Rottlerin: Structure Modifications and KCNQ1/KCNE1 Ion Channel Activity.

The slow delayed rectifier potassium current (IKs ) is formed by the KCNQ1 (Kv 7.1) channel, an ion channel of four α-subunits that modulates KCNE1 ß-subunits. IKs is central to the repolarization of the cardiac action potential. Loss of function mutation reducing ventricular cardiac IKs cause the long-QT syndrome (LQTS), a disorder that predisposes patients to arrhythmia and sudden death. Current therapy for LQTS is inadequate. Rottlerin, a natural product of the kamala tree, activates IKs and has the potential to provide a new strategy for rational drug therapy. In this study, we show that simple modifications such as penta-acetylation or penta-methylation of rottlerin blunts activation activity. Total synthesis was used to prepare side-chain-modified derivatives that slowed down KCNQ1/KCNE1 channel deactivation to different degrees. A binding hypothesis of rottlerin is provided that opens the way to improved IKs activators as novel therapeutics for the treatment of LQTS.

Polyunsaturated fatty acid-derived IKs channel activators shorten the QT interval ex-vivo and in-vivo.

AIM: We aimed to assess the ability of natural and modified polyunsaturated fatty acids (PUFAs) to shorten QT interval in ex-vivo and in-vivo guinea pig hearts. METHODS: The effect of one natural (docosahexaenoic acid [DHA]) and three modified (linoleoyl glycine [Lin-GLY], docosahexaenoyl glycine [DHA-GLY], N-arachidonoyl taurine [N-AT]) PUFAs on ventricular action potential duration (APD) and QT interval was studied in a E4031 drug-induced long QT2 model of ex-vivo guinea pig hearts. The effect of DHA-GLY on QT interval was also studied in in-vivo guinea pig hearts upon intravenous administration. The effect of modified PUFAs on IKs was studied using Xenopus laevis oocytes expressing human KCNQ1 and KCNE1. RESULTS: All tested PUFAs shortened ADP and QT interval in ex-vivo guinea pig hearts, however, with different ability in restoring baseline APD/QT interval with specific modified PUFAs being most efficacious. Despite comparable ability in activating the human KCNQ1/KCNE1 channel, Lin-GLY was not as effective in shortening APD/QT interval as DHA-GLY in ex-vivo hearts. By constructing a guinea pig-like KCNE1, we found Lin-GLY to induce less activating effect compared with DHA-GLY on human KCNQ1 co-expressed with guinea pig-like KCNE1. Docosahexaenoyl glycine was studied in more detail and was found to shorten QT interval in in-vivo guinea pig hearts. CONCLUSION: Our results show that specific PUFAs shorten QT interval in guinea pig hearts. The tendency of modified PUFAs with pronounced IKs channel activating effect to better restore QT interval suggests that modifying PUFAs to target the IKs channel is a means to improve the QT-shortening effect.

Reversal of a Treatment-Resistant, Depression-Related Brain State with the Kv7 Channel Opener Retigabine.

Neuroinflammation is associated with increased vulnerability to diverse psychiatric conditions, including treatment-resistant major depressive disorder (MDD). Here we assessed whether high fat diet (HFD) induced neuroinflammation may be suitable to model a treatment-resistant depressive-like brain state in mice. Male and female mice were fed a HFD for 18 weeks, followed by quantitation of glucose tolerance, inflammatory markers of brain tissue (TNFα, IL-6, IL-1ß, Iba-1), neural excitability in the prelimbic cortex (PLC), as well as assessment of emotional reactivity and hedonic behavior in a battery of behavioral tests. In addition, we assessed the behavioral responsiveness of mice to fluoxetine, desipramine, ketamine, and the Kv7 channel opener and anticonvulsant retigabine. HFD exposure led to glucose intolerance and neuroinflammation in male mice, with similar but non-significant trends in females. Neuroinflammation of males was associated with anxious-depressive-like behavior and defects in working memory, along with neural hyperexcitability and increased Ih currents of pyramidal cells in the PLC. The behavioral changes were largely resistant to chronic treatment with fluoxetine and desipramine, as well as ketamine. By contrast, retigabine (also known as ezogabine) normalized neural excitability and Ih currents recorded from slices of HFD-treated animals and significantly ameliorated most of the behavioral impairments, without effects in control diet exposed animals. Thus, treatment resistant depressive-like brain states that are associated with chronic neuroinflammation may involve hyperexcitability of pyramidal neurons and may be effectively treated by retigabine.

