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Histamine, acting via distinct histamine H1 , H2 , H3 , and H4 receptors, regulates various physiological and pathological processes, including pain. In the last two decades, there has been a particular increase in evidence to support the involvement of H3 receptor and H4 receptor in the modulation of neuropathic pain, which remains challenging in terms of management. However, recent data show contrasting effects on neuropathic pain due to multiple factors that determine the pharmacological responses of histamine receptors and their underlying signal transduction properties (e.g., localization on either the presynaptic or postsynaptic neuronal membranes). This review summarizes the most recent findings on the role of histamine and the effects mediated by the four histamine receptors in response to the various stimuli associated with and promoting neuropathic pain. We particularly focus on mechanisms underlying histamine-mediated analgesia, as we aim to clarify the analgesic potential of histamine receptor ligands in neuropathic pain. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1−/− mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1−/− mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1−/− mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1−/− neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1−/− mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress.

Objective This systematic review aims to explore the changes in expression of neuropeptides and/or their receptors following experimental trigeminal neuropathic pain in animals. Design MEDLINE, Embase, and Scopus were searched for publications up to 31st March 2021. Study selection and data extraction were completed by two independent reviewers based on the eligibility criteria. The quality of articles was judged based on the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk-of-bias tool. Results A total of 19 studies satisfied the eligibility criteria and were included for narrative synthesis. Methods of trigeminal neuropathic pain induction were nerve ligation, nerve compression/crush, nerve transection and dental pulp injury. Animal behaviours used for pain verification were evoked responses to mechanical and thermal stimuli. Non-evoked behaviours, including vertical exploration, grooming and food consumption, were also employed in some studies. Calcitonin gene-related peptide (CGRP) and substance P were the most frequently reported neuropeptides. Overall, unclear to high risk of bias was identified in the included studies. Conclusions Limited evidence has suggested the pro-nociceptive role of CGRP in trigeminal neuropathic pain. In order to further translational pain research, animal models of trigeminal neuropathic pain and pain validation methods need to be optimised. Complete reporting of future studies based on available guidelines to improve confidence in research is encouraged.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process – from pre-patterned neural progenitor to active neuron – takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na+ current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiatein vitrointo immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K+but not outward Na+currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

Background: Inhibitors of the extracellular calcium-sensing receptor (CaSR), termed calcilytics, were developed to evoke pulsatile changes in plasma parathyroid hormone (PTH) levels as a novel anti-osteoporotic treatment. Recently we have shown that they can suppress airway hyperresponsiveness (AHR) and inflammation in asthma models, providing a potential for a novel asthma treatment strategy1. Objective: To develop a new calcilytic specifically for the treatment of airway disease and test its efficacy in allergic asthma. Methods: The efficacy of a new chemical class of calcilytics to suppress CaSR signalling was tested in vitro in human embryonic kidney 293 cells expressing the CaSR. The most active stereoisomer of one compound (B2.1-E1) was tested for its biological activity in vivo by administering the calcilytic solution to mice via gavage, after which plasma PTH levels were measured. Finally, the efficacy of the inhaled calcilytics to suppress AHR and inflammation was tested in the poly-L-arginine-induced model of AHR and the ovalbumin-sensitised acute asthma model in mice1. Results: B2.1-E1 supressed CaSR signalling in vitro and increased PTH levels in vivo. Inhaled B2.1-E1 led to dose-dependent suppression of AHR evoked by poly-L-arginine, and diminished AHR and inflammation in the acute asthma model in vivo. Conclusions: The novel calcilytic B2.1-E1 suppressed CaSR in vitro, and elicited PTH release in vivo. In addition, B2.1-E1 suppressed AHR and inflammation in mice, providing a rationale for further development of this calcilytic and its chemical class for the treatment of inflammatory lung disease (IP GB1719023.2). References:1Yarova PL et al. (2015). Science TM 7: 284ra60.

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.

