Channel Opener Retigabine Research Articles

Kv7 channel opener retigabine reduces self-administration of cocaine but not sucrose in rats.

The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviours that can be modelled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that Kv7/KCNQ channels may contribute to the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drugs is not known. Here, we tested the ability of retigabine (ezogabine), a Kv7 channel opener, to regulate instrumental behaviour in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a conditioned place preference (CPP) assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine pretreatment attenuated the SA of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared with sucrose-SA, cocaine-SA was associated with reductions in the expression of the Kv7.5 subunit in the nucleus accumbens, without alterations in Kv7.2 and Kv7.3. Therefore, these studies reveal a reward-specific reduction in SA behaviour and support the notion that Kv7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.

The Kv7 channel opener Retigabine reduces neuropathology and alleviates behavioral deficits in APP/PS1 transgenic mice

Hyperexcitability of neuronal networks is central to the pathogenesis of Alzheimer's disease (AD). Pharmacological activation of Kv7 channels is an effective way to reduce neuronal firing. Our results showed that that pharmacologically activating the Kv7 channel with Retigabine (RTG) can alleviate cognitive impairment in mice without affecting spontaneous activity. RTG could also ameliorate damage to the Nissl bodies in cortex and hippocampal CA and DG regions in 9-month-old APP/PS1 mice. Additionally, RTG could reduce the Aβ plaque number in the hippocampus and cortex of both 6-month-old and 9-month-old mice. By recordings of electroencephalogram, we showed that a decrease in the number of abnormal discharges in the brains of the AD model mice when the Kv7 channel was opened. Moreover, Western blot analysis revealed a reduction in the expression of the p-Tau protein in both the hippocampus and cortex upon Kv7 channel opening. These findings suggest that Kv7 channel opener RTG may ameliorate cognitive impairment in AD, most likely by reducing brain excitability.

Glial KCNQ K+ channels control neuronal output by regulating GABA release from glia in C. elegans

KCNQs are voltage-gated K+ channels that control neuronal excitability and are mutated in epilepsy and autism spectrum disorder (ASD). KCNQs have been extensively studied in neurons, but their function in glia is unknown. Using voltage, calcium, and GABA imaging, optogenetics, and behavioral assays, we show here for the first time in Caenorhabditis elegans (C.elegans) that glial KCNQ channels control neuronal excitability by mediating GABA release from glia via regulation of the function of L-type voltage-gated Ca2+ channels. Further, we show that human KCNQ channels have the same role when expressed in nematode glia, underscoring conservation of function across species. Finally, we show that pathogenic loss-of-function and gain-of-function human KCNQ2 mutations alter glia-to-neuron GABA signaling in distinct ways and that the KCNQ channel opener retigabine exerts rescuing effects. This work identifies glial KCNQ channels as key regulators of neuronal excitability via control of GABA release from glia.

KV7 but not dual small and intermediate KCa channel openers inhibit the activation of colonic afferents by noxious stimuli.

In numerous subtypes of central and peripheral neurons, small and intermediate conductance Ca2+-activated K+ (SK and IK, respectively) channels are important regulators of neuronal excitability. Transcripts encoding SK channel subunits, as well as the closely related IK subunit, are coexpressed in the soma of colonic afferent neurons with receptors for the algogenic mediators ATP and bradykinin, P2X3 and B2, highlighting the potential utility of these channels as drug targets for the treatment of abdominal pain in gastrointestinal diseases such as irritable bowel syndrome. Despite this, pretreatment with the dual SK/IK channel opener SKA-31 had no effect on the colonic afferent response to ATP, bradykinin, or noxious ramp distention of the colon. Inhibition of SK or IK channels with apamin or TRAM-34, respectively, yielded no change in spontaneous baseline afferent activity, indicating these channels are not tonically active. In contrast to its lack of effect in electrophysiological experiments, comparable concentrations of SKA-31 abolished ongoing peristaltic activity in the colon ex vivo. Treatment with the KV7 channel opener retigabine blunted the colonic afferent response to all applied stimuli. Our data therefore highlight the potential utility of KV7, but not SK/IK, channel openers as analgesic agents for the treatment of abdominal pain.NEW & NOTEWORTHY Despite marked coexpression of small (Kcnn1, Kcnn2) and intermediate (Kcnn4) conductance calcium-activated potassium channel transcripts with P2X3 (P2rx3) or bradykinin B2 (Bdkrb2) receptors in colonic sensory neurons, pharmacological activation of these channels had no effect on the colonic afferent response to ATP, bradykinin or luminal distension of the colon. This is in contrast to the robust inhibitory effect of the KV7 channel opener, retigabine.

