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Background: 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., Objectives: 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., Methods: 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., Results: 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., Conclusion: Our results suggest that RTG treatment might robustly mitigate inflammation-induced altered excitability and preserve ascending information processing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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.

The mechanisms behind renal vasodilatation elicited by stimulation of β-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during β-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional K V 7.1 channels was examined in a wire-myograph. The β-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of K V 7 and BK Ca channels affected the β-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of K V 7 channels significantly decreased the β-adrenergic vasorelaxation. However, inhibiting BK Ca channels was equally effective in reducing the β-adrenergic vasorelaxation. The β-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking K V 7.1 channels. As opposed to rats, inhibition of K V 7 channels did not affect the murine β-adrenergic vasorelaxation. Although inhibition and activation of K V 7 channels or BK Ca channels significantly changed baseline RBF in vivo, none of the treatments affected β-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of K V 7 and BK Ca channels significantly reduced the β-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions., (© 2024 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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 (PIP 2 ) 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stott and Greenwood.)

Despite the increasing availability of genomic data and enhanced data analysis procedures, predicting the severity of associated diseases remains elusive in the absence of clinical descriptors. To address this challenge, we have focused on the K V 7.2 voltage-gated potassium channel gene ( KCNQ2 ), known for its link to developmental delays and various epilepsies, including self-limited benign familial neonatal epilepsy and epileptic encephalopathy. Genome-wide tools often exhibit a tendency to overestimate deleterious mutations, frequently overlooking tolerated variants, and lack the capacity to discriminate variant severity. This study introduces a novel approach by evaluating multiple machine learning (ML) protocols and descriptors. The combination of genomic information with a novel Variant Frequency Index (VFI) builds a robust foundation for constructing reliable gene-specific ML models. The ensemble model, MLe-KCNQ2, formed through logistic regression, support vector machine, random forest and gradient boosting algorithms, achieves specificity and sensitivity values surpassing 0.95 (AUC-ROC > 0.98). The ensemble MLe-KCNQ2 model also categorizes pathogenic mutations as benign or severe, with an area under the receiver operating characteristic curve (AUC-ROC) above 0.67. This study not only presents a transferable methodology for accurately classifying KCNQ2 missense variants, but also provides valuable insights for clinical counseling and aids in the determination of variant severity. The research context emphasizes the necessity of precise variant classification, especially for genes like KCNQ2 , contributing to the broader understanding of gene-specific challenges in the field of genomic research. The MLe-KCNQ2 model stands as a promising tool for enhancing clinical decision making and prognosis in the realm of KCNQ2 -related pathologies.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuan Huang reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background and Purpose: Cannabidiol (CBD) is used clinically as an anticonvulsant. Its precise mechanism of action has remained unclear. CBD was recently demonstrated to enhance the activity of the neuronal K V 7.2/7.3 channel, which may be one important contributor to CBD anticonvulsant effect. Curiously, CBD inhibits the closely related cardiac K V 7.1/KCNE1 channel. Whether and how CBD affects other K V 7 subtypes remains uninvestigated and the CBD interaction sites mediating these diverse effects remain unknown., Experimental Approach: Here, we used electrophysiology, molecular dynamics simulations, molecular docking and site-directed mutagenesis to address these questions., Key Results: We found that CBD modulates the activity of all human K V 7 subtypes and that the effects are subtype dependent. CBD enhanced the activity of K V 7.2-7.5 subtypes, seen as a V 50 shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the K V 7.1 and K V 7.1/KCNE1 channels, seen as a V 50 shift towards more positive voltages and reduced conductance. In K V 7.2 and K V 7.4, we propose a CBD interaction site at the subunit interface in the pore domain that overlaps with the interaction site of other compounds, notably the anticonvulsant retigabine. However, CBD relies on other residues for its effects than the conserved tryptophan that is critical for retigabine effects. We propose a similar, though not identical CBD site in K V 7.1, with a non-conserved phenylalanine being important., Conclusions and Implications: We identify novel targets of CBD, contributing to a better understanding of CBD clinical effects and provide mechanistic insights into how CBD modulates different K V 7 subtypes., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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., Competing Interests: Declaration of Competing Interest None. The authors have reported that they have no relationships with industry relevant to the contents of this paper to disclose. The funders played no role in the study design; nor in the collection, analyses, interpretation of data, nor in the writing of the manuscript., (Copyright © 2023. Published by Elsevier Masson SAS.)

