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The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.

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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.

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The development of cell-based fluorescent assays has resulted in an incredible tool for searching new ion channels' modulators with a biophysical and clinical profile. Among all the ion channels, potassium (K+)-permeable channels represent the most diverse and relevant for cell function, making them attractive targets for drug discovery. Some of the cell-based assays for K+ channels take advantage of a thallium-sensitive dye whose fluorescence increased upon the binding of thallium (Tl+), an ion able to move through K+ channels. We optimize the FLIPR Potassium Assay Kit based on thallium influx to measure the Kv10.1 activity.

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Plant metabolites from natural product extracts offer unique advantages against carcinogenesis in the development of drugs. The target-based virtual screening from food-derived compounds represents a promising approach for tumor therapy. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly potent Kv10.1 inhibitor, liensinine (Lien), which can inhibit the channel in a dose-dependent way with an IC50 of 0.24 ± 0.07 µM. Combining molecular dynamics simulations with mutagenesis experiments, our data show that Lien interacts with Kv10.1 by binding with Y539, T543, D551, E553, and H601 in the C-linker domain of Kv10.1. In addition, the interaction of sequence alignment and 3D structural modeling revealed differences between the C-linker domain of the Kv10.1 channel and the Kv11.1 channel. Furthermore, antitumor experiments revealed that Lien suppresses the proliferation and migration of HCC both in vitro and in vivo. In summary, the food-derived compound, Lien, may serve as a lead compound for antihepatoma therapeutic drugs targeting Kv10.1.

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Kv10.1 is a voltage-dependent K channel whose ectopic expression is associated with several human cancers. Additionally, Kv10.1 has structure-function properties which are not yet well understood. We are using drugs of clinical importance in an attempt to gain insight on the relationship between pharmacology and characteristic functional properties of this channel. Herein, we report the interaction of desethylamiodarone (desAd), the active metabolic product of the antiarrhythmic amiodarone with Kv10.1: desAd binds to both closed and open channels, with most inhibition taking place from the open state, with affinity ~ 5 times smaller than that of amiodarone. Current inhibition by desAd and amiodarone is not synergistic. Upon repolarization desAd becomes trapped in Kv10.1 and thereafter dissociates slowly from closed-and-blocked channels. The addition of the Cole-Moore shift plus desAd open-pore-block time courses yields an increasing phase on the steady-state inhibition curve (H∞) at hyperpolarized holding potentials. In contrast to amiodarone, desAd does not inhibit the Kv10.1 Cole-Moore shift, suggesting that a relevant hydrophobic interaction between amiodarone and Kv10.1 participates in the inhibition of the Cole-Moore shift, which is lost with desAd.

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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.

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Resting membrane potential is a bioelectric property of all cells. Multiple players govern this property, the ion channels being the most important. Ion channel dysfunction can affect cells' resting membrane potential and could be associated with numerous diseases. Therefore, the drug discovery focus on ion channels has increased yearly. In addition to patch-clamp, cell-based fluorescent assays have shown a rapid and reliable method for searching new ion channel modulators. Here, we used a cell-based membrane potential assay to search for new blockers of the Kv10.1, a potassium channel strongly associated with cancer progression and a promising target in anticancer therapy. We found that fluoxetine and miconazole can inhibit the Kv10.1 channel in the micromolar range. In contrast, BL-1249 potentiates Kv10.1 currents in a dose-dependent manner, becoming the first molecule described as an activator of the channel. These results demonstrate that cell-based membrane potential assay can accelerate the discovery of new Kv10.1 modulators.

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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.

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Expression of the voltage-gated potassium channel KV10.1 (Eag1) has been detected in over 70% of human cancers, making the channel a promising new target for new anticancer drug discovery. A new structural class of KV10.1 inhibitors was prepared by structural optimisation and exploration of the structure-activity relationship of the previously published hit compound ZVS-08 (1) and its optimised analogue 2. The potency and selectivity of the new inhibitors between KV10.1 and hERG were investigated using whole-cell patch-clamp experiments. We obtained two new optimised KV10.1 inhibitors, 17a and 18b, with improved nanomolar IC50 values of 568 nM and 214 nM, respectively. Compound 17a exhibited better ratio between IC50 values for hEAG1 and hERG than previously published diarylamine inhibitors. Compounds 17a and 18b moderately inhibited the growth of the KV10.1-expressing cell line MCF-7 in two independent assays. In addition, 17a and 18b also inhibited the growth of hERG-expressing Panc-1 cells with higher potency compared with MCF-7 cells. The main obstacle for newly developed diarylamine KV10.1 inhibitors remains the selectivity toward the hERG channel, which needs to be addressed with targeted drug design strategies in the future.