Inhibition of neuronal Kv7 channels ameliorates MK-801-induced cognitive dysfunction in mice via up-regulating NAMPT expression

PurposeAbnormal energy metabolism affects cognitive function in schizophrenia. Nicotinamide phosphoribosyltransferase (NAMPT), as the rate-limiting enzyme of nicotinamide adenine dinucleotide (NAD+), is involved in energy metabolism by regulating the synthesis of NAD+. This study aims to clarify whether inhibition of Kv7 channels improves cognitive impairment by up-regulating NAMPT expression to increase the level of NAD+. MethodsThe dominant negative pore mutation of KCNQ2 in transgenic mice was achieved by mutating residual 279-Gly to Ser (rQ2-G279S). A cognitive deficit model was established by injecting MK-801 into C57BL/6J mice. Y-maze and prepulse inhibition (PPI) tests were performed to evaluate cognitive ability. Gene and protein expression of NAMPT in the mouse hippocampus, cortex, and PC-12 cells were measured by qRT-PCR and Western blot. The level of NAD+ was measured by a WST-8 assay. ResultsThe Y-maze and PPI results showed that genetic or pharmacological inhibition of Kv7 channels by XE991 enhanced cognitive function in mice. Furthermore, inhibition of Kv7 channels increased the gene and protein expression of NAMPT and the level of NAD+ in the hippocampus and cortex of the above animal model. Similarly, XE991 treatment increased NAMPT expression and NAD+ levels in PC-12 cells. NAMPT inhibitor FK866 and Kv7 channel opener retigabine reversed the effects of XE991 in vivo and in vitro. In addition, XE991 increased pAMPK protein expression in PC-12 cells, while AMPK inhibitor Compound C counteracted the effect of XE991 on increasing NAMPT expression and NAD+ levels. ConclusionsSuppression of Kv7 channel function improved spatial working memory and PPI impairment. This result may be achieved by activating AMPK to up-regulate NAMPT expression and thus increase NAD+ levels.

Blockade of Kv7 channels reverses the inhibitory effects of exchange protein directly activated by cAMP activation on purinergic contractions of the murine detrusor.

Purinergic contractions of the detrusor are reduced by cAMP, but the underlying mechanisms are unclear. We examined the effects of BK and Kv7 channel modulators on purinergic contractions of the detrusor and tested if the inhibitory effects of activators of the cAMP effectors, PKA and EPAC, were reduced by blockade of BK or Kv7 channels. Purinergic contractions of the murine detrusor were induced by electric field stimulation (EFS) or application of the P2X receptor agonist α,β-MeATP. EFS responses were inhibited by the L-type Ca2+ channel blocker nifedipine, but not by the SERCA inhibitor CPA or the SOCE blocker GSK7975A. The Kv7 channel opener retigabine and BK channel activator compound X inhibited purinergic responses, while blockade of Kv7 or BK channels with XE991 or iberiotoxin, respectively, augmented these responses. Application of the EPAC activator 007-AM or PKA activator 6-MB-cAMP inhibited EFS responses. These effects were unaffected by iberiotoxin; however, XE991 reduced the effects of 007-AM, but not 6-MB-cAMP. Kv7.5 was the only Kv7 transcript detected in isolated detrusor myocytes. These data suggest that purinergic contractions of the detrusor are regulated by BK and Kv7 channels and the latter may also play a role in EPAC-dependent inhibition of this activity.