Significance: Ventilatory responses to hypoxia are initiated by the carotid body, where inhibition of specific K+ channels causes cell depolarization, voltage-gated Ca2+ influx, and neurotransmitter release. The identity of the upstream oxygen (O2) sensor is still controversial. Recent Advances: The activity of BKCa channels is regulated by O2, carbon monoxide (CO), and hydrogen sulfide (H2S), suggesting that integration of these signals may be crucial to the physiological response of this tissue. BKCa is colocalized with hemeoxygenase-2, an enzyme that generates CO in the presence of O2, and CO is a BKCa channel opener. Reduced CO during hypoxia results in channel closure, conferring a degree of O2 sensitivity to the BKCa channel. Conversely, H2S is a potent BKCa inhibitor. H2S is produced endogenously by cystathionine-β-synthase and cystathionine-γ-lyase in the rat carotid body, and its intracellular concentration is dependent upon the balance between its enzymatic generation and its mitochondrial breakdown. During hypoxia, mitochondrial oxidation of H2S in many tissues is reduced, leading to hypoxia-evoked rises in its concentration. This may be sufficient to inhibit K+ channels and lead to carotid body excitation. Critical Issues: Carotid body function is heavily dependent upon regulated production and breakdown of CO and H2S and integration of signals from these newly emerging gasotransmitters, in combination with several other proposed mechanisms, may refine, or even define, responses of this tissue to hypoxia. Future Directions: Since several other sensors have been postulated, the challenge of future research is to begin to integrate each in a unifying mechanism, as has been attempted for the first time herein.

Proteomic studies have suggested a biochemical interaction between α subunit of the large conductance, voltage- and Ca2+-activated potassium channel (BKCa α), and annexin A5 (ANXA5), which we verify here by coimmunoprecipitation and double labelling immunocytochemistry. The observation that annexin is flipped to the outer membrane leaflet of the plasma membrane during apoptosis, together with the knowledge that the intracellular C-terminal of BKCa α contains both Ca2+-binding and a putative annexin-binding motif, prompted us to investigate the functional consequences of this protein partnership to cell death. Membrane biotinylation demonstrated that ANXA5 was flipped to the outer membrane leaflet of HEK 293 cells early in serum deprivation-evoked apoptosis. As expected, serum deprivation caused caspase-3/7 activation and this was accentuated in BKCa α expressing HEK 293 cells. The functional consequences of ANXA5 partnership with BKCa α were striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BKCa channel Ca2+-sensitivity, measured in inside-out membrane patches by patch-clamp. Taken together, these data suggest a novel model of the early stages of apoptosis where membrane flippage results in removal of the inhibitory effect of ANXA5 on K+ channel activity with the consequent amplification of Ca2+ influx and augmented activation of caspases.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell‐derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro‐synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell‐derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process – from pre‐patterned neural progenitor to active neuron – takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

Recent evidence suggests that H(2)S contributes to activation of the carotid body by hypoxia by inhibiting K(+) channels. Here, we determine both the molecular identity of the K(+) channel target within the carotid body and the biophysical characteristics of the H(2)S-evoked inhibition by analyzing native rat and human recombinant BK(Ca) channel activity in voltage-clamped, inside-out membrane patches. Rat glomus cells express the enzymes necessary for the endogenous generation of H(2)S, cystathionine-beta-synthase and cystathionine-gamma-lyase. H(2)S inhibits native carotid body and human recombinant BK(Ca) channels with IC(50) values of around 275 microM. Inhibition by H(2)S is rapid and reversible, works by a mechanism which is distinct from that suggested for CO gas regulation of this channel and does not involve an interaction with either the "Ca bowl" or residues distal to this Ca(2+)-sensing domain. These data show that BK(Ca) is a K(+) channel target of H(2)S, and suggest a mechanism to explain the H(2)S-dependent component of O(2) sensing in the carotid body.

