KCNQ5 Potassium Channel Activation Underlies Vasodilation by Tea.

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.

Dexamethasone reduces airway epithelial Cl- secretion by rapid non-genomic inhibition of KCNQ1, KCNN4 and KATP K+ channels.

Basolateral membrane K+ channels play a key role in basal and agonist stimulated Cl- transport across airway epithelial cells by generating a favourable electrical driving force for Cl- efflux. The K+ channel sub-types and molecular mechanisms of regulation by hormones and secretagoues are still poorly understood. Here we have identified the type of K+ channels involved in cAMP and Ca2+ stimulated Cl- secretion and uncovered a novel anti-secretory effect of dexamethasone mediated by inhibition of basolateral membrane K+ channels in a human airway cell model of 16HBE14o- cells commonly used for ion transport studies. Dexamethasone produced a rapid inhibition of transepithelial chloride ion secretion under steady state conditions and after stimulation with cAMP agonist (forskolin) or a Ca2+ mobilizing agonist (ATP). Our results show three different types of K+ channels are targeted by dexamethasone to reduce airway secretion, namely Ca2+-activated secretion via KCNN4 (KCa3.1) channels and cAMP-activated secretion via KCNQ1 (Kv7.1) and KATP (Kir6.1,6.2) channels. The down-regulation of KCNN4 and KCNQ1 channel activities by dexamethasone involves rapid non-genomic activation of PKCα and PKA signalling pathways, respectively. Dexamethasone signal transduction for PKC and PKA activation was demonstrated to occur through a rapid non-genomic pathway that did not implicate the classical nuclear receptors for glucocorticoids or mineralocorticoids but occurred via a novel signalling cascade involving sequentially a Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation. The rapid, non-genomic, effects of dexamethasone on airway epithelial ion transport and cell signalling introduces a new paradigm for glucocorticoid actions in lung epithelia which may serve to augment the anti-inflammatory activity of the steroid and enhance its therapeutic potential in treating airway hypersecretion in asthma and COPD.

Effect of glucoraphanin and sulforaphane against chemotherapy-induced neuropathic pain: Kv7 potassium channels modulation by H2 S release in vivo.

The beneficial effects of isothiocyanate-based compounds, as well as their safety, have been shown in neuropathological disorders, such as neuropathic pain. Aim of the present work was to study the efficacy of the glucosinolate glucoraphanin (GRA) and the derived isothiocyanate sulforaphane (SFN), secondary metabolites occurring exclusively in Brassicales, on chemotherapy-induced neuropathic pain. Mice were repeatedly treated with oxaliplatin (2.4 mg kg-1 ip) for 14 days to induce neuropathic pain. GRA and SFN effects were evaluated after a single administration on Day 15 or after a daily repeated oral and subcutaneous treatment starting from the first day of oxaliplatin injection until the 14th day. Single subcutaneous and oral administrations of GRA (4.43-119.79 µmol kg-1 ) or SFN (1.33-13.31 µmol kg-1 ) reduced neuropathic pain in a dose-dependent manner. The repeated administration of GRA and SFN (respectively 13.31 and 4.43 µmol kg-1 ) prevented the chemotherapy-induced neuropathy. The co-administration of GRA and SFN in mixture with the H2 S binding molecule, haemoglobin, abolished their pain-relieving effect, which was also reverted by pretreating the animals with the selective blocker of Kv7 potassium channels, XE991. GRA and SFN reduce neuropathic pain by releasing H2 S and modulating Kv7 channels and show a protective effect on the chemotherapy-induced neuropathy.

A novel role of the antitumor agent tricyclodecan-9-yl-xanthogenate as an open channel blocker of KCNQ1/KCNE1.

Tricyclodecan-9-yl-xanthogenate (D609) is widely known for its antitumor and antiviral properties via the inhibition of phosphatidylcholine-specific phospholipase C and sphingomyelin synthase. Previously, we found that chronic application of D609 suppressed the K+ channel, KCNQ1/KCNE1, more drastically than expected from its actions on the enzymes, suggesting a direct action of D609 on the channel. Here, we aimed to test this possibility by studying the affinity, specificity, and mechanisms of D609 on KCNQ1/KCNE1. The effect of D609 on KCNQ1/KCNE1 was studied using an in vitro expression system and in native cells, using electrophysiological techniques. We found that D609 rapidly and reversibly inhibited KCNQ1/KCNE1 channels expressed in human embryonic kidney 293 T (HEK293T) cells, in a concentration-dependent manner with a high affinity. D609 neither suppressed endogenous K+ currents in HEK293T cells, nor inhibited the sustained and transient K+ currents of mouse neostriatal neurons, but blocked a KCNQ1/KCNE1-like current in neostriatal neurons. D609 potently blocked IKs, the cardiac KCNQ1/KCNE1 channel, in guinea pig cardiac muscle cells. The action of D609 on KCNQ1/KCNE1 depended on the usage of the channel, suggesting that D609 binds to the channel in the open state. We identified D609 as a potent and specific open channel blocker of KCNQ1/KCNE1. Because KCNQ1/KCNE1 is highly expressed in the heart, the inner ear and the pancreas, D609, when used as an antitumor or antiviral drug, may affect the function of a number of organs in vivo even when used at low concentrations.