The effects of certain TRP channels and voltage-gated KCNQ/Kv7 channel opener retigabine on calcitonin gene-related peptide release in the trigeminovascular system.

Calcitonin gene-related peptide release in trigeminovascular system is a pivotal component of neurogenic inflammation underlying migraine pathophysiology. Transient receptor potential channels and voltage-gated KCNQ/Kv7 potassium channels expressed throughout trigeminovascular system are important targets for modulation of calcitonin gene-related peptide release. We investigated the effects of certain transient receptor potential (TRP) channels the vanilloid 1 and 4 (TRPV1 and TRPV4), the ankyrin 1 (TRPA1), and metastatin type 8 (TRPM8), and voltage-gated potassium channel (Kv7) opener retigabine on calcitonin gene-related peptide release from peripheral (dura mater and trigeminal ganglion) and central (trigeminal nucleus caudalis) trigeminal components of rats. The experiments were carried out using well-established in-vitro preparations (hemiskull, trigeminal ganglion and trigeminal nucleus caudalis) from male Wistar rats. Agonists and antagonists of TRPV1, TRPV4, TRPA1 and TRPM8 channels, and also retigabine were tested on the in-vitro release of calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations were measured using enzyme-linked immunosorbent assay. Agonists of these transient receptor potential channels induced calcitonin gene-related peptide release from hemiskull, trigeminal ganglion and trigeminal nucleus caudalis, respectively. The transient receptor potential channels-induced calcitonin gene-related peptide releases were blocked by their specific antagonists and reduced by retigabine. Retigabine also decreased basal calcitonin gene-related peptide releases in all preparations. Our findings suggest that favorable antagonists of these transient receptor potential channels, or Kv7 channel opener retigabine may be effective in migraine therapy by inhibiting neurogenic inflammation that requires calcitonin gene-related peptide release.

The novel dual-mechanism Kv7 potassium channel/TSPO receptor activator GRT-X is more effective than the Kv7 channel opener retigabine in the 6-Hz refractory seizure mouse model

Epilepsy, one of the most common and most disabling neurological disorders, is characterized by spontaneous recurrent seizures, often associated with structural brain alterations and cognitive and psychiatric comorbidities. In about 30% of patients, the seizures are resistant to current treatments; so more effective treatments are urgently needed. Among the ∼30 clinically approved antiseizure drugs, retigabine (ezogabine) is the only drug that acts as a positive allosteric modulator (or opener) of voltage-gated Kv7 potassium channels, which is particularly interesting for some genetic forms of epilepsy. Here we describe a novel dual-mode-of-action compound, GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-carboxylic acid amide) that activates both Kv7 potassium channels and the mitochondrial translocator protein 18 kDa (TSPO), leading to increased synthesis of brain neurosteroids. TSPO activators are known to exert anti-inflammatory, neuroprotective, anxiolytic, and antidepressive effects, which, together with an antiseizure effect (mediated by Kv7 channels), would be highly relevant for the treatment of epilepsy. This prompted us to compare the antiseizure efficacy of retigabine and GRT-X in six mouse and rat models of epileptic seizures, including the 6-Hz model of difficult-to-treat focal seizures. Furthermore, the tolerability of the two compounds was compared in mice and rats. Potency comparisons were based on both doses and peak plasma concentrations. Overall, GRT-X was more effective than retigabine in three of the six seizure models used here, the most important difference being the high efficacy in the 6-Hz (32 mA) seizure model in mice. Based on drug plasma levels, GRT-X was at least 30 times more potent than retigabine in the latter model. These data indicate that GRT-X is a highly interesting novel anti-seizure drug with a unique (first-in-class) dual-mode mechanism of action.

KCNQ (Kv7) K<sup>+</sup> channel opener retigabine inhibits pain and itch.

KCNQ (Kv7) K<sup>+</sup> channel opener retigabine inhibits pain and itch.

