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Neuronal K V 7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca 2+ -binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca 2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of K V 7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca 2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca 2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca 2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca 2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves K V 7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling., Competing Interests: EN, FJ, AM, JU, AA, CM, GB, CD, OM, NG, AV No competing interests declared, (© 2023, Nuñez et al.)

Potassium channels facilitate and regulate physiological processes as diverse as electrical signaling, ion, solute and hormone secretion, fluid homeostasis, hearing, pain sensation, muscular contraction, and the heartbeat. Potassium channels are each formed by either a tetramer or dimer of pore-forming α subunits that co-assemble to create a multimer with a K + -selective pore that in most cases is capable of functioning as a discrete unit to pass K + ions across the cell membrane. The reality in vivo, however, is that the potassium channel α subunit multimers co-assemble with ancillary subunits to serve specific physiological functions. The ancillary subunits impart specific physiological properties that are often required for a particular activity in vivo; in addition, ancillary subunit interaction often alters the pharmacology of the resultant complex. In this chapter the modes of action of ancillary subunits on K + channel physiology and pharmacology are described and categorized into various mechanistic classes., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the K V 7.2/3 channel opening activity of 45 derivatives. Sulfide analogues were identified that are devoid of the risk of quinone formation, but possess potent K V 7.2/3 opening activity. For example, flupirtine analogue 3-(3,5-difluorophenyl)-N-(6-(isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl)propanamide (48) has 100-fold enhanced activity (EC 50 =1.4 nm), a vastly improved toxicity/activity ratio, and the same efficacy as retigabine in vitro., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The M-current (I M ) is a voltage-dependent, persistent K + current so termed because it is strongly inhibited by the cholinergic agonist muscarine. The I M main function is to limit neuronal excitability by contrasting action potential firing. Although motoneurons are sensitive to acetylcholine, the role of I M in modulating their excitability is still controversial. The aim of the present report was to examine the presence of I M in hypoglossal motoneurons (HMs) and its role in the modulation of firing properties using an in vitro model of rat brainstem slice. For this purpose, we employed the whole-cell patch-clamp technique to record HM responses upon stimulation with either a standard I M deactivation voltage protocol or depolarizing current steps. Voltage commands from depolarized potential induced inward relaxations with the common characteristics of I M , comprising inhibition by either muscarine (10μM) or the selective I M inhibitor linopirdine (30μM). I M was pharmacologically distinguished from the hyperpolarization-activated inward-rectifying current and, within the -20 to -50mV range, deactivated with >100-ms time constant. Current-clamp experiments demonstrated that I M strongly regulated HM action potential firing, since both muscarine and linopirdine increased spike frequency whereas the M-channel opener retigabine (20μM) reduced it. Conversely, I M seemed uninvolved in the generation of the medium afterhyperpolarizing potential. Our results suggest that HMs possess I M , whose pharmacological modulation is an important tool to up- or down-regulate excitability, to be explored in experimental models of neurodegeneration., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

• Induction of a cortical inflammatory lesion affects the excitability of ipsilateral thalamic neurons in the EAE model of MS. • K v 7.3 channels regulate the firing pattern of thalamic neurons and are crucial for auditory-stimulus discrimination. • RTG treatment rescues inflammation-induced hyperexcitability of vMGN neurons and preserves auditory information processing. Multiple Sclerosis (MS) is an autoimmune neurodegenerative disease, whose primary hallmark is the occurrence of inflammatory lesions in white and grey matter structures. Increasing evidence in MS patients and respective murine models reported an impaired ionic homeostasis driven by inflammatory-demyelination, thereby profoundly affecting signal propagation. However, the impact of a focal inflammatory lesion on single-cell and network functionality has hitherto not been fully elucidated. In this study, we sought to determine the consequences of a localized cortical inflammatory lesion on the excitability and firing pattern of thalamic neurons in the auditory system. Moreover, we tested the neuroprotective effect of Retigabine (RTG), a specific K v 7 channel opener, on disease outcome. To resemble the human disease, we focally administered pro-inflammatory cytokines, TNF-α and IFN-γ, in the primary auditory cortex (A1) of MOG 35-55 immunized mice. Thereafter, we investigated the impact of the induced inflammatory milieu on afferent thalamocortical (TC) neurons, by performing ex vivo recordings. Moreover, we explored the effect of K v 7 channel modulation with RTG on auditory information processing, using in vivo electrophysiological approaches. Our results revealed that a cortical inflammatory lesion profoundly affected the excitability and firing pattern of neighboring TC neurons. Noteworthy, RTG restored control-like values and TC tonotopic mapping. Our results suggest that RTG treatment might robustly mitigate inflammation-induced altered excitability and preserve ascending information processing. [ABSTRACT FROM AUTHOR]

This article explores the role of potassium channels in animal models of post-traumatic stress disorder (PTSD) and their potential therapeutic implications. Potassium channels are important in regulating brain function and have been associated with various neuropsychiatric disorders. Animal models have shown that different types of potassium channels play a role in fear-related behaviors and memory. Genome-wide association studies have identified genes involved in fear and stress responses that may contribute to vulnerability to PTSD. Specific potassium channel subtypes have been implicated in the development of certain PTSD symptoms and may be potential targets for treatment. Activation or inhibition of these channels can affect fear responses and consolidation of fear memories. The article acknowledges the need for further research in humans and the limitations of the available evidence. [Extracted from the article]