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The hEag1 (Kv10.1) K+ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC50 = 0.135 µM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K+ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 µM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

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Carcinogenesis is a multistage process involving the dysregulation of multiple genes, proteins, and pathways that make any normal cell acquire a cancer cell phenotype. Therefore, it is no surprise that numerous ion channels could be involved in this process. Since their discovery and subsequent cloning, ion channels have been established as therapeutic targets in excitable cell pathologies (e.g., cardiac arrhythmias or epilepsy); however, their involvement in non-excitable cell pathologies is relatively recent. Among all ion channels, the voltage-gated potassium channels Kv10.1 have been established as a promising target in cancer treatment due to their high expression in tumoral tissues compared to low levels in healthy tissues.

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Bioelectricity goes far beyond electrical signaling in the nervous system, but this was initially not obvious for me. This article describes the journey from studying the biophysics of ion channels in classical electrically excitable tissues to focusing on the pathogenic roles of the Kv10.1 potassium channel in cancers.

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Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

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The KV10.1 voltage-gated potassium channel is highly expressed in 70% of tumors, and thus represents a promising target for anticancer drug discovery. However, only a few ligands are known to inhibit KV10.1, and almost all also inhibit the very similar cardiac hERG channel, which can lead to undesirable side-effects. In the absence of the structure of the KV10.1-inhibitor complex, there remains the need for new strategies to identify selective KV10.1 inhibitors and to understand the binding modes of the known KV10.1 inhibitors. To investigate these binding modes in the central cavity of KV10.1, a unique approach was used that allows derivation and analysis of ligand-protein interactions from molecular dynamics trajectories through pharmacophore modeling. The final molecular dynamics-derived structure-based pharmacophore model for the simulated KV10.1-ligand complexes describes the necessary pharmacophore features for KV10.1 inhibition and is highly similar to the previously reported ligand-based hERG pharmacophore model used to explain the nonselectivity of KV10.1 pore blockers. Moreover, analysis of the molecular dynamics trajectories revealed disruption of the π-π network of aromatic residues F359, Y464, and F468 of KV10.1, which has been reported to be important for binding of various ligands for both KV10.1 and hERG channels. These data indicate that targeting the KV10.1 channel pore is also likely to result in undesired hERG inhibition, and other potential binding sites should be explored to develop true KV10.1-selective inhibitors as new anticancer agents.

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BACKGROUND: Ion channels have been proposed as therapeutic targets for different types of malignancies. One of the most studied ion channels in cancer is the voltage-gated potassium channel ether-à-go-go 1 or Kv10.1. Various studies have shown that Kv10.1 expression induces the proliferation of several cancer cell lines and in vivo tumor models, while blocking or silencing inhibits proliferation. Kv10.1 is a promising target for drug discovery modulators that could be used in cancer treatment. This work aimed to screen for new Kv10.1 channel modulators using a thallium influx-based assay. METHODS: Pharmacological effects of small molecules on Kv10.1 channel activity were studied using a thallium-based fluorescent assay and patch-clamp electrophysiological recordings, both performed in HEK293 stably expressing the human Kv10.1 potassium channel. RESULTS: In thallium-sensitive fluorescent assays, we found that the small molecules loperamide and amitriptyline exert a potent inhibition on the activity of the oncogenic potassium channel Kv10.1. These results were confirmed by electrophysiological recordings, which showed that loperamide and amitriptyline decreased the amplitude of Kv10.1 currents in a dose-dependent manner. Both drugs could be promising tools for further studies. CONCLUSIONS: Thallium-sensitive fluorescent assay represents a reliable methodological tool for the primary screening of different molecules with potential activity on Kv10.1 channels or other K+ channels.