Inhibition of M/Kv7 Currents Contributes to Chloroquine-Induced Itch in Mice.

M/Kv7 potassium channels play a key role in regulation of neuronal excitability. Modulation of neuronal excitability of primary sensory neurons determines the itch sensation induced by a variety of itch-causing substances including chloroquine (CQ). In the present study, we demonstrate that suppression of M/Kv7 channel activity contributes to generation of itch in mice. CQ enhances excitability of the primary sensory neurons through inhibiting M/Kv7 potassium currents in a Ca2+ influx-dependent manner. Specific M/Kv7 channel opener retigabine (RTG) or tannic acid (TA) not only reverses the CQ-induced enhancement of neuronal excitability but also suppresses the CQ-induced itch behavior. Systemic application of RTG or TA also significantly inhibits the itch behavior induced by a variety of pruritogens. Taken together, our findings provide novel insight into the molecular basis of CQ-induced itch sensation in mammals that can be applied to the development of strategies to mitigate itch behavior.

Abstract A143: Identification of the synergistic drugs in combination with EGFR therapy that can target ASCL1+ Lung Adenocarcinoma (A+ LUAD)

Abstract Introduction: Lung cancer is the leading cause of cancer death in the U.S. and in the world. In 2018, lung cancer alone resulted in 25.3% of all cancer deaths evaluated worldwide. Lung Adenocarcinoma (LUAD) is a major subtype of lung cancer and accounts for 40% of these tumors. Our group has recently discovered that about 20% of LUAD are characterized by abnormally high expression of the neuroendocrine transcription factor, ASCL1. In these tumors, ASCL1 orchestrates a network of genomic changes that create opportunities for treatment. We have shown that LUAD tumors expressing high ASCL1 have RET mRNA levels that are predictive of patient survival (p < 0.004), and, these tumors were susceptible to EGFR inhibitors, with having a functional interaction between RET and EGFR. We found EGFR inhibitor, Lapatinib, to have highest efficacy, indicating it to be a primary drug treatment for ASCL1+ (A+) LUAD. However, we and others have observed Lapatinib induces resistance to EGFR therapy over time, and, Lapatinib therapy associated by-pass signaling pathways have recently gained attention. Therefore, we hypothesize that for a long lasting therapy, it is important to identify additional drugs that in combination with EGFR inhibitor Lapatinib will eliminate A+ LUAD tumors. Methodology and Results: An in vitro preliminary screen of approx. 2000 FDA approved small molecule inhibitors done with A+ and A- LUAD cell lines in collaboration at NIH revealed potential candidates for alternative pathways, 80 of which showed a differential sensitivity to A+ LUAD as single agent. We identified best 28 drugs in total: 23 selected from this pre-screen (based on their effectiveness to A+LUAD lines) and 5 additional chosen based on their biological relevance to the disease, which may act upon putative compensatory pathways in EGFR therapy resistance. Our in vitro drug screening in HCC1833 ASCL1+ and ASCL1 knockdown lines has revealed 8 out of 28 drugs to synergize as per their IC50 and fixed ratios, with Lapatinib. From these 8 leads, we have confirmed a definite synergy of Lapatinib with DDR-1 7rh, an inhibitor of ECM receptor DDR-1, using Mac-Synergy-II software. DDR-1, Discoidin domain receptor-1, is a collagen receptor highly upregulated in NSCLC, and is associated with poor prognosis in LUAD. Our microarray data on lapatinib treated tumors from mice PDX has shown ECM signatures to be among top upregulated. Additionally, TM4SF1, a signaling molecule associated with DDR-1, was found to associate with poor outcome in A+LUAD (P=0.0001). Other synergistic drugs identified include, a K+ channel opener (Retigabine), Wnt signaling and Lyn kinase inhibitors (PRI-724, and Bafetinib), Tubulin inhibitor (Docetaxel) and 3 multi-tyrosine kinase inhibitors. Interestingly, Wnt pathway was found to be elevated in Lapatinib treated ASCL1+LUAD in our microarray data. Moreover, Wnt signaling in known to crosstalk with ECM and DDR-1 in other tumors. K+ channel dysfunction is associated with neuronal/neuroendocrine disorders and Lyn kinases are the predominant SRC kinases in brain tumors, indicating their possible involvement in ASCL1+ neuroendocrine tumor pathophysiology. Conclusion: Taken together this data reveals a potential involvement of ECM (DDR-1 receptor) and Wnt signaling in effect of Lapatinib, and the identified leads as path to novel combination therapies for A+ LUAD. Citation Format: Kshama Gupta, Farhad Kosari. Identification of the synergistic drugs in combination with EGFR therapy that can target ASCL1+ Lung Adenocarcinoma (A+ LUAD) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A143. doi:10.1158/1535-7163.TARG-19-A143