The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties. [ABSTRACT FROM AUTHOR]

The KCNQ family is comprised of five genes and the expression products form voltage-gated potassium channels (Kv7.1–7.5) that have a major impact upon cellular physiology in many cell types. Each functional Kv7 channel forms as a tetramer that often associates with proteins encoded by the KCNE gene family (KCNE1-5) and is critically reliant upon binding of phosphatidylinositol bisphosphate (PIP2) and calmodulin. Other modulators like A-kinase anchoring proteins, ubiquitin ligases and Ca-calmodulin kinase II alter Kv7 channel function and trafficking in an isoform specific manner. It has now been identified that for Kv7.4, G protein βγ subunits (Gβγ) can be added to the list of key regulators and is paramount for channel activity. This article provides an overview of this nascent field of research, highlighting themes and directions for future study. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium (Kv) channels each comprise four pore-forming α-subunits that orchestrate essential duties such as voltage sensing and K+ selectivity and conductance. In vivo, however, Kv channels also incorporate regulatory subunits-some Kv channel specific, others more general modifiers of protein folding, trafficking, and function. Understanding all the above is essential for a complete picture of the role of Kv channels in physiology and disease.

Potassium channels facilitate and regulate physiological processes as diverse as electrical signaling, ion, solute and hormone secretion, fluid homeostasis, hearing, pain sensation, muscular contraction, and the heartbeat. Potassium channels are each formed by either a tetramer or dimer of pore-forming α subunits that co-assemble to create a multimer with a K+-selective pore that in most cases is capable of functioning as a discrete unit to pass K+ ions across the cell membrane. The reality in vivo, however, is that the potassium channel α subunit multimers co-assemble with ancillary subunits to serve specific physiological functions. The ancillary subunits impart specific physiological properties that are often required for a particular activity in vivo; in addition, ancillary subunit interaction often alters the pharmacology of the resultant complex. In this chapter the modes of action of ancillary subunits on K+ channel physiology and pharmacology are described and categorized into various mechanistic classes.

KV7 channels exert a pivotal role regulating vascular tone in several vascular beds. In this context, KV7 channel agonists represent an attractive strategy for the treatment of pulmonary arterial hypertension (PAH). Therefore, in this study, we have explored the pulmonary vascular effects of the novel KV7 channel agonist URO-K10. Consequently, the vasodilator and electrophysiological effects of URO-K10 were tested in rat and human pulmonary arteries (PA) and PA smooth muscle cells (PASMC) using myography and patch-clamp techniques. Protein expression was also determined by Western blot. Morpholino-induced knockdown of KCNE4 was assessed in isolated PA. PASMC proliferation was measured by BrdU incorporation assay. In summary, our data show that URO-K10 is a more effective relaxant of PA than the classical KV7 activators retigabine and flupirtine. URO-K10 enhanced KV currents in PASMC and its electrophysiological and relaxant effects were inhibited by the KV7 channel blocker XE991. The effects of URO-K10 were confirmed in human PA. URO-K10 also exhibited antiproliferative effects in human PASMC. Unlike retigabine and flupirtine, URO-K10-induced pulmonary vasodilation was not affected by morpholino-induced knockdown of the KCNE4 regulatory subunit. Noteworthy, the pulmonary vasodilator efficacy of this compound was considerably increased under conditions mimicking the ionic remodelling (as an in vitro model of PAH) and in PA from monocrotaline-induced pulmonary hypertensive rats. Taking all together, URO-K10 behaves as a KCNE4-independent KV7 channel activator with much increased pulmonary vascular effects compared to classical KV7 channel activators. Our study identifies a promising new drug in the context of PAH.

Voltage-gated potassium (Kv) channels formed by Kv7 (KCNQ) α-subunits are recognized as crucial for vascular smooth muscle function, in addition to their established roles in the heart (Kv7.1) and the brain (Kv7.2-5). In vivo, Kv7 α-subunits are often regulated by KCNE subfamily ancillary (β) subunits. We investigated the effects of targeted germline Kcne4 deletion on mesenteric artery reactivity in adult male and female mice. Kcne4 deletion increased mesenteric artery contractility in response to α-adrenoceptor agonist methoxamine, and decreased responses to Kv7.2-7.5 channel activator ML213, in male but not female mice. In contrast, Kcne4 deletion markedly decreased vasorelaxation in response to isoprenaline in both male and female mice. Kcne4 expression was 2-fold lower in the female versus the male mouse mesenteric artery, and Kcne4 deletion elicited only moderate changes of other Kcne transcripts, with no striking sex-specific differences. However, Kv7.4 protein expression in females was twice that in males, and was reduced in both sexes by Kcne4 deletion. Our findings confirm a crucial role for KCNE4 in regulation of Kv7 channel activity to modulate vascular tone, and provide the first known molecular mechanism for sex-specificity of this modulation that has important implications for vascular reactivity and may underlie sex-specific susceptibility to cardiovascular diseases.