ω-6 and ω-9 polyunsaturated fatty acids with double bonds near the carboxyl head have the highest affinity and largest effects on the cardiac IKs potassium channel.

AIM: The IKs channel is important for termination of the cardiac action potential. Hundreds of loss-of-function mutations in the IKs channel reduce the K+ current and, thereby, delay the repolarization of the action potential, causing Long QT Syndrome. Long QT predisposes individuals to Torsades de Pointes which can lead to ventricular fibrillation and sudden death. Polyunsaturated fatty acids (PUFAs) are potential therapeutics for Long QT Syndrome, as they affect IKs channels. However, it is unclear which properties of PUFAs are essential for their effects on IKs channels. METHODS: To understand how PUFAs influence IKs channel activity, we measured effects on IKs current by two-electrode voltage clamp while changing different properties of the hydrocarbon tail. RESULTS: There was no, or weak, correlation between the tail length or number of double bonds in the tail and the effects on or apparent binding affinity for IKs channels. However, we found a strong correlation between the positions of the double bonds relative to the head group and effects on IKs channels. CONCLUSION: Polyunsaturated fatty acids with double bonds closer to the head group had higher apparent affinity for IKs channels and increased IKs current more; shifting the bonds further away from the head group reduced apparent binding affinity for and effects on the IKs current. Interestingly, we found that ω-6 and ω-9 PUFAs, with the first double bond closer to the head group, left-shifted the voltage dependence of activation the most. These results allow for informed design of new therapeutics targeting IKs channels in Long QT Syndrome.

Heterogeneity in Kv7 channel function in the cerebral and coronary circulation.

OBJECTIVES: Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels. METHODS AND RESULTS: PCR confirmed that basilar, Circle of Willis and LAD arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50 µM) was significantly greater in the basilar artery compared to the LAD artery. Similarly, the Kv7 channel inhibitor, linopirdine (10 µM) caused a stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than in the coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. CONCLUSIONS: This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction.

Characterization of a novel high-potency positive modulator of K(v)7 channels.

K(v)7 channel activators decrease neuronal excitability and might potentially treat neuronal hyperexcitability disorders like epilepsy and mania. Here we introduce NS15370 ((2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3-pyridyl]acetamide)hydrochloride, an in vitro high-potency chemical analogue of retigabine, without effects on GABA(A) receptors. NS15370 activates recombinant homo- and heteromeric K(v)7.2-K(v)7.5 channels in HEK293 cells at sub-micromolar concentrations (EC50~100 nM, as quantified by a fluorescence based Tl⁺-influx assay). In voltage clamp experiments NS15370 exhibits a complex, concentration-dependent mode-of-action: At low concentrations it accelerates voltage-dependent activation rates, slows deactivations, and increases steady-state current amplitudes. Quantified by the peak-tail current method, the V½ value of the steady-state activation curve is shifted towards hyperpolarized potentials at concentrations ~100 times lower than retigabine. However, in contrast to retigabine, NS15370 also introduces a distinct time-dependent current decrease, which eventually, at higher concentrations, causes suppression of the current at depolarized potentials, and an apparent "cross-over" of the voltage-activation curve. In brain slices, NS15370 hyperpolarizes and increases spike frequency adaptation of hippocampal CA1 neurons and the compound reduces the autonomous firing of dopaminergic neurons in the substantia-nigra pars compacta. NS15370 is effective in rodent models of hyperexcitability: (i) it yields full protection against mouse 6 Hz seizures and rat amygdala kindling discharges, two models of partial epilepsia; (ii) it reduces (+)-MK-801 hydrogen maleate (MK-801)-induced hyperactivity as well as chlordiazepoxide (CDP)+d-amphetamine (AMP)-induced hyperactivity, models sensitive to classic anti-psychotic and anti-manic treatments, respectively. Our findings with NS15370 consolidate neuronal K(v)7 channels as targets for anti-epileptic and psychiatric drug development.

Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator.

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.

Pharmacological dissection of K(v)7.1 channels in systemic and pulmonary arteries.

BACKGROUND AND PURPOSE: The aim of this study was to characterize the functional impact of KCNQ1-encoded voltage-dependent potassium channels (K(v)7.1) in the vasculature. EXPERIMENTAL APPROACH: Mesenteric arteries, intrapulmonary arteries and thoracic aortae were isolated from adult rats. K(v)7.1 channel expression was established by fluorescence immunocytochemistry. Wire myography determined functionality of these channels in response to selective blockers and activators. Xenopus oocytes expressing K(v)7.1 channels were used to assess the effectiveness of selective K(v)7.1 channel blockers. KEY RESULTS: K(v)7.1 channels were identified in arterial myocytes by immunocytochemistry. K(v)7.1 blockers HMR1556, L-768,673 (10 µM) and JNJ39490282 (JNJ282; 1 µM) had no contractile effects in arteries, whereas the pan-K(v)7 channel blocker linopirdine (10 µM) evoked robust contractions. Application of two compounds purported to activate K(v)7.1 channels, L-364 373 (R-L3) and mefenamic acid, relaxed mesenteric arteries preconstricted by methoxamine. These responses were reversed by HMR1556 or L-768,673 but not JNJ282. Similar effects were observed in the thoracic aorta and intrapulmonary arteries. CONCLUSIONS AND IMPLICATIONS: In contrast to previous assumptions, K(v)7.1 channels expressed in arterial myocytes are functional ion channels. Although these channels do not appear to contribute to resting vascular tone, K(v)7.1 activators were effective vasorelaxants.

Drosophila KCNQ channel displays evolutionarily conserved electrophysiology and pharmacology with mammalian KCNQ channels.

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac I(Ks) current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50-60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K(+) current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.

Ginsenoside Rg3 activates human KCNQ1 K+ channel currents through interacting with the K318 and V319 residues: a role of KCNE1 subunit.

The slowly activating delayed rectifier K(+) channels (I(Ks)) are one of the main pharmacological targets for development of drugs against cardiovascular diseases. Cardiac I(Ks) consists of KCNQ1 plus KCNE1 subunits. Ginsenoside, one of the active ingredient of Panax ginseng, enhances cardiac I(Ks) currents. However, little is known about the molecular mechanisms of how ginsenoside interacts with channel proteins to enhance cardiac I(Ks). In the present study, we investigated ginsenoside Rg(3) (Rg(3)) effects on human I(Ks) by co-expressing human KCNQ1 plus KCNE1 subunits in Xenopus oocytes. Rg(3) enhanced I(Ks) currents in concentration- and voltage-dependent manners. The EC(50) was 15.2+/-8.7 microM. However, in oocytes expressing KCNQ1 alone, Rg(3) inhibited the currents with concentration- and voltage-dependent manners. The IC(50) was 4.8+/-0.6 microM. Since Rg(3) acts opposite ways in oocytes expressing KCNQ1 alone or KCNQ1 plus KCNE1 subunits, we examined Rg(3) effects after co-expression of different ratios of KCNE1 and KCNQ1. The increase of KCNE1/KCNQ1 ratio converted I(Ks) inhibition to I(Ks) activations. One to ten ratio of KCNE1 and KCNQ1 subunit is required for Rg(3) activation of I(Ks). Mutations of K318 and V319 into K318Y and V319Y of KCNQ1 channel abolished Rg(3) effects on KCNQ1 or KCNQ1 plus KCNE1 channel currents. The docked modeling revealed that K318 residue plays a key role in stabilization between Rg(3) and KCNQ1 plus KCNE1 or KCNQ1 subunit. These results indicate that Rg(3)-induced activation of I(Ks) requires co-assembly of KCNQ1 and KCNE1 subunits and achieves this through interaction with residues K318 and V319 of KCNQ1 subunit.