The ability of ion channels to respond to an acute perturbation in oxygen tension is a widespread phenomenon, which encompasses many of the major ion channel families. Integral to the ability of several ion channels to respond to acute hypoxic challenge is modulation by upstream enzymatic reactions, suggesting that many ion channels sense oxygen via enzyme-linked processes. Several enzyme-linked oxygen sensing systems have been proposed, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent production of hydrogen peroxide, hemoxygenase-dependent generation of carbon monoxide, adenosine monophosphate (AMP) kinase-dependent channel phosphorylation, and src-Lck protein tyrosine kinase, via a currently undetermined mechanism. Each of these enzymes has been shown to endow specific ion channels with the ability to respond to changes in oxygen, with hypoxia exclusively evoking channel inhibition. This article reviews these proposed mechanisms and presents new insights into how one system, hemeoxygenase-2, confers oxygen sensitivity to large conductance, voltage- and calcium-activated potassium channels.

Increased pressure-induced (myogenic) tone in small uteroplacental arteries from late pregnant (LP) rats has been previously observed. In this study, we hypothesized that this response may result from a diminished activity of vascular smooth muscle cell (SMC) voltage-gated delayed-rectifier K+(Kv) channels, leading to membrane depolarization, augmented Ca2+influx, and vasoconstriction (tone). Elevation of intraluminal pressure from 10 to 60 and 100 mmHg resulted in a marked, diltiazem-sensitive rise in SMC cytosolic Ca2+concentration ([Ca2+]i) associated with a vasoconstriction of uteroplacental arteries of LP rats. In contrast, these changes were significantly diminished in uterine arteries from nonpregnant (NP) rats. Gestational augmentation of pressure-induced Ca2+influx through L-type Ca2+channels was associated with an enhanced SMC depolarization, the appearance of electrical and [Ca2+]ioscillatory activities, and vasomotion. Exposure of vessels from NP animals to 4-aminopyridine, which inhibits the activity of Kvchannels, mimicked the effects of pregnancy by increasing pressure-induced depolarization, elevation of [Ca2+]i, and development of myogenic tone. Furthermore, currents through Kvchannels were significantly reduced in myocytes dissociated from arteries of LP rats compared with those of NP controls. Based on these results, we conclude that decreased Kvchannel activity contributes importantly to enhanced pressure-induced depolarization, Ca2+entry, and increase in myogenic tone present in uteroplacental arteries from LP rats.

Voltage‐gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF‐induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS‐7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time‐lapse imaging analysis showed that EF‐induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS‐7 cells to EF stimulation more than their motility. Immunocytochemistry and pull‐down analyses demonstrated association of Kv1.2 channels with actin‐binding protein cortactin and its re‐localization to the cathode‐facing membrane at EF stimulation, which confirms the mechanism of EF‐induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF‐induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy, and various degenerative syndromes. J. Cell. Physiol. 231: 1375–1384, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Calcium signaling is essential to support erythroid proliferation and differentiation. Precise control of the intracellular Ca2+levels in erythroid precursor cells (EPCs) is afforded by coordinated expression and function of several cation channels, including the recently identified N-methyl-d-aspartate receptor (NMDAR). Here, we characterized the changes in Ca2+uptake and electric currents mediated by the NMDARs occurring during EPC differentiation using flow cytometry and patch clamp. During erythropoietic maturation, subunit composition and properties of the receptor changed; in proerythroblasts and basophilic erythroblasts, fast deactivating currents with high amplitudes were mediated by the GluN2A subunit-dominated receptors, while at the polychromatic and orthochromatic erythroblast stages, the GluN2C subunit was getting more abundant, overriding the expression of GluN2A. At these stages, the currents mediated by the NMDARs carried the features characteristic of the GluN2C-containing receptors, such as prolonged decay time and lower conductance. Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor. Despite this variability, NMDARs were essential for survival of EPCs in any subject tested. Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca2+homeostasis from the orthochromatic erythroblast stage to circulating red blood cells.