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(1) Background: The voltage-gated potassium channel KV10.1 (Eag1) is considered a near- universal tumour marker and represents a promising new target for the discovery of novel anticancer drugs. (2) Methods: We utilized the ligand-based drug discovery methodology using 3D pharmacophore modelling and medicinal chemistry approaches to prepare a novel structural class of KV10.1 inhibitors. Whole-cell patch clamp experiments were used to investigate potency, selectivity, kinetics and mode of inhibition. Anticancer activity was determined using 2D and 3D cell-based models. (3) Results: The virtual screening hit compound ZVS-08 discovered by 3D pharmacophore modelling exhibited an IC50 value of 3.70 µM against KV10.1 and inhibited the channel in a voltage-dependent manner consistent with the action of a gating modifier. Structural optimization resulted in the most potent KV10.1 inhibitor of the series with an IC50 value of 740 nM, which was potent on the MCF-7 cell line expressing high KV10.1 levels and low hERG levels, induced significant apoptosis in tumour spheroids of Colo-357 cells and was not mutagenic. (4) Conclusions: Computational ligand-based drug design methods can be successful in the discovery of new potent KV10.1 inhibitors. The main problem in the field of KV10.1 inhibitors remains selectivity against the hERG channel, which needs to be addressed in the future also with target-based drug design methods.

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It's now clearly established that the tumor microenvironment participates to tumor development. Among the different actors contributing to these processes, ion channels, located at the cancer cell surface, play a major role. We recently demonstrated that the association of Kv10.1, Orai1 and SPCA2 is crucial to promote the collagen-induced survival of MCF-7 breast cancer cells. By using siRNA directed against SPCA2, we shown that this protein is involved in the regulation of the activity, the expression and the sub-cellular localization of Kv10.1. In addition, it has been demonstrated that SPCA2 is involved in SICE in MCF-7 cells and that the N- and the C-terminal parts of this protein are necessary to interact and to produce Ca2+ entry. However, no information is available about the necessary SPCA2's important region to regulate Kv10.1. The aim of our work is to evaluate how SPCA2 could interact with Kv10.1 channel to induce survival promotion. By using different SPCA2 mutants, we evaluate the role of the N- and C-terminal sections on the expression and the activity of Kv10.1 channels. In addition, we analyzed the impact of these deletions on the collagen 1-induced cell survival. Our results bring out new information about the regulation of Kv10.1 channel through SPCA2. More specifically how the N- and C-terminus of this Ca2+ transporter regulate Kv10.1 expression, trafficking, and function suggesting new opportunities to target Kv10.1 channels in cancer progression.

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Here, we describe four patients suffering from a rather broad spectrum of epilepsy-related disorders, ranging from developmental and epileptic encephalopathy with intellectual disability (DEE) to genetic generalized epilepsy (GGE), which all harbor novel KCNH1 mutations. In one family, we found a weak association of a novel nonsense mutation with epilepsy, suggesting reduced penetrance, and which shows, in agreement with previous findings, that gain-of-function effects rather than haploinsufficiency are important for the pathogenicity of mutations. De novo missense variants in the pore region of the channel result in severe phenotypes presenting usually with DEE with various malformations. The potential pathogenicity of a novel KCNH1 germline mutation located outside of the critical pore domain observed in a GGE patient with a milder phenotype is supported by the fact that the very same amino acid exchange was detected as a somatic mutation in the resected brain tissue of a patient suffering from a focal cortical dysplasia type IIb. Thus, our case series broadens the phenotypic spectrum of KCNH1-associated diseases.

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Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression-downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.

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Antibody-based therapies hold promise for a safe and efficient treatment of cancer. The identification of target tumor cells through a specific antigen enriched on their surface and the subsequent delivery of the therapeutic agent only to those cells requires, besides the efficacy of the therapeutic agent itself, the identification of an antigen enriched on the surface of tumor cells, the generation of high affinity antibodies against that antigen. We have generated single-domain antibodies (nanobodies) against the voltage-gated potassium channel Kv10.1, which outside of the brain is detectable almost exclusively in tumor cells. The nanobody with highest affinity was fused to an improved form of the tumor necrosis factor-related apoptosis inducing ligand TRAIL, to target this cytokine to the surface of tumor cells. The resulting construct, VHH-D9-scTRAIL, shows rapid and strong apoptosis induction in different tumor models in cell culture. The construct combines two sources of specificity, the expression of the antigen restricted to tumor cells and the tumor selectivity of TRAIL. Such specificity combined with the high affinity obtained through nanobodies make the novel agent a promising concept for cancer therapy.