K+ channels. CNBD superfamily channels contain the transmembrane pore domain (PD) and voltage sensor domain (VSD) found in all voltage-gated cation channels but connected to an intracellular cyclic nucleotide binding (homology) domain (CNBD, CNBHD). Animal and plant CNBD channels can be gated by depolarization (EAG, SKOR, e.g.) or hyperpolarization (HCN, KAT1, e.g.). We show that extracellular heme is a highly effective modulator of voltage-gating in these CNBD superfamily channels, promoting opening of depolarization-gated channels and closing of hyperpolarization-gated channels. These seemingly opposite effects on gating can be explained by a single mechanism in which heme interaction favors an “outward” or “up” conformation for S4 in the VSD. In the mammalian depolarization-gated K+ channel Elk1, heme modulation is pH-dependent and the maximal GV shift of approximately −60 mV occurs at pH 8. Mutations to a highly conserved S5 motif (HXXXC) in the external PD/VSD interface of CNBD superfamily channels, and also to the VSD's extracellular acidic charge cluster in S2 and S3, alter heme modulation. We propose that heme could promote outward movement of S4 in Elk1 and other CNBD superfamily channels by oxidizing the acidic charge cluster or by altering PD/VSD interactions.

Regulation of Eag by Ca2+/calmodulin controls presynaptic excitability in Drosophila.

Drosophila ether-à-go-go ( eag) is the founding member of a large family of voltage-gated K+ channels, the KCNH family, which includes Kv10, 11, and 12. Concurrent binding of calcium/calmodulin (Ca2+/CaM) to NH2- and COOH-terminal sites inhibits mammalian EAG1 channels at submicromolar Ca2+ concentrations, likely by causing pore constriction. Although the Drosophila EAG channel was believed to be Ca2+-insensitive (Schönherr R, Löber K, Heinemann SH. EMBO J 19: 3263-3271, 2000.), both the NH2- and COOH-terminal sites are conserved. In this study we show that Drosophila EAG is inhibited by high Ca2+ concentrations that are only present at plasma membrane Ca2+ channel microdomains. To test the role of this regulation in vivo, we engineered mutations that block CaM-binding to the major COOH-terminal site of the endogenous eag locus, disrupting Ca2+-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca2+ influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K+ conductance. These results are predicted by a conductance-based multicompartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca2+ channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults. NEW & NOTEWORTHY Regulation of excitability is critical to tuning the nervous system for complex behaviors. We demonstrate in this article that the EAG family of voltage-gated K+ channels exhibit conserved gating by Ca2+/CaM. Disruption of this inhibition in Drosophila results in decreased evoked neurotransmitter release due to truncated Ca2+ influx in presynaptic terminals. In adults, disrupted Ca2+ dynamics cripples memory formation. These data demonstrate that the biophysical details of channels have important implications for cell function and behavior.

Kv10.1 potassium channel: from the brain to the tumors.

The KCNH1 gene encodes the Kv10.1 (Eag1) ion channel, a member of the EAG (ether-à-go-go) family of voltage-gated potassium channels. Recent studies have demonstrated that KCHN1 mutations are implicated in Temple-Baraitser and Zimmermann-Laband syndromes and other forms of developmental deficits that all present with mental retardation and epilepsy, suggesting that Kv10.1 might be important for cognitive development in humans. Although the Kv10.1 channel is mainly expressed in the mammalian brain, its ectopic expression occurs in 70% of human cancers. Cancer cells and tumors expressing Kv10.1 acquire selective advantages that favor cancer progression through molecular mechanisms that involve several cellular pathways, indicating that protein-protein interactions may be important for Kv10.1 influence in cell proliferation and tumorigenesis. Several studies on transcriptional and post-transcriptional regulation of Kv10.1 expression have shown interesting mechanistic insights about Kv10.1 role in oncogenesis, increasing the importance of identifying the cellular factors that regulate Kv10.1 expression in tumors.

Ginsenoside Rg3, a Gating Modifier of EAG Family K+ Channels.