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on outward transmembrane ion current in isolated smooth muscle cells (SMC) of the main pulmonary artery of the rabbit. We also studied the characteristics of unitary high-conductance Ca2+-dependent K+ channels (KCa channels) in the SMC membrane in the cell-attached and outside-out configurations. Nitroglycerin in a 10 μM concentration increased the amplitude and intensified oscillations of outward transmembrane current induced by step depolarization. In this case, the threshold of activation of the current (–40 mV) did not change. If the potential was +70 mV, the transmembrane current in the presence of NG increased, as compared with the control, by 32.6 ± 19.4% (n = 6), on average. Simultaneous addition of 10 μM NG and 1 mM tetraethylammonium chloride (TEA), a blocker of KCa channels, to the external solution at the potential of +70 mV decreased the amplitude of outward transmembrane current with respect to the control by 25.2 ± 11% (n = 6) and suppressed oscillations of this current. In the series of experiments carried out in the outside-out configuration (concentration of K+ ions in the external solution was 5.9 mM), we calculated the conductance of a single KCa channel, which was approximately 150 pS. In the case where the potential was equal to +40 mV, 1 mM TEA suppressed completely the current through unitary KCa channels. In the series of experiments performed in the cell-attached configuration, 100 μM NG to a considerable extent intensified the activity of unitary high-conductance KCa channels by increasing the probability of the channel open state (P 0), on average, by 80 ± 1%, as compared with the control. In this case, NG did not influence the conductance of single KCa channels. We concluded that the NO donor NG increases the amplitude of outward transmembrane current in SMC of the rabbit main pulmonary artery by stimulation of the activity of TEA-sensitive high-conductance KCa channels. Our experiments carried out on single KCa channels demonstrated that the activating effect of NG on KCa channels is realized at the expense of an increase in the P 0 of these channels, but not of a change in the conductance of single channels.

Using a sucrose-bridge technique, we studied electrical and mechanical responses of smooth muscle ring strips of the rabbit main pulmonary artery to applications of blockers of voltage-operated (including Ca2+-dependent) K+ channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), as well to application of nitric oxide (NO); nitroglycerin (NG) was used as a donor of the latter. All experiments were carried out under conditions of blockade of the adreno- and cholinoreceptors in the preparation. Both TEA and 4-AP evoked dose-dependent effects: depolarization of smooth muscle cells (SMC) and their contraction. Simultaneous addition of TEA and 4-AP to the normal superfusate (Krebs solution) resulted in intensification of depolarization and initiated generation of action potentials (AP); contractions became rather intensive and possessed a tetanic pattern. Addition of NG to TEA- and 4-AP-containing Krebs solution effectively suppressed AP generation and contractions, whereas the depolarization level underwent only mild modifications. These findings show that Ca2+-dependent high-conductance K+ channels (KCa channels) and 4-AP-sensitive voltage-operated K+ channels (KV channels) are involved in the formation of the resting membrane potential (RMP) in SMC of the rabbit main pulmonary artery. The impact of the KCa channels is greater than that of the KV channels. We suppose that the effects of NO on SMC are related to inhibition of the activity of high-threshold voltage-operated L-type Ca2+ channels and, probably, to lowering of the sensitivity of the contractile SMC apparatus to Ca2+.

All Kv7 potassium channels require membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P2 activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % (P