Anomalous Kv 7 channel activity in human malignant hyperthermia syndrome unmasks a key role for H2 S and persulfidation in skeletal muscle.

Human malignant hyperthermia (MH) syndrome is induced by volatile anaesthetics and involves increased levels of cystathionine β-synthase (CBS)-derived H2 S within skeletal muscle. This increase contributes to skeletal muscle hypercontractility. Kv 7 channels, expressed in skeletal muscle, may be a molecular target for H2 S. Here, we have investigated the role of Kv 7 channels in MH. Skeletal muscle biopsies were obtained from MH-susceptible (MHS) and MH-negative (MHN) patients. Immunohistochemistry, RT-PCR, Western blot, and in vitro contracture test (IVCT) were carried out. Development and characterization of primary human skeletal muscle cells (PHSKMC) and evaluation of cell membrane potential were also performed. The persulfidation state of Kv 7 channels and polysulfide levels were measured. Kv 7 channels were similarly expressed in MHN and MHS biopsies. The IVCT revealed an anomalous contractility of MHS biopsies following exposure to the Kv 7 channel opener retigabine. Incubation of negative biopsies with NaHS, prior to retigabine addition, led to an MHS-like positive response. MHS-derived PHSKMC challenged with retigabine showed a paradoxical depolarizing effect, compared with the canonical hyperpolarizing effect. CBS expression and activity were increased in MHS biopsies, resulting in a major polysulfide bioavailability. Persulfidation of Kv 7.4 channels was significantly higher in MHS than in MHN biopsies. In skeletal muscle of MHS patients, CBS-derived H2 S induced persulfidation of Kv 7 channels. This post-translational modification switches the hyperpolarizing activity into depolarizing. This mechanism can contribute to the pathological skeletal muscle hypercontractility typical of MH syndrome. This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

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-1β, 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.

The Kv7/KCNQ channel blocker XE991 protects nigral dopaminergic neurons in the 6-hydroxydopamine rat model of Parkinson’s disease

The excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) that supply the striatum with dopamine (DA) determines the function of the nigrostriatal system for motor coordination. We previously showed that 4-pyridinylmethyl-9(10H)-anthracenone (XE991), a specific blocker of Kv7/KCNQ channels, enhanced the excitability of nigral DA neurons and resulted in attenuation of haloperidol-induced catalepsy in a Parkinson’s disease (PD) rat model. However, whether XE991 exhibits neuroprotective effects towards DA neuron degeneration remains unknown. The aim of this study was to investigate the effects of Kv7/KCNQ channel blocker, XE991, on 6-hydroxydopamine (6-OHDA)-induced nigral DA neuron degeneration and motor dysfunction. Using immunofluorescence staining and western blotting, we showed that intracerebroventricular administration of XE991 prevented the 6-OHDA-induced decrease in tyrosine hydroxylase (TH)-positive neurons and TH protein expression in the SNc. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) also revealed that XE991 partly restored the levels of DA and its metabolites in the striatum. Moreover, XE991 decreased apomorphine (APO)-induced contralateral rotations, enhanced balance and coordination, and attenuated muscle rigidity in 6-OHDA-treated rats. Importantly, all neuroprotective effects by XE991 were abolished by co-application of Kv7/KCNQ channel opener retigabine and XE991. Thus, Kv7/KCNQ channel inhibition by XE991 can exert neuroprotective effects against 6-OHDA-induced degeneration of the nigrostriatal DA system and motor dysfunction.