Ginsenoside 20(S)-Rg3 (Rg3) is a steroid glycoside that induces human ether-à-go-go-related gene type 1 (hERG1, Kv11.1) channels to activate at more negative potentials and to deactivate more slowly than normal. However, it is unknown whether this action is unique to hERG1 channels. Here we compare and contrast the mechanisms of actions of Rg3 on hERG1 with three other members of the ether-à-go-go (EAG) K(+) channel gene family, including EAG1 (Kv10.1), ERG3 (Kv11.3), and ELK1 (Kv12.1). All four channel types were heterologously expressed in Xenopus laevis oocytes, and K(+) currents were measured using the two-microelectrode voltage-clamp technique. At a maximally effective concentration, Rg3 shifted the half-point of voltage-dependent activation of currents by -14 mV for ERG1 (EC50 = 414 nM), -20 mV for ERG3 (EC50 = 374 nM), -28 mV for EAG1 (EC50 = 1.18 μM), and more than -100 mV for ELK1 (EC50 = 197 nM) channels. Rg3 also induced slowing of ERG1, ERG3, and ELK1 channel deactivation and accelerated the rate of EAG1 channel activation. A Markov model was developed to simulate gating and the effects of Rg3 on the voltage dependence of activation of hELK1 channels. Understanding the mechanism underlying the action of Rg3 may facilitate the development of more potent and selective EAG family channel activators as therapies for cardiovascular and neural disorders.

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.

Epigenome-wide ovarian cancer analysis identifies a methylation profile differentiating clear-cell histology with epigenetic silencing of the HERG K+ channel

Ovarian cancer remains the leading cause of death in women with gynecologic malignancies, despite surgical advances and the development of more effective chemotherapeutics. As increasing evidence indicates that clear-cell ovarian cancer may have unique pathogenesis, further understanding of molecular features may enable us to begin to understand the underlying biology and histology-specific information for improved outcomes. To study epigenetics in clear-cell ovarian cancer, fresh frozen tumor DNA (n = 485) was assayed on Illumina Infinium HumanMethylation450 BeadChips. We identified a clear-cell ovarian cancer tumor methylation profile (n = 163) which we validated in two independent replication sets (set 1, n = 163; set 2, n = 159), highlighting 22 CpG loci associated with nine genes (VWA1, FOXP1, FGFRL1, LINC00340, KCNH2, ANK1, ATXN2, NDRG21 and SLC16A11). Nearly all of the differentially methylated CpGs showed a propensity toward hypermethylation among clear-cell cases. Several loci methylation inversely correlated with tumor gene expression, most notably KCNH2 (HERG, a potassium channel) (P = 9.5 × 10(-7)), indicating epigenetic silencing. In addition, a predicted methylation class mainly represented by the clear-cell cases (20 clear cell out of 23 cases) had improved survival time. Although these analyses included only 30 clear-cell carcinomas, results suggest that loss of expression of KCNH2 (HERG) by methylation could be a good prognostic marker, given that overexpression of the potassium (K(+)) channel Eag family members promotes increased proliferation and results in poor prognosis. Validation in a bigger cohort of clear-cell tumors of the ovary is warranted.

Deletion of the Amino-Terminus Uncovers an Inactivated State in EAG1 Channels

Ether-a-go-go (Eag) family channels are voltage-gated K+ channels that are important in cardiac and neural function and have a well-documented role in disease. One of eight members of the mammalian Eag family, EAG1 channels expression is confined to the central nervous system of the healthy adult. EAG1 channels are also expressed in cancer cells and are implicated in tumor progression and regulation of the cell cycle. Most members of the Eag channel family exhibit voltage-dependent inactivation, yet wild-type EAG1 channels exhibit only voltage-gated activation and deactivation, with no apparent inactivation. Here we report that deletion of the entire intracellular amino-terminal domain uncovers an inactivated channel gating state at depolarizing potentials. We characterized this inactivated state in excised patches from Xenopus oocytes expressing mutant EAG1 channels, and recorded their currents in the inside-out configuration of the patch clamp technique. Similar to wild-type channels, EAG1 with a deleted amino-terminus begins to activate around −80 mV, however, these channels also inactivate at potentials higher than −40 mV. The rate of inactivation becomes faster with increasingly depolarizing potentials. Additionally, the kinetics of the EAG1 channels with a deleted amino-terminus are quite slow. Other members of the Eag family of channels exhibit C-type inactivation, which results from collapse of the selectivity filter. To determine whether the inactivation found in this EAG1 mutant was also C-type, we mutated a pore-lining residue known to alter C-type inactivation in both Shaker and hERG1 channels. As a second approach, we asked whether triethylammonium applied to the external surface of the channel altered the rate of inactivation. Results from both experiments are inconsistent with C-type inactivation. Instead, we hypothesize that inactivation in the EAG1 channel lacking their amino-terminus is simply a reclosure of the activation gate.