Downregulation of Long Non-Coding RNA Kcnq1ot1: An Important Mechanism of Arsenic Trioxide-Induced Long QT Syndrome.

BACKGROUND/AIMS: Arsenic trioxide (ATO) is a known anti-acute promyelocytic leukemia (APL) reagent, whose clinical applications are limited by its serious cardiac toxicity and fatal adverse effects, such as sudden cardiac death resulting from long QT syndrome (LQTS). The mechanisms of cardiac arrhythmia due to ATO exposure still need to be elucidated. Long non-coding RNAs (lncRNAs) are emerging as major regulators of various pathophysiological processes. This study aimed to explore the involvement of lncRNAs in ATO-induced LQTS in vivo and in vitro. METHODS: For in vivo experiments, mice were administered ATO through the tail vein. For in vitro experiments, ATO was added to the culture medium of primary cultured neonatal mouse cardiomyocytes. To evaluate the effect of lncRNA Kcnq1ot1, siRNA and lentivirus-shRNA were synthesized to knockdown lncRNA Kcnq1ot1. RESULTS: After ATO treatment, the Kcnq1ot1 and Kcnq1 expression levels were down regulated. lncRNA Kcnq1ot1 knockdown prolonged the action potential duration (APD) in vitro and exerted LQTS in vivo. Correspondingly, Kcnq1 expression was decreased after silencing lncRNA Kcnq1ot1. However, the knockdown of Kcnq1 exerted no effect on lncRNA Kcnq1ot1 expression. CONCLUSIONS: To our knowledge, this report is the first to demonstrate that lncRNA Kcnq1ot1 downregulation is responsible for QT interval prolongation induced by ATO at least partially by repressing Kcnq1 expression. lncRNA Kcnq1ot1 has important pathophysiological functions in the heart and could become a novel antiarrhythmic target.

Discovery of three toxin peptides with Kv1.3 channel and IL-2 cytokine-inhibiting activities from Non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai.

Non-Buthidae venomous scorpions are huge natural sources of toxin peptides; however, only a few studies have been done to understand their toxin peptides. Herein, we describe three new potential immunomodulating toxin peptides, Ctri18, Ctry68 and Ctry2908, from two non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai. Sequence alignment analyses showed that Ctri18, Ctry68 and Ctry2908 are three new members of the scorpion toxin α-KTx15 subfamily. Electrophysiological experiments showed that Ctri18, Ctry68 and Ctry2908 blocked the Kv1.3 channel at micromole to nanomole levels, but had weak effects on potassium channel KCNQ1 and sodium channel Nav1.4, which indicated that Ctri18, Ctry68 and Ctry2908 might have specific inhibiting effects on the Kv1.3 channel. ELISA experiments showed that Ctri18, Ctry68 and Ctry2908 inhibited IL-2 cytokine secretions of activated T lymphocyte in human PBMCs. Excitingly, consistent with the good Kv1.3 channel inhibitory activity, Ctry2908 inhibited cytokine IL-2 secretion in nanomole level, which indicated that Ctry2908 might be a new lead drug template toward Kv1.3 channels. Together, these studies discovered three new toxin peptides, Ctri18, Ctry68 and Ctry2908, with Kv1.3 channel and IL-2 cytokine-inhibiting activities from two scorpions, C. tricostatus and C. tryznai, and highlighted that non-Buthidae venomous scorpions are new natural toxin peptide sources.

Engineering a peptide inhibitor towards the KCNQ1/KCNE1 potassium channel (IKs).