Do KV 7.1 channels contribute to control of arterial vascular tone?

KV 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (VSMC) of diverse arteries, including mesenteric arteries. Based on pharmacological evidence using R-L3 (KV 7.1 channel opener), HMR1556, chromanol 293B (KV 7.1 channel blockers), stimulation of these channels has been suggested to evoke profound relaxation in various vascular beds of rats. However, the specificity of these drugs in vivo is uncertain. We used Kcnq1-/- mice and pharmacological tools to determine whether KV 7.1 channels play a role in the regulation of arterial tone. R-L3 produced similar concentration-dependent relaxations (EC50 ~1.4μM) of arteries from wild-type (Kcnq1+/+ ) and Kcnq1-/- mice, pre-contracted with either phenylephrine or 60mM KCl. This relaxation was not affected by 10μM chromanol 293B, 10μM HMR1556 or 30μM XE991 (pan-KV 7 channel blocker). The anti-contractile effects of the perivascular adipose tissue (PVAT) were normal in Kcnq1-/- arteries. Chromanol 293B and HMR1556 did not affect the anti-contractile effects of (PVAT). Isolated VSMCs from Kcnq1-/- mice exhibited normal peak KV currents. The KV 7.2-5 channel opener retigabine caused similar relaxations in Kcnq1-/- and wild-type vessels. We conclude that KV 7.1 channels were apparently not involved in the control of arterial tone by α1 -adrenoceptor agonists and PVAT. In addition, R-L3 is an inappropriate pharmacological tool for studying the function of native vascular KV 7.1 channels in mice.

The pan-Kv7 (KCNQ) Channel Opener Retigabine Inhibits Striatal Excitability by Direct Action on Striatal Neurons In Vivo.

Central Kv7 (KCNQ) channels are voltage-dependent potassium channels composed of different combinations of four Kv7 subunits, being differently expressed in the brain. Notably, striatal dopaminergic neurotransmission is strongly suppressed by systemic administration of the pan-Kv7 channel opener retigabine. The effect of retigabine likely involves the inhibition of the activity in mesencephalic dopaminergic neurons projecting to the striatum, but whether Kv7 channels expressed in the striatum may also play a role is not resolved. We therefore assessed the effect of intrastriatal retigabine administration on striatal neuronal excitability in the rat determined by c-Fos immunoreactivity, a marker of neuronal activation. When retigabine was applied locally in the striatum, this resulted in a marked reduction in the number of c-Fos-positive neurons after a strong excitatory striatal stimulus induced by acute systemic haloperidol administration in the rat. The relative mRNA levels of Kv7 subunits in the rat striatum were found to be Kv7.2 = Kv7.3 = Kv7.5 > >Kv7.4. These data suggest that intrastriatal Kv7 channels play a direct role in regulating striatal excitability in vivo.

Intracellular Zinc Potentiates KCNQ Channels through Modulation of their Sensitivity to PIP2