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. [ABSTRACT FROM AUTHOR]

• Muscarinic acetylcholine receptors (mAChR) inhibit KCNQ channels in calyces. • Activation of mAChRs has an excitatory effect on calyx terminals. • Activation of mAChRs decreases the first spike latency during step depolarizations. • Activation of mAChRs decreases the action potential threshold. • mAChR activation by efferents can be used to modulate calyx activity. Vestibular nerve afferents are divided into regular and irregular groups based on the variability of interspike intervals in their resting discharge. Most afferents receive inputs from bouton terminals that contact type II hair cells as well as from calyx terminals that cover the basolateral walls of type I hair cells. Calyces have an abundance of different subtypes of KCNQ (Kv7) potassium channels and muscarinic acetylcholine receptors (mAChRs) and receive cholinergic efferent inputs from neurons in the brainstem. We investigated whether mAChRs affected membrane properties and firing patterns of calyx terminals through modulation of KCNQ channel activity. Patch clamp recordings were performed from calyx terminals in central regions of the cristae of the horizontal and anterior canals in 13–26 day old Sprague-Dawley rats. KCNQ mediated currents were observed as voltage sensitive currents with slow kinetics (activation and deactivation), resulting in spike frequency adaptation so that calyces at best fired a single action potential at the beginning of a depolarizing step. Activation of mAChRs by application of oxotremorine methiodide or inhibition of KCNQ channels by linopirdine dihydrochloride decreased voltage activated currents by ∼30%, decreased first spike latencies by ∼40%, resulted in action potential generation in response to smaller current injections and at lower (i.e., more hyperpolarized) membrane potentials, and increased the number of spikes fired during depolarizing steps. Interestingly, some of the calyces showed spontaneous discharge in the presence of these drugs. Together, these findings suggest that cholinergic efferents can modulate the response properties and encoding of head movements by afferents. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium channels of the KV7 family are expressed in many tissues. The physiological importance of KV7 channels is evident from specific forms of disorders linked to dysfunctional KV7 channels, including variants of epilepsy, cardiac arrhythmia and hearing impairment. Thus, understanding how KV7 channels are regulated in the body is of great interest. This Mini Review focuses on the effects of polyunsaturated fatty acids (PUFAs) on KV7 channel activity and possible underlying mechanisms of action. By summarizing reported effects of PUFAs on KV7 channels and native KV7-mediated currents, we conclude that the generally observed effect is a PUFA-induced increase in current amplitude. The increase in current is commonly associated with a shift in the voltage-dependence of channel opening and in some cases with increased maximum conductance. Auxiliary KCNE subunits, which associate with KV7 channels in certain tissues, may influence PUFA effects, though findings are conflicting. Both direct and indirect activating PUFA effects have been described, direct effects having been most extensively studied on KV7.1. The negative charge of the PUFA head-group has been identified as critical for electrostatic interaction with conserved positively charged amino acids in transmembrane segments 4 and 6. Additionally, the localization of double bonds in the PUFA tail tunes the apparent affinity of PUFAs to KV7.1. Indirect effects include those mediated by PUFA metabolites. Indirect inhibitory effects involve KV7 channel degradation and re-distribution from lipid rafts. Understanding how PUFAs regulate KV7 channels may provide insight into physiological regulation of KV7 channels and bring forth new therapeutic strategies. [ABSTRACT FROM AUTHOR]

The voltage-gated Kv7.2/Kv7.3 potassium channel is a critical regulator of neuronal excitability. It is strategically positioned at the axon initial segment (AIS) of neurons, where it effectively inhibits repetitive action potential firing. While the selective accumulation of Kv7.2/Kv7.3 channels at the AIS requires binding to the adaptor protein ankyrin G, it is currently unknown if additional molecular mechanisms contribute to the localization and fine-tuning of channel numbers at the AIS. Here, we utilized a chimeric approach to pinpoint regions within the Kv7.3 C-terminal tail with an impact upon AIS localization. This strategy identified two domains with opposing effects upon the AIS localization of Kv7.3 chimeras expressed in cultured hippocampal neurons. While a membrane proximal domain reduced AIS localization of Kv7.3 chimeras, helix D increased and stabilized chimera AIS localization. None of the identified domains were required for AIS localization. However, the domains modulated the relative efficiency of the localization raising the possibility that the two domains contribute to the regulation of Kv7 channel numbers and nanoscale organization at the AIS. [ABSTRACT FROM AUTHOR]

KCNQ (Kv7) channels are voltage-gated, phosphatidylinositol 4,5-bisphosphate- (PIP 2 -) modulated potassium channels that play essential roles in regulating the activity of neurons and cardiac myocytes. Hundreds of mutations in KCNQ channels are closely related to various cardiac and neurological disorders, such as long QT syndrome, epilepsy, and deafness, which makes KCNQ channels important drug targets. During the past several years, the application of single-particle cryo-electron microscopy (cryo-EM) technique in the structure determination of KCNQ channels has greatly advanced our understanding of their molecular mechanisms. In this review, we summarize the currently available structures of KCNQ channels, analyze their special voltage gating mechanism, and discuss their activation mechanisms by both the endogenous membrane lipid and the exogenous synthetic ligands. These structural studies of KCNQ channels will guide the development of drugs targeting KCNQ channels. • Currently available structures of KCNQ channels are summarized. • KCNQs utilize a special voltage activation without rotation of the S4–S5 linker. • The endogenous membrane lipid PIP 2 binds to two sites to activate KCNQs. • Exogenous synthetic ligands bind to multiple sites to potentiate KCNQ activation. [ABSTRACT FROM AUTHOR]