For the characterization of K v 7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for K v 7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known K v 7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of K v 7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z'-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study., (© 2023 The Authors. Archiv der Pharmazie published by Wiley-VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Ion channels are highly diverse in the cnidarian model organism Nematostella vectensis (Anthozoa), but little is known about the evolutionary origins of this channel diversity and its conservation across Cnidaria. Here, we examined the evolution of voltage-gated K+ channels in Cnidaria by comparing genomes and transcriptomes of diverse cnidarian species from Anthozoa and Medusozoa. We found an average of over 40 voltage-gated K+ channel genes per species, and a phylogenetic reconstruction of the Kv, KCNQ, and Ether-a-go-go (EAG) gene families identified 28 voltage-gated K+ channels present in the last common ancestor of Anthozoa and Medusozoa (23 Kv, 1 KCNQ, and 4 EAG). Thus, much of the diversification of these channels took place in the stem cnidarian lineage prior to the emergence of modern cnidarian classes. In contrast, the stem bilaterian lineage, from which humans evolved, contained no more than nine voltage-gated K+ channels. These results hint at a complexity to electrical signaling in all cnidarians that contrasts with the perceived anatomical simplicity of their neuromuscular systems. These data provide a foundation from which the function of these cnidarian channels can be investigated, which will undoubtedly provide important insights into cnidarian physiology., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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.)

K V 7 channel openers have proven their therapeutic value in the treatment of pain as well as epilepsy and, moreover, they hold the potential to expand into additional indications with unmet medical needs. However, the clinically validated but meanwhile discontinued K V 7 channel openers flupirtine and retigabine bear an oxidation-sensitive triaminoraryl scaffold, which is suspected of causing adverse drug reactions via the formation of quinoid oxidation products. Here, we report the design and synthesis of nicotinamide analogs and related compounds that remediate the liability in the chemical structure of flupirtine and retigabine. Optimization of a nicotinamide lead structure yielded analogs with excellent K V 7.2/3 opening activity, as evidenced by EC 50 values approaching the single-digit nanomolar range. On the other hand, weighted K V 7.2/3 opening activity data including inactive compounds allowed for the establishment of structure-activity relationships and a plausible binding mode hypothesis verified by docking and molecular dynamics simulations., (© 2022 The Authors. Archiv der Pharmazie published by Wiley-VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

K + channels play potent roles in the process of neurotransmitter release by influencing the action potential waveform and modulating neuronal excitability and release probability. These diverse effects of K + channel activation are ensured by the wide variety of K + channel genes and their differential expression in different cell types. Accordingly, a variety of K + channels have been implicated in regulating neurotransmitter release, including the Ca 2+ - and voltage-gated K + channel Slo1 (also known as BK channel), voltage-gated K + channels of the Kv3 (Shaw-type), Kv1 (Shaker-type), and Kv7 (KCNQ) families, G-protein-gated inwardly rectifying K + (GIRK) channels, and SLO-2 (a Ca 2+ -. Cl - , and voltage-gated K + channel in C. elegans). These channels vary in their expression patterns, subcellular localization, and biophysical properties. Their roles in neurotransmitter release may also vary depending on the synapse and physiological or experimental conditions. This chapter summarizes key findings about the roles of K + channels in regulating neurotransmitter release., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Epilepsy is a neurological disease affected by an imbalance of inhibitory and excitatory signaling in the brain., Introduction: In this disease, the targets are active in pathophysiology and thus can be used as a focus for pharmacological treatment., Methods: Several studies demonstrated the antiepileptic effect of drugs acting on the following targets: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-gated calcium channel (Cav), Gamma aminobutyric acid transporter type 1 (GAT1), voltage-gated sodium channels (Nav), voltage-gated potassium channel of the Q subfamily (KCNQ) and Gamma aminobutyric acid type A (GABAA) receiver., Results: These studies highlight the importance of molecular docking., Conclusion: Quantitative Structure-Activity Relationship (QSAR) and computer aided drug design (CADD) in predicting of possible pharmacological activities of these targets., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Tree and shrub barks have been used as folk medicine by numerous cultures across the globe for millennia, for a variety of indications, including as vasorelaxants and antispasmodics. Here, using electrophysiology and myography, we discovered that the KCNQ5 voltage-gated potassium channel mediates vascular smooth muscle relaxant effects of barks used in Native American folk medicine. Bark extracts (1%) from Birch, Cramp Bark, Slippery Elm, White Oak, Red Willow, White Willow, and Wild Cherry each strongly activated KCNQ5 expressed in Xenopus oocytes. Testing of a subset including both the most and the least efficacious extracts revealed that Red Willow, White Willow, and White Oak KCNQ-dependently relaxed rat mesenteric arteries; in contrast, Black Haw bark neither activated KCNQ5 nor induced vasorelaxation. Two compounds common to the active barks (gallic acid and tannic acid) had similarly potent and efficacious effects on both KCNQ5 activation and vascular relaxation, and this together with KCNQ5 modulation by other tannins provides a molecular basis for smooth muscle relaxation effects of Native American folk medicine bark extracts., (© 2022 Federation of American Societies for Experimental Biology.)