KCNQ potassium channels which underlie neuronal M current play dominant roles in controlling neuronal excitability. Inhibition or genetic loss of M channels are implicated in many excitability disorders (e.g. epilepsy, pain, deafness, arrhythmia), while M channel openers retigabine and flupirtine are used clinically as anticonvulsant drugs and analgesics. Zinc pyrithione (ZnPy) is one of the recently discovered M channel openers which activity was previously attributed to the pyrithione moiety; ZnPy is also used as zinc ionophore. Here we show that the likely mechanism of ZnPy action on KCNQ channels is elevation of intracellular zinc which, in turn, activates the channels. Using patch-clamp recordings we show that 10μM ZnPy increased the recombinant KCNQ4 and KCNQ2/3 currents as well as native M current in DRG neurons by 2.4±0.4, 1.6±0.1 and 1.6±0.2 fold respectively; these effects were completely reversed by zinc chelator TPEN (20μM) added in the presence of ZnPy. Fluorescent zinc imaging with FluoZin3TM in HEK293 cells and DRG neurons demonstrated that ZnPy induced robust intracellular zinc rises which were completely reversed by TPEN. Four other chemically unrelated zinc ionophores PDTC, DEDTC, DIQ and Zinc ionophore I had similar to ZnPy effects: all four compounds augmented KCNQ4 and KCNQ2/3 currents and induced intracellular zinc rises; TPEN completely abolished these effects. Using voltage sensitive phosphatase CiVSP we found that the Ci-VSP-dependent PIP2 hydrolysis strongly inhibited recombinant KCNQ4 and KCNQ2/3, however, ZnPy and PDTC completely abolished these effect rendering channels insensitive to Ci-VSP activation. TIRF imaging using optical PIP2 reporter PLCδPH-GFP confirmed that neither ZnPy nor PDTC affected the ability of CiVSP to hydrolyze PIP2, as determined by the depolarization-induced PLCδPH-GFP translocation. In sum, our data suggest that intracellular zinc potently augment KCNQ channel activity by reducing channel requirement for PIP2.

Lower KV7.5 Potassium Channel Subunit Expression in an Animal Model of Paroxysmal Dystonia.

Dystonia is a hyperkinetic disabling movement disorder. In the dt(sz) hamster, a model of paroxysmal dystonia, pronounced antidystonic effects of the KV7.2-5 potassium channel opener retigabine and aggravation of dystonia by a selective KV7.2-5 blocker indicated a pathophysiological role of an abnormal expression of KV7 channels. We therefore investigated the expression of KV7 subunits in brains of dystonic hamsters. While KV7.2 and KV7.3 subunits were unaltered, lower KV7.5 mRNA levels became evident in motor areas and in limbic structures of dystonic hamsters. The KV7.2/3 subunit-preferring channel opener N-(6-chloropyridin-3-yl)-3,4- difluorobenzamide (ICA 27243; 10-30 mg/kg i.p.) failed to reduce the severity of dystonia in mutant hamsters, suggesting that the previously observed antidystonic action of retigabine is mediated by the activation of KV7.5 channels. The experiments indicate a functional relevance for KV7.5 channels in paroxysmal dystonia. We suggest that compounds highly selective for subtypes of KV7 channels, i.e. for KV7.5, may provide new therapeutic approaches.

The Kv7 potassium channel activator retigabine decreases alcohol consumption in rats

Background: Activation of Kv7 potassium channels may decrease the reactivity of mesolimbic dopaminergic neurons that are implicated in mediating the reinforcing effects of ethanol. Objectives: The objective of this study was to determine whether the administration of the Kv7 potassium channel opener retigabine would decrease ethanol intake in Long Evans rats. Methods: A limited access two-bottle choice model of alcohol (10% solution) consumption was used in this study. A separate group of animals was tested to evaluate the actions of retigabine on sucrose (5% solution) consumption to determine whether this drug might produce non-selective impairment of the ability of rats to drink liquids. Animals were treated with either vehicle or increasing doses (2.5–7.5 mg/kg SC) of retigabine administered over a 3-day period. Results: Compared to vehicle, retigabine at a dose of 7.5 mg/kg produced a reduction in the amount of ethanol consumed. These effects did not occur in association with significant changes in water consumption. A significant time effect was found for the actions of retigabine in sucrose-drinking rats with a trend for an increase in sucrose intake with the highest dose of retigabine administered. Conclusions: These results indicate that the administration of retigabine may produce a decrease in ethanol consumption by rats at doses that do not significantly reduce the drinking of either water or a sucrose solution. These findings are consistent with the hypothesis that activation of Kv7 channels facilitates the reduction of alcohol consumption in the rat.

M1-Muscarinic Receptors Promote Fear Memory Consolidation via Phospholipase C and the M-Current

Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with β2-adrenergic (β2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating β2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.