Opioid-induced respiratory depression (OIRD) is the major cause of death associated with opioid analgesics and drugs of abuse, but the underlying cellular and molecular mechanisms remain poorly understood. We investigated opioid action in vivo in unanesthetized mice and in in vitro medullary slices containing the preBötzinger Complex (preBötC), a locus critical for breathing and inspiratory rhythm generation. Although hypothesized as a primary mechanism, we found that mu-opioid receptor (MOR1)-mediated GIRK activation contributed only modestly to OIRD. Instead, mEPSC recordings from genetically identified Dbx1- derived interneurons, essential for rhythmogenesis, revealed a prevalent presynaptic mode of action for OIRD. Consistent with MOR1-mediated suppression of presynaptic release as a major component of OIRD, Cacna1a KO slices lacking P/Q-type Ca2+ channels enhanced OIRD. Furthermore, OIRD was mimicked and reversed by KCNQ potassium channel activators and blockers, respectively. In vivo whole-body plethysmography combined with systemic delivery of GIRK- and KCNQ-specific potassium channel drugs largely recapitulated these in vitro results, and revealed state-dependent modulation of OIRD. We propose that respiratory failure from OIRD results from a general reduction of synaptic efficacy, leading to a state-dependent collapse of rhythmic network activity. [ABSTRACT FROM AUTHOR]

Relief from chronic pain continues to represent a large unmet need. The voltage-gated potassium channel Kv7.2/7.3, also known as KCNQ2/3, is a key contributor to the control of resting membrane potential and excitability in nociceptive neurons and represents a promising target for potential therapeutics. In this study, we present a medium throughput electrophysiological assay for the identification and characterization of modulators of Kv7.2/7.3 channels, using the IonWorks Barracuda™ automated voltage clamp platform. The assay combines a family of voltage steps used to construct conductance curves with a unique analysis method. Kv7.2/7.3 modulators shift the activation voltage and/or change the maximal conductance of the current, and both parameters have been used to quantify compound mediated effects. Both effects are expected to modulate neuronal excitability in vivo. The analysis method described assigns a single potency value that combines changes in activation voltage and maximal conductance and is expected to predict compound mediated changes in excitability. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Novel pan-Kv7 openers for the treatment of epilepsy are discussed. • Compounds from two series of Kv7 openers show efficacy in PTZ and MES-T seizure models. • Bladder contraction is associated with pan-Kv7 activity, but perhaps not with homomeric Kv7.4 activity as reported. Abstract Neuronal voltage-gated potassium channels, K V 7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K V 7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K V 7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K V 7 isoforms was investigated in human detrusor tissue using a panel of K V 7 openers with distinct activity profiles among K V 7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K V 7.4 did not reduce detrusor contraction. This may suggest that the homomeric K V 7.4 channel plays a less significant role in bladder contraction and further investigation is needed. [ABSTRACT FROM AUTHOR]

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery. [ABSTRACT FROM AUTHOR]

Centipedes can subdue giant prey by using venom, which is metabolically expensive to synthesize and thus used frugally through efficiently disrupting essential physiological systems. Here,we show that a centipede (Scolopendra subspinipes mutilans, ∼3 g) can subdue a mouse (∼45 g) within 30 seconds. We found that this observation is largely due to a peptide toxin in the venom, SsTx and further established that SsTx blocks KCNQ potassium channels to exert the lethal toxicity.We also demonstrated that a KCNQ opener, retigabine, neutralizes the toxicity of a centipede's venom. The study indicates that centipede's venom has evolved to simultaneously disrupt cardiovascular, respiratory, muscular and nervous systems by targeting the broadly distributed KCNQ channels, thus providing a therapeutic strategy for centipede envenomation. [ABSTRACT FROM AUTHOR]

K V 7 channels exert a pivotal role regulating vascular tone in several vascular beds. In this context, K V 7 channel agonists represent an attractive strategy for the treatment of pulmonary arterial hypertension (PAH). Therefore, in this study, we have explored the pulmonary vascular effects of the novel K V 7 channel agonist URO-K10. Consequently, the vasodilator and electrophysiological effects of URO-K10 were tested in rat and human pulmonary arteries (PA) and PA smooth muscle cells (PASMC) using myography and patch-clamp techniques. Protein expression was also determined by Western blot. Morpholino-induced knockdown of KCNE4 was assessed in isolated PA. PASMC proliferation was measured by BrdU incorporation assay. In summary, our data show that URO-K10 is a more effective relaxant of PA than the classical K V 7 activators retigabine and flupirtine. URO-K10 enhanced K V currents in PASMC and its electrophysiological and relaxant effects were inhibited by the K V 7 channel blocker XE991. The effects of URO-K10 were confirmed in human PA. URO-K10 also exhibited antiproliferative effects in human PASMC. Unlike retigabine and flupirtine, URO-K10-induced pulmonary vasodilation was not affected by morpholino-induced knockdown of the KCNE4 regulatory subunit. Noteworthy, the pulmonary vasodilator efficacy of this compound was considerably increased under conditions mimicking the ionic remodelling (as an in vitro model of PAH) and in PA from monocrotaline-induced pulmonary hypertensive rats. Taking all together, URO-K10 behaves as a KCNE4-independent K V 7 channel activator with much increased pulmonary vascular effects compared to classical K V 7 channel activators. Our study identifies a promising new drug in the context of PAH. [Display omitted] • K V 7 agonists could be potentially useful in pulmonary arterial hypertension. • URO-K10 is a novel K V 7 agonist with augmented pulmonary vascular effects compared to other K V 7 agonists. • URO-K10 acts independently of KCNE4 regulatory subunit. • URO-K10′s effects are enhanced in pulmonary arteries from experimental pulmonary arterial hypertension. • URO-K10 can exert a potential benefit over other K V 7 agonists in pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP2). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP2. Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP. In HEK cells stably expressing Kv7.4, PIP or Gβγ increased open probability in a concentration dependent manner. Depleting PIP prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP-induced current enhancement. A combination of PIP and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ. [ABSTRACT FROM AUTHOR]