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.

A single sub-anesthetic dose of ketamine produces a rapid and sustained antidepressant response, yet the molecular mechanisms responsible for this remain unclear. Here, we identified cell-type-specific transcriptional signatures associated with a sustained ketamine response in mice. Most interestingly, we identified the Kcnq2 gene as an important downstream regulator of ketamine action in glutamatergic neurons of the ventral hippocampus. We validated these findings through a series of complementary molecular, electrophysiological, cellular, pharmacological, behavioral, and functional experiments. We demonstrated that adjunctive treatment with retigabine, a KCNQ activator, augments ketamine's antidepressant-like effects in mice. Intriguingly, these effects are ketamine specific, as they do not modulate a response to classical antidepressants, such as escitalopram. These findings significantly advance our understanding of the mechanisms underlying the sustained antidepressant effects of ketamine, with important clinical implications., Competing Interests: Declaration of interests A.C. and J.P.L. are named on a patent pending for the combined use of retigabine (ezogabine) and ketamine to amplify antidepressant effects to treat depression and related conditions. F.J.T. reports receiving consulting fees from Roche Diagnostics GmbH and Cellarity Inc. and ownership interest in Cellarity, Inc. and Dermagnostix. All other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The K V 7.4 and K V 7.5 subtypes of voltage-gated potassium channels play a role in important physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate K V 7.4 and K V 7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human K V 7.4 and K V 7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hK V 7.5 by shifting the V 50 of the conductance versus voltage (G(V)) curve toward more negative voltages. This response depends on the head group charge, as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V 50 shifts. In contrast, PUFAs inhibit activation of hK V 7.4 by shifting V 50 toward more positive voltages. No effect on V 50 of hK V 7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hK V 7.5 channel's response to PUFAs is analogous to the one previously observed in hK V 7.1-7.3 channels, whereas the hK V 7.4 channel response is opposite, revealing subtype-specific responses to PUFAs. We identify a unique inner PUFA interaction site in the voltage-sensing domain of hK V 7.4 underlying the PUFA response, revealing an unconventional mechanism of modulation of hK V 7.4 by PUFAs., Competing Interests: DF, KC, JN, SL No competing interests declared, LD Reviewing editor, eLife, (© 2022, Frampton et al.)

The previous studies revealed that many types of ion channels have sensitivity to PtdIns(4,5)P 2 , which has been mainly shown using heterologous expression system. On the other hand, there remains few evidence showing that PtdIns(4,5)P 2 natively regulate the ion channel activities in physiological context. Our group recently discovered that a sperm specific K + channel, Slo3, is natively regulated by PtdIns(4,5)P 2 in sperm flagellum. Very interestingly, a principal piece, to which Slo3 specifically localized, had extremely low density of PtdIns(4,5)P 2 compared to the regular cell plasma membrane. Furthermore, our studies and the previous ones also revealed that Slo3 had much stronger PtdIns(4,5)P 2 affinity than KCNQ2/3 channels, which are widely regulated by endogenous PtdIns(4,5)P 2 in neurons. Thus, the high-PtdIns(4,5)P 2 affinity of Slo3 is well-adapted to the specialized PtdIns(4,5)P 2 environment in the principal piece. This study sheds light on the relationship between PtdIns(4,5)P 2 -affinity of ion channels and their PtdIns(4,5)P 2 environment in native cells. We discuss the current understanding about PtdIns(4,5)P 2 affinity of diverse ion channels and their possible regulatory mechanism in native cellular environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kawai and Okamura.)

K V 7 channels, the voltage-gated K + channels encoded by KCNQ genes, mediate heterogeneous vascular responses in rodents. Postnatal changes in the functional expression of K V 7 channels have been reported in rodent saphenous arteries, but their physiological function in the neonatal renal vascular bed is unclear. Here, we report that, unlike adult pigs, only KCNQ1 (K V 7.1) out of the five members of KCNQ genes was detected in neonatal pig renal microvessels. KCNQ1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21 . Activation of renal vascular smooth muscle cell (SMC) K V 7.1 stimulated whole cell currents, inhibited by HMR1556 (HMR), a selective K V 7.1 blocker. HMR did not change the steady-state diameter of isolated renal microvessels. Similarly, intrarenal artery infusion of HMR did not alter mean arterial pressure, renal blood flow, and renal vascular resistance in the pigs. An ∼20 mmHg reduction in mean arterial pressure evoked effective autoregulation of renal blood flow, which HMR inhibited. We conclude that 1 ) the expression of KCNQ isoforms in porcine renal microvessels is dependent on kidney maturation, 2 ) K V 7.1 is functionally expressed in neonatal pig renal vascular SMCs, 3 ) a decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs, and 4 ) SMC K V 7.1 does not control basal renal vascular tone but contributes to neonatal renal autoregulation triggered by a step decrease in arterial pressure. NEW & NOTEWORTHY K V 7.1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21 . K V 7.1 is functionally expressed in neonatal pig renal vascular smooth muscle cells (SMCs). A decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs. Although SMC K V 7.1 does not control basal renal vascular resistance, its inhibition blunts neonatal renal autoregulation engendered by a step decrease in arterial pressure.