A shared mechanism for lipid- and beta-subunit-coordinated stabilization of the activated K+ channel voltage sensor.

The low-dielectric plasma membrane provides an energy barrier hindering transmembrane movement of charged particles. The positively charged, voltage-sensing fourth transmembrane domain (S4) of voltage-gated ion channels must surmount this energy barrier to initiate channel activation, typically necessitating both membrane depolarization and interaction with membrane lipid phospho-head groups (MLPHGs). In contrast, and despite containing S4, the KCNQ1 K(+) channel alpha subunit exhibits predominantly constitutive activation when in complexes with transmembrane beta subunits, MinK-related peptide (MiRP) 1 (KCNE2) or MiRP2 (KCNE3). Here, using a 2-electrode voltage clamp and scanning mutagenesis of channels heterologously expressed in Xenopus laevis oocytes, we discovered that 2 of the 8 MiRP2 extracellular domain acidic residues (D54 and D55) are important for KCNQ1-MiRP2 constitutive activation. Double-mutant thermodynamic cycle analysis revealed energetic coupling of D54 and D55 to R237 in KCNQ1 S4 but not to 10 other native or introduced polar residues in KCNQ1 S4 and surrounding linkers. MiRP2-D54 and KCNQ1-R237 also similarly dictated susceptibility to the inhibitory effects of MLPHG hydrolysis, whereas other closely situated polar residues did not. Thus, by providing negative charge near the plasma membrane extracellular face, MiRP2 uses a lipomimetic mechanism to constitutively stabilize the activated KCNQ1 voltage sensor.

Evaluation of channel function after alteration of amino acid residues at the pore center of KCNQ1 channel.

The effect of the electrical charge or the size of the amino acid residue at the pore center of a slowly activation component of the delayed rectifier potassium channel: KCNQ1 was studied. K(+) currents were measured after transfection of one of four KCNQ1 mutants: substituting Isoleucine with Lysine, Glutamate, Valine or Glycine and then transfected in COS-7 cells. Both the negatively- and positive charged residue I313K and I313E showed a loss of function when expressed alone and a dominant negative suppression when co-expressed with wild type KCNQ1. When the site was substituted with the smallest neutral amino acid residue: I313G, there was a small reduction of current when transfected alone and a gain of function when co-transfected with the wild type. I313V showed no difference from the wild type. Changes of amino acid residue at the pore center of KCNQ1 may alter the channel function but this depends on the electrical charge or the size of amino acid residue.

Bimodal effects of the Kv7 channel activator retigabine on vascular K+ currents.

BACKGROUND AND PURPOSE: This study investigated the functional and electrophysiological effects of the Kv7 channel activator, retigabine, on murine portal vein smooth muscle. EXPERIMENTAL APPROACH: KCNQ gene expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical experiments. Whole cell voltage clamp and current clamp were performed on isolated myocytes from murine portal vein. Isometric tension recordings were performed on whole portal veins. K+ currents generated by KCNQ4 and KCNQ5 expression were recorded by two-electrode voltage clamp in Xenopus oocytes. KEY RESULTS: KCNQ1, 4 and 5 were expressed in mRNA derived from murine portal vein, either as whole tissue or isolated myocytes. Kv7.1 and Kv7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2-20 microM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at -20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the K(v)7 blocker XE991 (10 muM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine. CONCLUSIONS AND IMPLICATIONS: The Kv7 channel activators retigabine and flupirtine have bimodal effects on vascular potassium currents, which are not seen with recombinant KCNQ channels. These results provide support for KCNQ4- or KCNQ5-encoded channels having an important functional impact in the vasculature.