Facilitating activation, or delaying inactivation, of the native Kv7 channel reduces neuronal excitability, which may be beneficial in controlling spontaneous electrical activity during epileptic seizures. In an effort to identify a compound with such properties, the structure–activity relationship (SAR) and in vitro ADME for a series of heterocyclic Kv7.2–7.5 channel openers was explored. PF-05020182 ( 2 ) demonstrated suitable properties for further testing in vivo where it dose-dependently decreased the number of animals exhibiting full tonic extension convulsions in response to corneal stimulation in the maximal electroshock (MES) assay. In addition, PF-05020182 ( 2 ) significantly inhibited convulsions in the MES assay at doses tested, consistent with in vitro activity measure. The physiochemical properties, in vitro and in vivo activities of PF-05020182 ( 2 ) support further development as an adjunctive treatment of refractory epilepsy. [ABSTRACT FROM AUTHOR]

Aim We tested the hypothesis that KV7 channels contribute to basal renal vascular tone and that they participate in agonist-induced renal vasoconstriction or vasodilation. Methods KV7 channel subtypes in renal arterioles were characterized by immunofluorescence. Renal blood flow ( RBF) was measured using an ultrasonic flow probe. The isometric tension of rat interlobar arteries was examined in a wire myograph. Mice afferent arteriolar diameter was assessed utilizing the perfused juxtamedullary nephron technique. Results Immunofluorescence revealed that KV7.4 channels were expressed in rat afferent arterioles. The KV7 blocker XE991 dose-dependently increased the isometric tension of rat interlobar arteries and caused a small (approx. 4.5%) RBF reduction in vivo. Nifedipine abolished these effects. Likewise, XE991 reduced mouse afferent arteriolar diameter by approx. 5%. The KV7.2-5 stimulator flupirtine dose-dependently relaxed isolated rat interlobar arteries and increased (approx. 5%) RBF in vivo. The RBF responses to NE or Ang II administration were not affected by pre-treatment with XE991 or flupirtine. XE991 pre-treatment caused a minor augmentation of the acetylcholine-induced increase in RBF, while flupirtine pre-treatment did not affect this response. Conclusion It is concluded that KV7 channels, via nifedipine sensitive channels, have a role in the regulation of basal renal vascular tone. There is no indication that KV7 channels have an effect on agonist-induced renal vasoconstriction while there is a small effect on acetylcholine-induced vasodilation. [ABSTRACT FROM AUTHOR]

High dose sodium salicylate causes moderate, reversible hearing loss and tinnitus. Salicylate-induced hearing loss is believed to arise from a reduction in the electromotile response of outer hair cells (OHCs) and/or reduction of KCNQ4 potassium currents in OHCs, which decreases the driving force for the transduction current. Therefore, enhancing OHC potassium currents could potentially prevent salicylate-induced temporary hearing loss. In this study, we tested whether opening voltage-gated potassium channels using ICA-105665, a novel small molecule that opens KCNQ2/3 and KCNQ3/5 channels, can reduce salicylate-induced hearing loss.We found that systemic application of ICA-105665 at 10 mg/kg prevented the salicylate-induced amplitude reduction and threshold shift in the compound action potentials recorded at the round window of the cochlea. ICA-105665 also prevented the salicylate-induced reduction of distortion-product otoacoustic emission. These results suggest that ICA-105665 partially compensates for salicylate-induced cochlear hearing loss by enhancing KCNQ2/3 and KCNQ3/5 potassium currents and the motility of OHCs. [ABSTRACT FROM AUTHOR]

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K+ channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease. [ABSTRACT FROM AUTHOR]

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na+ but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K+ currents were observed at potentials above -60 mV. K+ currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K+ channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K+ channel blocker 4-aminopyridine (10-50 µM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K+ conductances may contribute to different firing responses in calyx afferents. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins. [ABSTRACT FROM AUTHOR]

Background Refractory chronic pain dramatically reduces the quality of life of patients. Existing drugs cannot fully achieve effective chronic pain control because of their lower efficacy and/or accompanying side effects. Voltage-gated potassium channels (KCNQ) openers have demonstrated their analgesic effect in preclinical and clinical studies, and are thus considered to be a potential therapeutic target as analgesics. However, these drugs exhibit a narrow therapeutic window due to their imposed central nerve system (CNS) side effects. To clarify the analgesic effect by peripheral KCNQ channel activation, we investigated whether the analgesic effect of the KCNQ channel opener, retigabine, is inhibited by intracerebroventricular (i.c.v.) administration of the KCNQ channel blocker, 10, 10-bis (4- Pyridinylmethyl)-9(10H) -anthracenone dihydrochloride (XE-991) in rats. Results Oral administration (p.o.) of retigabine showed an anticonvulsant effect on maximal electronic seizures and an analgesic effect on complete Freund's adjuvant-induced thermal hyperalgesia. However, impaired motor coordination and reduced exploratory behavior were also observed at the analgesic doses of retigabine. Administration (i.c.v.) of XE-991 reversed the retigabine-induced anticonvulsant effect, impaired motor coordination and reduced exploratory behavior but not the analgesic effect. Moreover, intraplantar administration of retigabine or an additional KCNQ channel opener, N-(6-Chloro-pyridin-3-yl)-3,4-difluorobenzamide (ICA-27243), inhibited formalin-induced nociceptive behavior. Conclusions Our findings suggest that the peripheral sensory neuron is the main target for KCNQ channel openers to induce analgesia. Therefore, peripheral KCNQ channel openers that do not penetrate the CNS may be suitable analgesic drugs as they would prevent CNS side effects. [ABSTRACT FROM AUTHOR]