Anhedonia - the reduced ability to experience or respond to pleasure - is an important symptom domain for many psychiatric disorders. It is particularly relevant to depression and other mood disorders and it is a diagnostic criterion of a major depressive episode. Developing safe and effective pharmacological interventions for anhedonia is a critical public health need. The current chapter will review the state of the field with respect to both the efficacy of currently available pharmacotherapies for anhedonia and the recent clinical research focusing on new brain targets, including the kappa-opioid receptor and the KCNQ2/3 receptors. The evidence for anti-anhedonic effects of ketamine and psychedelic agents will be reviewed, as well., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martin-Batista, Manville, Rivero-Pérez, Bartolomé-Martín, Alvarez de la Rosa, Abbott and Giraldez.)

Migraine and major depression are debilitating disorders with high lifetime prevalence rates. Interestingly these disorders are highly comorbid and show significant heritability, suggesting shared pathophysiological mechanisms. Non-homeostatic function of ion channels and neuroinflammation may be common mechanisms underlying both disorders: The excitation-inhibition balance of microcircuits and their modulation by monoaminergic systems, which depend on the expression and function of membrane located K + , Na + , and Ca +2 channels, have been reported to be disturbed in both depression and migraine. Ion channels and energy supply to synapses not only change excitability of neurons but can also mediate the induction and maintenance of inflammatory signaling implicated in the pathophysiology of both disorders. In this respect, Pannexin-1 and P2X7 large-pore ion channel receptors can induce inflammasome formation that triggers release of pro-inflammatory mediators from the cell. Here, the role of ion channels involved in the regulation of excitation-inhibition balance, synaptic energy homeostasis as well as inflammatory signaling in migraine and depression will be reviewed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Eren-Koçak and Dalkara.)

Recent studies in our lab have shown that the K V 7 channel activator, flupirtine, inhibits colonic epithelial Cl - secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na + absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22 Na + fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na + transport in the rat colon. Flupirtine stimulated electroneutral Na + absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (I SC ; i.e., Cl - secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na + /H + exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na + absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na + channel (ENaC)-mediated Na + absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na + absorption in the proximal colon. Finally, the parallel effects of flupirtine on I SC (Cl - secretion) and Na + absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na + absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons. NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal K V 7 K + channels markedly stimulates Na + absorption and inhibits Cl - secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).

In arterial smooth muscle cells, changes in availability of integral membrane proteins influence the regulation of blood flow and blood pressure, which is critical for human health. However, the mechanisms that coordinate the trafficking and membrane expression of specific receptors and ion channels in vascular smooth muscle are poorly understood. In the vasculature, very little is known about microtubules, which form a road network upon which proteins can be transported to and from the cell membrane. This review article summarizes the impact of the microtubule network on arterial contractility, highlighting the importance of the network, with an emphasis on our recent findings regarding the trafficking of the voltage-dependent Kv7 channels., (© 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

The exercise pressor reflex (EPR) originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease or heart failure to increase the incidence of myocardial infarction. We seek to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Bradykinin (BK) is released within contracting muscle and can evoke the EPR. However, the mechanism is incompletely understood. K V 7 channels strongly regulate neuronal excitability and are inhibited by BK. We have identified K V 7 currents in muscle afferent neurons by their characteristic activation/deactivation kinetics, enhancement by the K V 7 activator retigabine, and block by K V 7 specific inhibitor XE991. The blocking of K V 7 current by different XE991 concentrations suggests that the K V 7 current is generated by both K V 7.2/7.3 (high affinity) and K V 7.5 (low affinity) channels. The K V 7 current was inhibited by 300 nM BK in neurons with diameters consistent with both group III and group IV afferents. The inhibition of K V 7 by BK could be a mechanism by which this metabolic mediator generates the EPR. Furthermore, our results suggest that K V 7 channel activators such as retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease. NEW & NOTEWORTHY K V 7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that K V 7 current is generated by K V 7.2/3 and K V 7.5 channels. The BK inhibition of K V 7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance K V 7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.