The KCNQ1/KCNE1 channel (IKs) plays important roles in the physiological and pathological process of heart, but no potent peptide acting on this channel has been reported. In this work, we found that the natural scorpion venom hardly inhibited KCNQ1/KCNE1 channel currents. Based on this observation, we attempted to use three natural scorpion toxins ChTX, BmKTX and OmTx2 with two different structural folds as templates to engineer potent peptide inhibitors towards the KCNQ1/KCNE1 channel. Pharmacological experiments showed that when we screen with 1µM MT2 peptide, an analog derived from BmKTX toxin, KCNQ1/KCNE1 channel currents could be effectively inhibited. Concentration-dependent experiments showed that MT2 inhibited the KCNQ1/KCNE1 channel with an IC50 value of 4.6±1.9µM. The mutagenesis experiments indicated that MT2 peptide likely used Lys26 residue to interact with the KCNQ1/KCNE1 channel. With MT2 as a new template, we further designed a more potent MT2-2 peptide, which selectively inhibited the KCNQ1/KCNE1 channel with an IC50 of 1.51±0.62µM. Together, this work provided a much potent KCNQ1/KCNE1 channel peptide inhibitor so far, and highlighted the role of molecular strategy in developing potent peptide inhibitors for the natural toxin-insensitive orphan receptors.

SjAPI-2 is the first member of a new neurotoxin family with Ascaris-type fold and KCNQ1 inhibitory activity.

Peptides with Ascaris-type fold are a new kind of toxins founded from venomous animals recently. Functionally, these unique toxin peptides had been identified as potent protease inhibitors, which was similar to other known Ascaris-type peptides from non-venomous animals. Whether Ascaris-type peptides from venom animals have neurotoxin activities remains unclear. Here, a scorpion toxin SjAPI-2 with Ascaris-type fold was characterized to have a neurotoxin activity, which can selectively inhibit KCNQ1 potassium channel. SjAPI-2 had 62 amino acid residues, including 10 cysteine residues. Charged residue analyses showed that two acidic residues of SjAPI-2 were regionally distributed, and 10 basic residues were distributed widely throughout the whole peptide, which was similar to classical potassium channel toxins. Pharmacological studies confirmed that SjAPI-2 was a selective KCNQ1 potassium channel inhibitor with weak effects on other potassium channels, such as Kv1.1, Kv1.2, Kv1.3, SKCa2, SKCa3, and IKCa channels. Concentration-dependent studies showed that SjAPI-2 inhibited the KCNQ1 potassium channel with an IC50 of 771.5±169.9 nM. To the best of our knowledge, SjAPI-2 is the first neurotoxin with a unique Ascaris-type fold, providing novel insights into the divergent evolution of neurotoxins from venomous animals.

Differential effects of the ß-adrenoceptor blockers carvedilol and metoprolol on SQT1- and SQT2-mutant channels.

BACKGROUND: N588K-KCNH2 and V307L-KCNQ1 mutations lead to a gain-of-function of IKr and IKs thus causing short-QT syndromes (SQT1, SQT2). Combined pharmacotherapies using K(+) -channel-blockers and ß-blockers are effective in SQTS. Since ß-blockers can block IKr and IKs , we aimed at determining carvedilol's and metoprolol's electrophysiological effects on N588K-KCNH2 and V307L-KCNQ1 channels. METHODS: Wild-type (WT)-KCNH2, WT-KCNQ1 and mutant N588K-KCNH2 and V307L-KCNQ1 channels were expressed in CHO-K1 or HEK-293T cells and IKs and IKr were recorded at baseline and during ß-blocker exposure. RESULTS: Carvedilol (10 µM) reduced IKs tail in WT- and V307L-KCNQ1 by 36.5 ± 5% and 18.6 ± 9% (P < 0.05). IC50 values were 16.3 µM (WT) and 46.1 µM (V307L), indicating a 2.8-fold decrease in carvedilol's IKs -blocking potency in V307L-KCNQ1. Carvedilol's (1 µM) inhibition of the IKr tail was attenuated in N588K-KCNH2 (4.5 ± 3% vs 50.3 ± 4%, WT, P < 0.001) with IC50 values of 2.8 µM (WT) and 25.4 µM (N588K). Carvedilol's IKr end-pulse inhibition, however, was increased in N588K-KCNH2 (10 µM, 60.7 ± 6% vs 36.5 ± 5%, WT, P < 0.01). Metoprolol (100 µM) reduced IKr end-pulse by 0.23 ± 3% (WT) and 74.1 ± 7% (N588K, P < 0.05), IKr tail by 32.9 ± 10% (WT) and 68.8 ± 7% (N588K, P < 0.05), and reduced IKs end-pulse by 18.3 ± 5% (WT) and 57.1 ± 11% (V307L, P < 0.05) and IKs tail by 3.3 ± 1% (WT) and 45.1 ± 13 % (V307L, P < 0.05), indicating an increased sensitivity to metoprolol in SQT mutated channels. CONCLUSIONS: N588K-KCNH2 and V307L-KCNQ1 mutations decrease carvedilol's inhibition of the IKs or IKr tail but increase carvedilol's IKr end-pulse inhibition and metoprolol's inhibition of tail and end-pulse currents. These different effects on SQT1 and SQT2 mutated channels should be considered when using ß-blocker therapy in SQTS patients.

Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.

Ion channels function as multi-protein complexes made up of ion-conducting α-subunits and regulatory ß-subunits. To detect, identify, and quantitate the regulatory ß-subunits in functioning K(+) channel complexes, we have chemically derivatized peptide-toxins that specifically react with strategically placed cysteine residues in the channel complex. Two protein labeling approaches have been developed to derivatize the peptide-toxin, charybdotoxin, with hydrophilic and hydrophobic bismaleimides, and other molecular probes. Using these cysteine-reactive peptide-toxins, we have specifically targeted KCNQ1-KCNE1 K(+) channel complexes expressed in both Xenopus oocytes and mammalian cells. The modular design of the reagents should permit this approach to be applied to the many ion channel complexes involved in electrical excitability as well as salt and water homoeostasis.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Purinergic signalling - a possible mechanism for KCNQ1 channel response to cell volume challenges.

AIM: A number of K(+) channels are regulated by small, fast changes in cell volume. The mechanisms underlying cell volume sensitivity are not known, but one possible mechanism could be purinergic signalling. Volume activated ATP release could trigger signalling pathways that subsequently lead to ion channel stimulation and cell volume back-regulation. Our aim was to investigate whether volume sensitivity of the voltage-gated K(+) channel, KCNQ1, is dependent on ATP release and regulation by purinergic signalling. METHODS: We used Xenopus oocytes heterologously expressing human KCNQ1, KCNE1, water channels (AQP1) and P2Y2 receptors. ATP release was monitored by a luciferin-luciferase assay and ion channel conductance was recorded by two-electrode voltage clamp. RESULTS: The luminescence assay showed that oocytes released ATP in response to mechanical, hypoosmotic stimuli and hyperosmotic stimuli. Basal ATP release was approx. three times higher in the KCNQ1 + AQP1 and KCNQ1 injected oocytes compared to the non-injected ones. Exogenously added ATP (0.1 mm) did not have any substantial effect on volume-induced KCNQ1 currents. Nevertheless, apyrase decreased all currents by about 50%. Suramin inhibited about 23% of the KCNQ1 volume sensitivity. Expression of P2Y2 receptors stimulated endogenous Cl(-) channels, but it also led to 68% inhibition of the KCNQ1 currents. Adenosine (0.1 mm) also inhibited the KCNQ1 currents by about 56%. CONCLUSION: Xenopus oocytes release ATP in response to mechanical stimuli and cell volume changes. Purinergic P2 and P1 receptors confer some of the KCNQ1 channel volume sensitivity, although endogenous adenosine receptors and expressed P2Y2 receptors do so in the negative direction.

An allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1.

The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac potassium channel KCNQ1. A total of 48 amino acid residues in the S6 segment, S4-S5 linker, and the proximal C-terminus of the KCNQ1 channel were mutated individually to alanine; alanines were mutated to cysteines. Residues modulating drug block were identified when mutant channels displayed <50% block on exposure to drug concentrations that inhibited wild-type current by ≥90%. Homology modeling of the KCNQ1 channel based on the Kv1.2 structure unexpectedly predicted that the key residue modulating drug block (F351) faces away from the permeating pore. In the open-state channel model, F351 lines a pocket that also includes residues L251 and V254 in S4-S5 linker. Docking calculations indicated that this pocket is large enough to accommodate quinidine. To test this hypothesis, L251A and V254A mutants were generated that display a reduced sensitivity to blockage with quinidine. Thus, our data support a model in which open state block of this channel occurs not via binding to a site directly in the pore but rather by a novel allosteric mechanism: drug access to a side pocket generated in the open-state channel configuration and lined by S6 and S4-S5 residues.

Disease characterization using LQTS-specific induced pluripotent stem cells.

AIMS: Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. CONCLUSIONS: This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

Galantamine (Reminyl) delays cardiac ventricular repolarization and prolongs the QT interval by blocking the HERG current.