KCNQ1-5 α-subunits assemble to form K+ channels that play critical roles in the function of numerous tissues. The channels are tetramers of subunits containing six transmembrane domains. Each subunit consists of a pore region (S5-pore-S6) and a voltage sensor domain (S1-S4). Despite similar structures, KCNQ2 and KCNQ3 homomers yield small current amplitudes compared to other KCNQ homomers and KCNQ2/3 heteromers. Two major mechanisms have been suggested as governing functional expression. The first involves control of channel trafficking to the plasma membrane by the distal part of the C-terminus, containing two coiled-coiled domains, required for channel trafficking and assembly. The proximal half of the C-terminus is the crucial region for channel modulation by signaling molecules such as calmodulin, which may mediate C- and N-terminal interactions. The N-terminus of KCNQ channels has also been postulated as critical for channel surface expression. The second mechanism suggests networks of interactions between the pore helix and the selectivity filter, and between the pore helix and the S6 domain that govern KCNQ current amplitudes. Here, we summarize the role of these different regions in expression of functional KCNQ channels.

KCNQ (Kv7) channels underlie a voltage-gated K+ current best known for control of neuronal excitability, and its inhibition by Gq/11-coupled, muscarinic signaling. Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K+ current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K+ channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K+ channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators. [ABSTRACT FROM AUTHOR]

Background and Purpose Retigabine is a recently approved antiepileptic agent which activates Kv7.2-7.5 potassium channels. It is emerging that these channels have an important role in vascular regulation, but the vascular effects of retigabine in the conscious state are unknown. Hence, in the present study we assessed the regional haemodynamic responses to retigabine in conscious rats. Experimental Approach Male Sprague Dawley rats were chronically instrumented with pulsed Doppler flow probes to measure regional haemodynamic responses to retigabine under control conditions and during acute hypertension induced by infusion of angiotensin II and arginine vasopressin. Further experiments were performed, using the β-adrenoceptor antagonists CGP 20712A, ICI 118551 and propranolol, to elucidate the roles of β-adrenoceptors in the responses to retigabine in vivo and in vitro. Key Results Under normotensive conditions, retigabine induced dose-dependent hypotension and hindquarters vasodilatation, with small, transient renal and mesenteric vasodilatations. In the acutely hypertensive state, the renal and mesenteric, but not hindquarters, vasodilatations were enhanced. The response of the hindquarters vascular bed to retigabine was mediated, in part, by β2-adrenoceptors. However, in vitro experiments confirmed that retigabine did not act as a β-adrenoceptor agonist. Conclusions and Implications We demonstrated that retigabine causes regionally specific vasodilatations, which are different under normotensive and hypertensive conditions, and are, in part, mediated by β2-adrenoceptors in some vascular beds but not in others. These results broadly support previous findings and further indicate that Kv7 channels are a potential therapeutic target for the treatment of vascular diseases associated with inappropriate vasoconstriction. [ABSTRACT FROM AUTHOR]

Highlights: [•] Retigabine, a KV7.2–5 opener, shows antidyskinetic effects in a rat model of LID. [•] We examined if this effect was mainly mediated by KV7.2/3 channels. [•] Indeed, the KV7.2/3 preferring channel opener ICA 27243 attenuated LID. [•] We suggest an antidyskinetic action mainly mediated by striatal KV7.2/3 channels [ABSTRACT FROM AUTHOR]

Abstract: There is strong evidence that M-currents modulate peripheral sensory afferent excitability and that altered M-current efficacy may underpin aspects of pain-induced nociceptor sensitization. Less clear is the role of the M-current in regulating central excitability within spinal dorsal horn nociceptive circuitry. In this study, an in vitro model of central hyperexcitability that uses the potassium channel blocker 4-aminopyridine (4-AP) to induce large amplitude population spikes and 4–12Hz oscillatory activity within rat spinal substantia gelatinosa (SG) has been used to determine the impact of pharmacological modulation of the M-current on central excitability. The M-current enhancers Retigabine (10 and 30μM) and Flupirtine (30μM) had a depressant effect on 4-AP-induced excitation in SG such that the frequency of large amplitude population spikes and the power of 4–12Hz oscillatory activity were both significantly reduced. In contrast, the M-current blockers XE911 (5μM) or Linopirdine (20μM) significantly potentiated 4–12Hz oscillatory activity as evidenced by significant increases in the parameters of power amplitude and power area but had no effect on large amplitude population spikes. These data indicate that pharmacological modulation of the M-current can influence excitability of nociceptive circuitry especially under conditions of central hyperexcitability, as may occur in chronic pain conditions. It is not clear whether these effects reflect a direct effect on interneurones localized to SG or indirectly via sensory afferent terminals. Nonetheless, these central actions should be taken into account alongside peripheral actions in terms of evaluating the potential therapeutic analgesic potency of novel M-current enhancers. [Copyright &y& Elsevier]