Mesocorticolimbic dopaminergic (DA) neurons have been implicated in regulating nociception in chronic pain, yet the mechanisms are barely understood. Here, we found that chronic constructive injury (CCI) in mice increased the firing activity and decreased the KCNQ channel-mediated M-currents in ventral tegmental area (VTA) DA neurons projecting to the nucleus accumbens (NAc). Chemogenetic inhibition of the VTA-to-NAc DA neurons alleviated CCI-induced thermal nociception. Opposite changes in the firing activity and M-currents were recorded in VTA DA neurons projecting to the medial prefrontal cortex (mPFC) but did not affect nociception. In addition, intra-VTA injection of retigabine, a KCNQ opener, while reversing the changes of the VTA-to-NAc DA neurons, alleviated CCI-induced nociception, and this was abolished by injecting exogenous BDNF into the NAc. Taken together, these findings highlight a vital role of KCNQ channel-mediated modulation of mesolimbic DA activity in regulating thermal nociception in the chronic pain state.

The pre-Bötzinger complex (preBötC) of the ventral medulla generates the mammalian inspiratory breathing rhythm. When isolated in explants and deprived of synaptic inhibition, the preBötC continues to generate inspiratory-related rhythm. Mechanisms underlying burst generation have been investigated for decades, but cellular and synaptic mechanisms responsible for burst termination have received less attention. KCNQ-mediated K + currents contribute to burst termination in other systems, and their transcripts are expressed in preBötC neurons. Therefore, we tested the hypothesis that KCNQ channels also contribute to burst termination in the preBötC. We recorded KCNQ-like currents in preBötC inspiratory neurons in neonatal rat slices that retain respiratory rhythmicity. Blocking KCNQ channels with XE991 or linopirdine (applied via superfusion or locally) increased inspiratory burst duration by 2- to 3-fold. By contrast, activation of KCNQ with retigabine decreased inspiratory burst duration by ~35%. These data from reduced preparations suggest that the KCNQ current in preBötC neurons contributes to inspiratory burst termination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Revill, Katzell, Del Negro, Milsom and Funk.)

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., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Kv7.1-Kv7.5 (KCNQ1-5) K + channels are voltage-gated K + channels with major roles in neurons, muscle cells and epithelia where they underlie physiologically important K + currents, such as neuronal M current and cardiac I Ks . Specific biophysical properties of Kv7 channels make them particularly well placed to control the activity of excitable cells. Indeed, these channels often work as 'excitability breaks' and are targeted by various hormones and modulators to regulate cellular activity outputs. Genetic deficiencies in all five KCNQ genes result in human excitability disorders, including epilepsy, arrhythmias, deafness and some others. Not surprisingly, this channel family attracts considerable attention as potential drug targets. Here we will review biophysical properties and tissue expression profile of Kv7 channels, discuss recent advances in the understanding of their structure as well as their role in various neurological, cardiovascular and other diseases and pathologies. We will also consider a scope for therapeutic targeting of Kv7 channels for treatment of the above health conditions., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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.)

Activation of the voltage-gated Kv7 channels holds therapeutic promise in several neurological and psychiatric disorders, including epilepsy, schizophrenia, and depression. Here, we present a pharmacological characterization of Lu AA41178, a novel, pan-selective Kv7.2-7.5 opener, using both in vitro assays and a broad range of in vivo assays with relevance to epilepsy, schizophrenia, and depression. Electrophysiological characterization in Xenopus oocytes expressing human Kv7.2-Kv7.5 confirmed Lu AA41178 as a pan-selective opener of Kv7 channels by significantly left-shifting the activation threshold. Additionally, Lu AA41178 was tested in vitro for off-target effects, demonstrating a clean Kv7-selective profile, with no impact on common cardiac ion channels, and no potentiating activity on GABAA channels. Lu AA41178 was evaluated across preclinical in vivo assays with relevance to neurological and psychiatric disorders. In the maximum electroshock seizure threshold test and PTZ seizure threshold test, Lu AA41178 significantly increased the seizure thresholds in mice, demonstrating anticonvulsant efficacy. Lu AA41178 demonstrated antipsychotic-like activity by reducing amphetamine-induced hyperlocomotion in mice as well as lowering conditioned avoidance responses in rats. In the mouse forced swim test, a model with antidepressant predictivity, Lu AA41178 significantly reduced immobility. Additionally, behavioral effects typically observed with Kv7 openers was also characterized. In vivo assays were accompanied by plasma and brain exposures, revealing minimum effective plasma levels <1000 ng/ml. Lu AA41178, a potent opener of neuronal Kv7 channels demonstrate efficacy in assays of epilepsy, schizophrenia and depression and might serve as a valuable tool for exploring the role of Kv7 channels in both neurological and psychiatric disorders., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells., Conclusions: SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.

