Use of Surface Plasmon Resonance Technique for Studies of Inter-domain Interactions in Ion Channels.

Ion channels are transmembrane proteins essential for cellular functions and are important drug targets. Surface plasmon resonance (SPR) is a powerful technique for investigating protein-protein and protein-small molecule ligand interactions. SPR has been underutilized for studies of ion channels, even though it could provide a wealth of information on the mechanisms of ion channel regulation and aid in ion channel drug discovery. Here we provide a detailed description of the use of SPR technology for investigating inter-domain interactions in KCNH potassium-selective and voltage-gated ion channels.

Chlorpromazine inhibits EAG1 channels by altering the coupling between the PAS, CNBH and pore domains.

EAG1 depolarization-activated potassium selective channels are important targets for treatment of cancer and neurological disorders. EAG1 channels are formed by a tetrameric subunit assembly with each subunit containing an N-terminal Per-Arnt-Sim (PAS) domain and C-terminal cyclic nucleotide-binding homology (CNBH) domain. The PAS and CNBH domains from adjacent subunits interact and form an intracellular tetrameric ring that regulates the EAG1 channel gating, including the movement of the voltage sensor domain (VSD) from closed to open states. Small molecule ligands can inhibit EAG1 channels by binding to their PAS domains. However, the allosteric pathways of this inhibition are not known. Here we show that chlorpromazine, a PAS domain small molecule binder, alters interactions between the PAS and CNBH domains and decreases the coupling between the intracellular tetrameric ring and the pore of the channel, while having little effect on the coupling between the PAS and VSD domains. In addition, chlorpromazine binding to the PAS domain did not alter Cole-Moore shift characteristic of EAG1 channels, further indicating that chlorpromazine has no effect on VSD movement from the deep closed to opened states. Our study provides a framework for understanding global pathways of EAG1 channel regulation by small molecule PAS domain binders.

Molecular mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain.

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels.

Effects of spike proteins on angiotensin converting enzyme 2 (ACE2).

The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.

Comparison of tumor growth assessment using GFP fluorescence and DiI labeling in a zebrafish xenograft model.

DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance.

Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 µM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 µM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.

Multifaceted effect of chlorpromazine in cancer: implications for cancer treatment.

Since its discovery in 1951, chlorpromazine (CPZ) has been one of the most widely used antipsychotic medications for treating schizophrenia and other psychiatric disorders. In addition to its antipsychotic effect, many studies in the last several decades have found that CPZ has a potent antitumorigenic effect. These studies have shown that CPZ affects a number of molecular oncogenic targets through multiple pathways, including the regulation of cell cycle, cancer growth and metastasis, chemo-resistance and stemness of cancer cells. Here we review studies on molecular mechanisms of CPZ's action on key proteins involved in cancer, including p53, YAP, Ras protein, ion channels, and MAPKs. We discuss common and overlapping signaling pathways of CPZ's action, its cancer-type specificity, antitumorigenic effects of CPZ reported in animal models and population studies on the rate of cancer in psychiatric patients. We also discuss the potential benefits and limitations of repurposing CPZ for cancer treatment.

The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker-CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker-CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.

Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity.

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per-Arnt-Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.

Identification of undecylenic acid as EAG channel inhibitor using surface plasmon resonance-based screen of KCNH channels.

BACKGROUND: KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development. METHODS: Here we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders. RESULTS: Using the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 µM. CONCLUSION: Our results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.

Protein Redox State Monitoring Studies of Thiol Reactivity.

Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H2O2) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H2O2 treatment and treating denatured human Prx6 with H2O2 results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H2O2 in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H2O2 eliminated these Prx6 multimers. In contrast, treating cell lysates with H2O2 promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H2O2 promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H2O2 and determines whether H2O2 acts as a facilitator or a disrupter of disulfide bonds.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

Flavonoid regulation of HCN2 channels.

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 µM. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.

Structure of the C-terminal region of an ERG channel and functional implications.