Background and Purpose KCNQ2-5 channels are voltage-gated potassium channels that regulate neuronal excitability and represent suitable targets for the treatment of hyperexcitability disorders. The effect of Chlor- N-(6-chlor-pyridin-3-yl)-benzamid was tested on KCNQ subtypes for its ability to alter neuronal excitability and for its anticonvulsant potential. Experimental Approach The effect of 4- Chlor- N-(6-chlor-pyridin-3-yl)-benzamid was evaluated using whole-cell voltage-clamp recordings from CHO cells and Xenopus laevis oocytes expressing different types of KCNQ channels. Epileptiform afterdischarges were recorded in fully amygdala-kindled rats in vivo. Neuronal excitability was assessed using field potential and whole cell recording in rat hippocampus in vitro. Key Results 4- Chlor- N-(6-chlor-pyridin-3-yl)-benzamid caused a hyperpolarizing shift of the activation curve and a pronounced slowing of deactivation in KCNQ2-mediated currents, whereas KCNQ3/5 heteromers remained unaffected. The effect was also apparent in the Retigabine-insensitive mutant KCNQ2- W236L. In fully amygdala-kindled rats, it elevated the threshold for induction of afterdischarges and reduced seizure severity and duration. In hippocampal CA1 cells, 4- Chlor- N-(6-chlor-pyridin-3-yl)-benzamid strongly damped neuronal excitability caused by a membrane hyperpolarization and a decrease in membrane resistance and induced an increase of the somatic resonance frequency on the single cell level, whereas synaptic transmission was unaffected. On the network level, 4- Chlor- N-(6-chlor-pyridin-3-yl)-benzamid caused a significant reduction of γ and θ oscillation peak power, with no significant change in oscillation frequency. Conclusion and Implications Our data indicate that 4- Chlor- N-(6-chlor-pyridin-3-yl)-benzamid is a potent KCNQ activator with a selectivity for KCNQ2 containing channels. It strongly reduces neuronal excitability and displays anticonvulsant activity in vivo. [ABSTRACT FROM AUTHOR]

KCNQ1-5 α-subunits assemble to form K+ channels that play critical roles in the function of numerous tissues. The channels are tetramers of subunits containing six transmembrane domains. Each subunit consists of a pore region (S5-pore-S6) and a voltage-sensor domain (S1-S4). Despite similar structures, KCNQ2 and KCNQ3 homomers yield small current amplitudes compared to other KCNQ homomers and KCNQ2/3 heteromers. Two major mechanisms have been suggested as governing functional expression. The first involves control of channel trafficking to the plasma membrane by the distal part of the C-terminus, containing two coiled-coiled domains, required for channel trafficking and assembly. The proximal half of the C-terminus is the crucial region for channel modulation by signaling molecules such as calmodulin (CaM), which may mediate C- and N-terminal interactions. The N-terminus of KCNQ channels has also been postulated as critical for channel surface expression. The second mechanism suggests networks of interactions between the pore helix and the selectivity filter (SF), and between the pore helix and the S6 domain that govern KCNQ current amplitudes. Here, we summarize the role of these different regions in expression of functional KCNQ channels. [ABSTRACT FROM AUTHOR]

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 μM XE991 sensitized Ad- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991. [ABSTRACT FROM AUTHOR]

Abstract: To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC50 values of the positive control compound (retigabine) determined by the thallium and 86rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC50 values of less than 20μmol/L and 2 demonstrated EC50 values of less than 1μmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels. [Copyright &y& Elsevier]

Abstract: Currently, there are no effective pharmacological therapies for chronic tinnitus despite a number of efforts from clinical studies and more recently, studies in animals using compounds to enhance endogenous inhibition or reduce central hyperactivity. The purpose of the current study was to evaluate the therapeutic efficacy of a novel anxiolytic with potassium channel activity in suppressing salicylate induced tinnitus in animals. Kv7 potassium channels are present in the peripheral and central auditory system where they are believed to modulate neural activity. Maxipost, a compound which attenuates hyperexcitability via positive modulation of Kv7.2–Kv7.5 channels, was administered to rats with behavioral evidence of salicylate induced tinnitus. Tinnitus was measured using our previously established animal model, Schedule Induced Polydipsia Avoidance Conditioning, a paradigm where rats were conditioned to drink only during quiet and suppress drinking in the presence of sound. Salicylate alone significantly suppressed licks in quiet but had no effect on licks in sound; results consistent with the presence of tinnitus. Maxipost at 10mg/kg suppressed behavioral evidence of tinnitus as it completely reversed salicylate''s suppression of licks in quiet. Unexpectedly, the R-enantiomer of Maxipost, R-Maxipost, which has no anxiolytic effects and negatively modulates Kv7.2–Kv7.5, also suppressed behavioral evidence of tinnitus. Our original hypothesis was that Kv7.2–Kv7.5 channels might play a key role in tinnitus generation and that Maxipost but not R-Maxipost would suppress tinnitus; however, it appears that a shared mechanism between Maxipost and R-xMaxipost, such as inhibition of Kv7.1 channels or activation of BK channels or some novel mechanism common to both compounds, underlies salicylate induced tinnitus as both compounds completely abolished behavioral evidence of tinnitus in a dose-dependent manner. Further studies with specific BK channel agonists/antagonists are necessary to determine the contribution of these channels to other forms of tinnitus or determine novel targets that could be related to tinnitus. [Copyright &y& Elsevier]