Relaxation and contraction of the urinary bladder smooth muscle, also known as the detrusor smooth muscle (DSM), facilitate the micturition cycle. DSM contractility depends on cell excitability, which is established by the synchronized activity of multiple diverse ion channels. K + channels, the largest family of channels, control DSM excitability by maintaining the resting membrane potential and shaping the action potentials that cause the phasic contractions. Among the members of the voltage-gated K + (K V ) channel superfamily, K V type 7 (K V 7) channels - K V 7.1-K V 7.5 members encoded by KCNQ1-KCNQ5 genes - have been recently identified as functional regulators in various cell types including vascular, cardiac, and neuronal cells. Their regulatory roles in DSM, however, are just now emerging and remain to be elucidated. To address this gap, our research group has initiated the systematic investigation of human DSM K V 7 channels in collaboration with clinical urologists. In this comprehensive review, we summarize the current understanding of DSM Kv7 channels and highlight recent discoveries in the field. We describe K V 7 channel expression profiles at the mRNA and protein levels, and further elaborate on functional effects of K V 7 channel selective modulators on DSM excitability, contractility, and intracellular Ca 2+ dynamics in animal species along with in vivo studies and the limited data on human DSM. Within each topic, we highlight the main observations, current gaps in knowledge, and most pressing questions and concepts in need of resolution. We emphasize the lack of systematic studies on human DSM K V 7 channels that are now actively ongoing in our laboratory., (Copyright © 2020 Malysz and Petkov.)

Pathogenic variants in KCNQ2 and KCNQ3 , paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K + channel subunits, are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early-onset developmental and epileptic encephalopathy (DEE). KCNQ2 variants account for the majority of pedigrees with BFNE and KCNQ3 variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel KCNQ3 variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S 4 transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S 4 segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits., (Copyright © 2020 Miceli, Carotenuto, Barrese, Soldovieri, Heinzen, Mandel, Lippa, Bier, Goldstein, Cooper, Cilio, Taglialatela and Sands.)

Voltage-gated potassium channels of the K V 7 family are expressed in many tissues. The physiological importance of K V 7 channels is evident from specific forms of disorders linked to dysfunctional K V 7 channels, including variants of epilepsy, cardiac arrhythmia and hearing impairment. Thus, understanding how K V 7 channels are regulated in the body is of great interest. This Mini Review focuses on the effects of polyunsaturated fatty acids (PUFAs) on K V 7 channel activity and possible underlying mechanisms of action. By summarizing reported effects of PUFAs on K V 7 channels and native K V 7-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 K V 7 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 K V 7.1. 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 K V 7.1. Indirect effects include those mediated by PUFA metabolites. Indirect inhibitory effects involve K V 7 channel degradation and re-distribution from lipid rafts. Understanding how PUFAs regulate K V 7 channels may provide insight into physiological regulation of K V 7 channels and bring forth new therapeutic strategies., (Copyright © 2020 Larsson, Frampton and Liin.)

Nearly three million people in the USA suffer traumatic brain injury (TBI) yearly; however, there are no pre- or post-TBI treatment options available. KCNQ2-5 voltage-gated K + channels underlie the neuronal "M current", which plays a dominant role in the regulation of neuronal excitability. Our strategy towards prevention of TBI-induced brain damage is predicated on the suggested hyper-excitability of neurons induced by TBIs, and the decrease in neuronal excitation upon pharmacological augmentation of M/KCNQ K + currents. Seizures are very common after a TBI, making further seizures and development of epilepsy disease more likely. Our hypothesis is that TBI-induced hyperexcitability and ischemia/hypoxia lead to metabolic stress, cell death and a maladaptive inflammatory response that causes further downstream morbidity. Using the mouse controlled closed-cortical impact blunt TBI model, we found that systemic administration of the prototype M-channel "opener", retigabine (RTG), 30 min after TBI, reduces the post-TBI cascade of events, including spontaneous seizures, enhanced susceptibility to chemo-convulsants, metabolic stress, inflammatory responses, blood-brain barrier breakdown, and cell death. This work suggests that acutely reducing neuronal excitability and energy demand via M-current enhancement may be a novel model of therapeutic intervention against post-TBI brain damage and dysfunction.