Galantamine is a reversible inhibitor of acetylcholinesterase and an allosteric-potentiating ligand of the nicotinic acetylcholine receptors. It is used for treating mild-to-moderate Alzheimer's disease. Interestingly, QT interval prolongation on the electrocardiogram (ECG), malignant ventricular arrhythmias and syncope have been reported with galantamine. Our objective was to evaluate the effects of galantamine on cardiac ventricular repolarization. Three sets of experiments were undertaken: 1) Whole cell patch-clamp experiments: HERG- or KCNQ1+KCNE1-transfected cells were exposed to galantamine 0.1-1000 µmol/l (n=25 cells, total) to assess drug effect on HERG and KCNQ1+KCNE1 currents. 2) Langendorff perfusion experiments: Isolated hearts from male Hartley guinea pigs (n=9) were exposed to galantamine 1 µmol/l to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD(90)). 3) Cardiac telemetry experiments: Guinea pigs (n=7) implanted with wireless transmitters were injected a single intraperitoneal (i.p.) dose of galantamine 3mg/kg and 24h ECG recordings were made. 1) The estimated IC(50) for galantamine on HERG current was 760.2 µmol/l. Moreover, galantamine 10 µmol/l had a small inhibiting effect on KCNQ1+KCNE1 current (12.17 ± 2.19% inhibition, n=10 cells). 2) While pacing at cycle lengths of 150, 200 or 250 ms, galantamine 1 µmol/l prolonged MAPD(90) by respectively 5.1 ± 1.6 ms, 9.4 ± 1.9 ms and 12.1 ± 2.1 ms. 3) Galantamine 3 mg/kgi.p. caused a maximal 11.9 ± 2.7 ms prolongation of the corrected QT (QTc). Galantamine is a weak HERG blocker. This contributes to its mild QT-prolonging effect. Patients could be at risk of cardiac proarrhythmia during drug overdosage or interactions involving cytochrome 2D6 drug-metabolizing enzyme.

Extracellular potassium inhibits Kv7.1 potassium channels by stabilizing an inactivated state.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1-50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC(50) of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2-5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

A multiscale simulation system for the prediction of drug-induced cardiotoxicity.

The preclinical assessment of drug-induced ventricular arrhythmia, a major concern for regulators, is typically based on experimental or computational models focused on the potassium channel hERG (human ether-a-go-go-related gene, K(v)11.1). Even if the role of this ion channel in the ventricular repolarization is of critical importance, the complexity of the events involved make the cardiac safety assessment based only on hERG has a high risk of producing either false positive or negative results. We introduce a multiscale simulation system aiming to produce a better cardiotoxicity assessment. At the molecular scale, the proposed system uses a combination of docking simulations on two potassium channels, hERG and KCNQ1, plus three-dimensional quantitative structure-activity relationship modeling for predicting how the tested compound will block the potassium currents IK(r) and IK(s). The obtained results have been introduced in electrophysiological models of the cardiomyocytes and the ventricular tissue, allowing the direct prediction of the drug effects on electrocardiogram simulations. The usefulness of the whole method is illustrated by predicting the cardiotoxic effect of several compounds, including some examples in which classic hERG-based models produce false positive or negative results, yielding correct predictions for all of them. These results can be considered a proof of concept, suggesting that multiscale prediction systems can be suitable for being used for preliminary screening in lead discovery, before the compound is physically available, or in early preclinical development when they can be fed with experimentally obtained data.

Increased cardiac risk in concomitant methadone and diazepam treatment: pharmacodynamic interactions in cardiac ion channels.

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 µM), hNav1.5 (11.2 µM tonic block; 5.5 µM phasic block), and hCav1.2 (26.7 µM tonic block; 7.7 µM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 µM) and hKvLQT1/hminK (53.3 µM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 µM. However, coadministration of 1-µM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 µM and 26.6 µM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na channel block may leave hERG K channel block uncompensated, thereby increasing cardiac risk.

The selective estrogen receptor modulator raloxifene inhibits cardiac delayed rectifier potassium currents and voltage-gated sodium current without QTc interval prolongation.

Raloxifene is widely used in the treatment of postmenopausal osteoporosis and also has been shown to be cardioprotective. The effect of raloxifene on cardiac ion channels is not fully understood. The present study investigated whether raloxifene could affect the cloned hERG channel (I(hERG)) and recombinant human cardiac KCNQ1/KCNE1 channel (I(Ks)) stably expressed in HEK 293 cells using a patch-clamp technique. Raloxifene blocked I(hERG) with an IC(50) of 1.1 µM and decreased I(Ks) (IC(50): 4.8 µM) without affecting activation kinetics. In addition, raloxifene significantly decreased I(Na) (IC(50): 2.8 µM) in guinea pig ventricular myocytes. However, this drug (1 µM) did not increase QRS and QTc interval in isolated guinea pig hearts. These results demonstrate that raloxifene, despite its inhibitory action on delayed rectifier potassium currents, does not prolong ECG QTc interval, suggesting that raloxifene is likely a safe selective estrogen receptor modulator with less cardiac toxicity.