Abstract: Objectives: The purpose of this study was to build a large reference database of excitability measures in normal subjects and to examine the effects of age, sex, and BMI. Methods: One hundred and five healthy subjects had median motor nerve excitability testing performed at the wrist using the automated threshold-tracking program, QTRAC. Statistical linear regression was used to explore relationships between nerve excitability and the independent variables. Results: The main effect of age is a reduced superexcitability. Lesser effects are flattening of the normalized stimulus response curve and reduction in threshold change following strong hyperpolarizing currents. Females have lower thresholds than males and small but significant differences in voltage-gated potassium channel (KCNQ) mediated properties (late subexcitability, accommodation half time, and threshold undershoot following depolarizing electrotonus), as well as a small increase in superexcitability. BMI has no influence on nerve excitability data and does not explain sex-related differences in threshold. Conclusions: Age and sex have few and small effects on excitability parameters. Significance: The expression of nodal KCNQ channels appears to be greater in females. Age-related increases in subexcitability may be attributable to changes in the muscle fibre and not the nerve. [Copyright &y& Elsevier]

Abstract: KCNQ2/3 voltage-gated potassium channels conduct low-threshold, slowly activating and non-inactivating currents to repolarize the neuronal resting membrane potential. The channels negatively regulate neuronal excitability and KCNQ2/3 openers are efficacious in hyperexcited states such as epilepsy and pain. We developed and utilized thallium influx assays to profile novel KCNQ2/3 channel openers with respect to selectivity across KCNQ subtypes and on requirement for tryptophan 236 of KCNQ2, a critical residue for activity of the KCNQ opener retigabine. Using distinct chemical series of openers, a quinazolinone series showed relatively poor selectivity across multiple KCNQ channels and lacked activity at the KCNQ2(W236L) mutant channel. In contrast, several novel benzimidazole openers showed selectivity for KCNQ2/3 and KCNQ2 and retain activity at KCNQ2(W236L). Profiling of several hundred KCNQ2/3 openers across multiple diverse chemical series revealed that openers show differential degrees of selectivity across subtypes, with selectivity most difficult to achieve against KCNQ2. In addition, we report the significant finding that KCNQ openers can pharmacologically differentiate between homomeric and heteromeric channels containing subtypes in common. Moreover, most openers assayed were dependent on the W236 for activity, whereas only a small number appear to use a distinct mechanism. Collectively, we provide novel insights into the molecular pharmacology of KCNQ channels by demonstrating differential selectivity and site of action for KCNQ2/3 openers. The high-throughput thallium influx assays should prove useful for rapid characterization of KCNQ openers and in guiding efforts to identify selective compounds for advancement towards the clinic. [Copyright &y& Elsevier]

The voltage-gated M-type potassium channel, encoded mainly by the KCNQ2/3 genes, plays an important role in the control of neuronal excitability. Mutations in the KCNQ2 gene lead to a form of neonatal epilepsy in humans termed 'benign familial neonatal convulsions', which is characterized by hyperexcitability of neurons. KCNQ openers or activators are expected to decrease the firing of overactive neurons and are thus conducive to the treatment of epilepsy and pain. Here, we report that four novel synthesized derivatives of pyrazolo[1,5-a]pyrimidin-7(4H)-one (PPO) named QO-26, QO-28, QO-40 and QO-41 potently augmented KCNQ2/3 channels expressed in Chinese hamster ovary cells and shifted the half-maximal activation voltage (V1/2) in the hyperpolarizing direction. The V1/2 was negatively shifted in a concentration-dependent manner. The compounds markedly slowed both KCNQ2/3 channel activation and deactivation kinetics. Structure-activity relationship studies suggest that trifluoromethyl at the C-2 position, phenyl or naphthyl at the C-3 position, and trifluoromethyl or chloromethyl at the C-5 position are essential for the activity. These results suggest the four PPO derivatives act as KCNQ2/3 channel openers, providing a new dimension for the design and development of more potent channel openers. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: With age, cardiac performance declines progressively and the risk of heart disease, a primary cause of mortality, rises dramatically. As the elderly population continues to increase, it is critical to gain a better understanding of the genetic influences and modulatory factors that impact cardiac aging. In an attempt to determine the relevance and utility of the Drosophila heart in unraveling the genetic mechanisms underlying cardiac aging, a variety of heart performance assays have recently been developed to quantify Drosophila heart performance that permit the use of the fruit fly to investigate the heart''s decline with age. As for the human heart, Drosophila heart function also deteriorates with age. Notably, with progressive age the incidence of cardiac arrhythmias, myofibrillar disorganization and susceptibility to heart dysfunction and failure all increase significantly. We review here the evidence for an involvement of the insulin-TOR pathway, the KATP channel subunit dSur, the KCNQ potassium channel, as well as Dystrophin and Myosin in fly cardiac aging, and discuss the utility of the Drosophila heart model for cardiac aging studies. [Copyright &y& Elsevier]