Working memory relies on the dorsolateral prefrontal cortex (dlPFC), where microcircuits of pyramidal neurons enable persistent firing in the absence of sensory input, maintaining information through recurrent excitation. This activity relies on acetylcholine, although the molecular mechanisms for this dependence are not thoroughly understood. This study investigated the role of muscarinic M1 receptors (M1Rs) in the dlPFC using iontophoresis coupled with single-unit recordings from aging monkeys with naturally occurring cholinergic depletion. We found that M1R stimulation produced an inverted-U dose response on cell firing and behavioral performance when given systemically to aged monkeys. Immunoelectron microscopy localized KCNQ isoforms (Kv7.2, Kv7.3, and Kv7.5) on layer III dendrites and spines, similar to M1Rs. Iontophoretic manipulation of KCNQ channels altered cell firing and reversed the effects of M1R compounds, suggesting that KCNQ channels are one mechanism for M1R actions in the dlPFC. These results indicate that M1Rs may be an appropriate target to treat cognitive disorders with cholinergic alterations., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca 2+ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K + , and direct permeation of this cation through specialized channels. Calmodulin (CaM), through direct binding to these proteins, participates in setting the membrane potential and the overall permeability to Ca 2+ . Over the past years many structures of complete channels in complex with CaM at near atomic resolution have been resolved. In combination with mutagenesis-function, structural information of individual domains and functional studies, different mechanisms employed by CaM to control channel gating are starting to be understood at atomic detail. Here, new insights regarding four types of tetrameric channels with six transmembrane (6TM) architecture, Eag1, SK2/SK4, TRPV5/TRPV6 and KCNQ1-5, and its regulation by CaM are described structurally. Different CaM regions, N-lobe, C-lobe and EF3/EF4-linker play prominent signaling roles in different complexes, emerging the realization of crucial non-canonical interactions between CaM and its target that are only evidenced in the full-channel structure. Different mechanisms to control gating are used, including direct and indirect mechanical actuation over the pore, allosteric control, indirect effect through lipid binding, as well as direct plugging of the pore. Although each CaM lobe engages through apparently similar alpha-helices, they do so using different docking strategies. We discuss how this allows selective action of drugs with great therapeutic potential.

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., (Copyright © 2020 Hefting, D’Este, Arvedsen, Benned-Jensen and Rasmussen.)

Phosphoinositide lipids regulate many cellular processes and are synthesized by lipid kinases. Type I phosphatidylinositol phosphate 5-kinases (PIP5KIs) generate phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ]. Several phosphoinositide-sensitive readouts revealed the nonequivalence of overexpressing PIP5KIβ, PIP5KIγ or Ras association domain family 4 (RASSF4), believed to activate PIP5KIs. Mass spectrometry showed that each of these three proteins increased total cellular phosphatidylinositol bisphosphates (PtdIns P 2 ) and trisphosphates (PtdIns P 3 ) at the expense of phosphatidylinositol phosphate (PtdIns P ) without changing lipid acyl chains. Analysis of KCNQ2/3 channels and PH domains confirmed an increase in plasma membrane PtdIns(4,5) P 2 in response to PIP5KIβ or PIP5KIγ overexpression, but RASSF4 required coexpression with PIP5KIγ to increase plasma membrane PtdIns(4,5) P 2 Effects on the several steps of store-operated calcium entry (SOCE) were not explained by plasma membrane phosphoinositide increases alone. PIP5KIβ and RASSF4 increased STIM1 proximity to the plasma membrane, accelerated STIM1 mobilization and speeded onset of SOCE; however, PIP5KIγ reduced STIM1 recruitment but did not change induced Ca 2+ entry. These differences imply actions through different segregated pools of phosphoinositides and specific protein-protein interactions and targeting.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The group of KCNQ-encoded voltage-gated potassium (Kv7) channels includes five family members (Kv7.1-7.5). We examined the molecular expression and functional roles of Kv7 channels in corporal smooth muscle (CSM). Isolated rabbit CSM strips were mounted in an organ bath system to characterize Kv7 channels during CSM relaxation. Intracellular Ca 2+ levels were measured in the CSM using the Ca 2+ dye Fluo-4 AM. The expression of the KCNQ1-5 (the encoding genes for Kv7.1-7.5) and KCNE1-5 subtypes was determined by quantitative real-time PCR. Electrophysiological recordings and an in situ proximity ligation assay (PLA) were also performed. ML213 (a Kv7.2/7.4/7.5 activator) exhibited the most potent relaxation effect. XE911 (a Kv7.1-7.5 blocker) significantly inhibited the relaxation caused by ML213. Removal of the endothelium from the CSM did not affect the relaxation effect of ML213. H-89 (a protein kinase A inhibitor) and ESI-09 (an exchange protein directly activated by cAMP inhibitor) significantly inhibited ML213-induced relaxation (H-89: 31.3%; ESI-09: 52.7%). XE991 significantly increased basal [Ca 2+ ] i in hCSM cells. KCNQ4 (the Kv7.4-encoding gene) and KCNE4 in CSM were the most abundantly expressed subtypes in humans and rats, respectively. KCNQ4 and KCNE4 expression was significantly decreased in diabetes mellitus rats. ML213 significantly increased the outward current amplitude. XE991 inhibited the ML213-induced outward currents. ML213 hyperpolarized the hCSM cell membrane potential. Subsequent addition of XE991 completely reversed the ML213-induced hyperpolarizing effects. A combination of Kv7.4 and Kv7.5 antibodies generated a strong PLA signal. We found that the Kv7.4 channel is a potential target for ED treatment.