The human ether-à-go-go-related gene (hERG) encodes a K(+) channel crucial for repolarization of the cardiac action potential. EAG-related gene (ERG) channels contain a C-terminal cyclic nucleotide-binding homology domain coupled to the pore of the channel by a C-linker. Here, we report the structure of the C-linker/cyclic nucleotide-binding homology domain of a mosquito ERG channel at 2.5-Å resolution. The structure reveals that the region expected to form the cyclic nucleotide-binding pocket is negatively charged and is occupied by a short ß-strand, referred to as the intrinsic ligand, explaining the lack of direct regulation of ERG channels by cyclic nucleotides. In hERG channels, the intrinsic ligand harbors hereditary mutations associated with long-QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. Mutations in the intrinsic ligand affected hERG channel gating and LQTS mutations abolished hERG currents and altered trafficking of hERG channels, which explains the LQT phenotype. The structure also reveals a dramatically different conformation of the C-linker compared with the structures of the related ether-à-go-go-like K(+) and hyperpolarization-activated cyclic nucleotide-modulated channels, suggesting that the C-linker region may be highly dynamic in the KCNH, hyperpolarization-activated cyclic nucleotide-modulated, and cyclic nucleotide-gated channels.

Flavonoid regulation of EAG1 channels.

The voltage-gated, K(+)-selective ether á go-go 1 (EAG1) channel is expressed throughout the brain where it is thought to regulate neuronal excitability. Besides its normal physiological role in the brain, EAG1 is abnormally expressed in several cancer cell types and promotes tumor progression. Like all other channels in the KCNH family, EAG1 channels have a large intracellular carboxy-terminal region that shares structural similarity with cyclic nucleotide-binding homology domains (CNBHDs). EAG1 channels, however, are not regulated by the direct binding of cyclic nucleotides and have no known endogenous ligands. In a screen of biological metabolites, we have now identified four flavonoids as potentiators of EAG1 channels: fisetin, quercetin, luteolin, and kaempferol. These four flavonoids shifted the voltage dependence of activation toward more hyperpolarizing potentials and slowed channel deactivation. All four flavonoids regulated channel gating with half-maximal concentrations of 2-8 µM. The potentiation of gating did not require the amino-terminal or post-CNBHD regions of EAG1 channels. However, in fluorescence resonance energy transfer and anisotropy-based binding assays, flavonoids bound to the purified CNBHD of EAG1 channels. The CNBHD of KCNH channels contains an intrinsic ligand, a conserved stretch of residues that occupy the cyclic nucleotide-binding pocket. Mutations of the intrinsic ligand in EAG1 (Y699A) potentiated gating similar to flavonoids, and flavonoids did not further potentiate EAG1-Y699A channels. Furthermore, the Y699A mutant CNBHD bound to flavonoids with higher affinity than wild-type CNBHD. These results suggest that the flavonoids identified here potentiated EAG1 channels by binding to the CNBHD, possibly by displacing their intrinsic ligand. EAG1 channels should be considered as a possible target for the physiological effects of flavonoids.

Structure of the carboxy-terminal region of a KCNH channel.

The KCNH family of ion channels, comprising ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like (ELK) K(+)-channel subfamilies, is crucial for repolarization of the cardiac action potential, regulation of neuronal excitability and proliferation of tumour cells. The carboxy-terminal region of KCNH channels contains a cyclic-nucleotide-binding homology domain (CNBHD) and C-linker that couples the CNBHD to the pore. The C-linker/CNBHD is essential for proper function and trafficking of ion channels in the KCNH family. However, despite the importance of the C-linker/CNBHD for the function of KCNH channels, the structural basis of ion-channel regulation by the C-linker/CNBHD is unknown. Here we report the crystal structure of the C-linker/CNBHD of zebrafish ELK channels at 2.2-Å resolution. Although the overall structure of the C-linker/CNBHD of ELK channels is similar to the cyclic-nucleotide-binding domain (CNBD) structure of the related hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channels, there are marked differences. Unlike the CNBD of HCN, the CNBHD of ELK displays a negatively charged electrostatic profile that explains the lack of binding and regulation of KCNH channels by cyclic nucleotides. Instead of cyclic nucleotide, the binding pocket is occupied by a short ß-strand. Mutations of the ß-strand shift the voltage dependence of activation to more depolarized voltages, implicating the ß-strand as an intrinsic ligand for the CNBHD of ELK channels. In both ELK and HCN channels the C-linker is the site of virtually all of the intersubunit interactions in the C-terminal region. However, in the zebrafish ELK structure there is a reorientation in the C-linker so that the subunits form dimers instead of tetramers, as observed in HCN channels. These results provide a structural framework for understanding the regulation of ion channels in the KCNH family by the C-linker/CNBHD and may guide the design of specific drugs.

Identifying regulators for EAG1 channels with a novel electrophysiology and tryptophan fluorescence based screen.

BACKGROUND: Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands. METHODOLOGY/PRINCIPAL FINDINGS: Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region. CONCLUSIONS/SIGNIFICANCE: Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels.

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.