Role of the N-Terminal PAS Domain of hERG K Channels in Gating, Disease and Rescue

Human ERG (Kv11 or KCNH2) channels are voltage-activated K channels in the eag family of channels. hERG is similar to other eag family member as they are homologous to voltage- activated K channels but also share homology with cyclic nucleotide-gated (CNG) channels. hERG shares important structural features with other members of the eag family including an N-terminal PAS domain (Per-Arnt-Sim) and C-terminal cyclic nucleotide-binding homology domain that is similar to the CNB domain of CNG, however eag family channels are not regulated by cyclic nucleotide. We have recently shown several new properties for the hERG PAS domain and CNBHD. Recently, we showed that the PAS domain made a direct interaction with the rest of the channel and markedly regulated slow deactivation, which is a characteristic of wild-type hERG. The CNBHD also plays a role in deactivation gating; it is necessary for regulation of deactivation as channels with the CNBHD deleted lost slow deactivation. Biochemical pull-down interactions and electrophysiology experiments suggest that the PAS domain makes a direct interaction with the CNBHD and that this interaction underlies slow deactivation. Mutations in hERG-PAS that are linked to LQTS disrupt the interaction of the PAS domain with the rest of the channel, indicating that disruption of this interaction may be the mechanism behind some instances of LQTS. The PAS domain itself can be used to fix aberrant gating in channels with PAS domain mutations, as if the recombinant PAS domain substitutes for the covalently attached, but mutated PAS domain. Here we will also discuss new data regarding the regulatory effect of the PAS domain on other gating transitions besides deactivation and the role of the region that connects the PAS domain to the transmembrane domain of the channel.

Association of Kv10.1 to Different Plasma Membrane Domains and its Interaction with other Membrane Associated Proteins in Endogenous Expression Systems

Kv10.1 is voltage gated potassium channel of the EAG family, which is mainly expressed in the Central Nervous System (CNS) under physiological conditions. In the past years, a causal association between Kv10.1 and tumor growth has also been proven, nevertheless the precise mechanism by which this occurs remains unknown. In this work we compare the direct association of membrane-associated proteins to Kv10.1 in different membrane domains of brain and cancer cells. Plasma membranes of brain tissue of adult mice (C57BL/6) and of the human prostate cancer cell line DU-145 were isolated. Detergent resistant membranes (DRM) were extracted through Triton X-100 solubilization and discontinuous gradient centrifugation. IP-assay for Kv10.1 was performed in the different membrane domains; protein expression was analyzed through the Colloidal Coomasie method. Kv10.1 associates partially to the DRM in brain tissue whereas in cancer cells no association can be observed. The protein pattern associated to Kv10.1 in the different systems also varies. We conclude that the different domain association of Kv10.1 is important for different protein interactions and could be involved in it's role as a tumor promoting protein.

Flavonoids Regulate Eag1 Channels

Ether-a-go-go (Eag) family channels, which include hErg1 channels, are voltage-gated K+ channels that are important in cardiac and neural function. From the amino acid sequence of Eag family channels, we identified two probable ligand-binding sites based on their similarities with well-characterized ligand-binding domains. The first putative ligand-binding domain is in the carboxy-terminal region and shares sequence similarity with the cyclic nucleotide-binding domain (CNBD) of proteins modulated by cyclic nucleotides.

Absence of Direct Cyclic Nucleotide Modulation of mEAG1 and hERG1 Channels Revealed with Fluorescence and Electrophysiological Methods

Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (K(d) > or = 51 microm) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K(+) channels might harbor a binding site for a ligand yet to be uncovered.