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 Ca 2+ 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., (Copyright © 2019 Wei and Ramirez.)

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.

Effective treatments of neuropathic pain have been a focus of many discovery programs. KCNQ (kv7) are voltage gated potassium channel openers that have the potential for the treatment of CNS disorders including neuropathic pain. Clinical studies have suggested agents such as Retigabine to be a modulator of pain-like effects such as hyperalgesia and allodynia. In this paper, we describe the discovery and evaluation of a series of novel pyrazolopyrimidines and their affinity for potassium channels KCNQ2/3. These pyrazolopyrimidines have also shown good efficacy in the capsaicin-induced acute and secondary mechanical allodynia model and excellent pharmacokinetic properties, which may be superior to Retigabine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The prefrontal cortex (PFC) mediates higher cognition but is impaired by stress exposure when high levels of catecholamines activate calcium-cAMP-protein kinase A (PKA) signaling. The current study examined whether stress and increased cAMP-PKA signaling in rat medial PFC (mPFC) reduce pyramidal cell firing and impair working memory by activating KCNQ potassium channels. KCNQ2 channels were found in mPFC layers II/III and V pyramidal cells, and patch-clamp recordings demonstrated KCNQ currents that were increased by forskolin or by chronic stress exposure, and which were associated with reduced neuronal firing. Low dose of KCNQ blockers infused into rat mPFC improved cognitive performance and prevented acute pharmacological stress-induced deficits. Systemic administration of low doses of KCNQ blocker also improved performance in young and aged rats, but higher doses impaired performance and occasionally induced seizures. Taken together, these data demonstrate that KCNQ channels have powerful influences on mPFC neuronal firing and cognitive function, contributing to stress-induced PFC dysfunction., (© 2019 Published by Elsevier Inc.)

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.)

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (I SC ) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted I SC in a negative direction, but did not attenuate the activity of epithelial Na + channels (ENaC). The application of bumetanide, a Na + -K + -2Cl - cotransporter inhibitor, to the basolateral side reduced the negative I SC shift induced by butyrate. The application of XE991, a KCNQ-type K + channel inhibitor, to the apical side decreased the I SC shift induced by butyrate in a dose-dependent manner. The I SC shift was independent of HCO 3 - and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H + -coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H + secretion through KCNQ-type K + channels on the apical membrane in rat rectal colon. KCNQ-type K + channels on the apical membrane in rat rectal colon. KCNQ-type K + channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.

Benign familial neonatal epilepsy (BFNE) is caused, in about 5% of families, by mutations in the KCNQ3 gene encoding voltage-gated potassium channel subunits. Usually, newborns with BFNE show a normal neurological outcome, but recently, refractory seizures and/or developmental disability have been reported suggesting phenotype variability associated with KCNQ3-related BFNE. Here, we describe a proband from a BFNE family carrying a novel variant in the KCNQ3 gene. Regarding the paucity of data in the literature, we describe the presented case with a view to further establishing: (1) a genotype/phenotype correlation in order to define a BFNE phenotype associated with favourable outcome; (2) an electroclinical pattern associated with BFNE based on video-EEG recording; (3) appropriate first-line AEDs; and (4) the duration of AED treatment. The presented case from Day 3 exhibited a cluster of ictal events, identified as epileptic seizures on Day 10 based on continuous video-EEG polygraphy. The seizures were characterized by asymmetric tonic posturing, associated with a generalized decrease in EEG amplitude, and followed by bilateral asynchronous clonic movements associated with bicentral sharp-wave discharges. The seizures were refractory to intravenous pyridoxine, whereas levetiracetam resulted in rapid total seizure control which has remained to date. This study demonstrates that the novel heterozygous KCNQ3 (c. 914A>T; p.Asp305Val) variant, affecting residues in the pore region, is associated with a specific electroclinical pattern and favourable neurodevelopmental outcome. [Published with video sequence on www.epilepticdisorders.com].