M-type (Kv7.2/7.3) channels are activated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) [Li et al., 2005: J.Neurosci., 25,9825], through interaction with a cluster of basic residues in the C-terminus [Hernandez et al, 2008: J.Gen.Physiol., 132,361]. However, little is known of the phospholipid specificity and requirements for this activation. We have explored this using inside-out membrane patches from CHO cells stably-expressing Kv7.2 and Kv7.3 subunits and held at a constant voltage ca. −20 mV. The dioctanoyl mono-, di- and tri-phosphatidylinositides DiC8-PI(4)P, DiC8-PI(4,5)P2 and DiC8-PI(3,4,5)P3 all produced biphasic Popen-concentration curves, maximizing at Popen ∼0.8. EC50s for the ‘high-affinity’ component 1 (maximum Popen ∼0.2) were similar at ∼1 μM; ‘low-affinity’ EC50s were inversely proportional to the number of phosphates (DiC8-PI(4)P ∼100 μM, DiC8-PI(4,5)P2 ∼50 μM, DiC8-PI(3,4,5)P3 ∼35 μM). In contrast, the inositol phosphates I(1,4,5)P3 and I(4,5)P2 neither activated nor inhibited the channels up to 300 μM, suggesting a crucial role for the lipophilic moiety. This was tested further using sphingosine-1-phosphate (S-1-P), fingolimod phosphate (FTY720-P) and 1-oleoyl lysophosphatidic acid (LPA). All three activated the Kv7.2/7.3 channels, to Popen values at 100 μM of ∼0.8 (LPA), 0.15 (S-1-P) and 0.022 (FTY720-P). In each case ‘high’ and ‘low’ affinity components to the activation curves could be discerned, with EC50s of 1.5 and 40 μM (LPA), 3 and 160 μM (S-1-P) and 0.5 and 61 μM (FTY720-P). No channel activation was observed using membrane lipids devoid of phosphate groups (D-erythrosphingosine, fingolimod, phosphatidylglycerol and phosphatidylcholine, all at 100 μM). Thus, M-channels can be activated in a rather similar manner by a range of membrane lipids, the minimal requirements at concentrations tested being one or more terminal phosphates and a lipophilic domain.Supported by Wellcome Trust grant 085419.

Hydrogen sulfide (H2S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H2S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H2S on the large conductance, calcium-sensitive potassium channel (BKCa). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BKCa channel α subunit, using patch-clamp in the inside-out configuration. The H2S donor, NaSH (100µM–10 mM), inhibited BKCa channels in a concentration-dependent manner with an IC50 of ca. 670μM. In contrast to the known effects of CO donors, the H2S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H2S-induced channel inhibition, suggesting that the effects of CO and H2S were non-competitive. RT-PCR showed that mRNA for both of the H2S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H2S. In conclusion, we have shown that H2S and CO have opposing effects on BKCachannels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BKCaαsubunit.

In the presence of oxygen (O2), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BKCa channels. This transduction pathway has been proposed to contribute to cellular O2 sensing in rat carotid body. In the present study we have explored the role that four cysteine residues (C820, C911, C995 and C1028), located in the vicinity of the “calcium bowl” of C-terminal of human BKCa-αsubunit, have on channel CO sensitivity. Mutant BKCa-αsubunits were generated by site-directed mutagenesis (single, double and triple cysteine residue substitutions with glycine residues) and were transiently transfected into HEK 293 cells before subsequent analysis in inside-out membrane patches. Potassium cyanide (KCN) completely abolished activation of wild type BKCa channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100μM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC50 of ca. 50μM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC50 values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC50 ca. 100 M). Furthermore, all double and triple mutants which contained the C911G substitution exhibited reduced CO sensitivity, whilst those which did not contain this mutation displayed essentially unaltered CO EC50 values. These data highlight that a single cysteine residue is crucial to the activation of BKCa by CO. We suggest that CO may bind to this channel subunit in a manner similar to the transition metal-dependent co-ordination which is characteristic of several enzymes, such as CO dehydrogenase.

Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK Ca channel alpha1-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside-out, excised membrane patches. Activation by CO was concentration dependent (EC50 approximately 20 microM), rapid, reversible, and evoked a shift in the V 0.5 of -20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca2+]i in the physiological range (100-1,000 nM). Importantly, CO "super-stimulated" BK Ca activity even in saturating [Ca2+]i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9-S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9-S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK Ca channel by redox-independent changes in gating.

M-currents, encoded by Kv7.2/7.3 genes, are low-threshold, voltage-gated potassium channels which contribute to resting membrane potential (Vm) and regulate neuronal excitability. Here, we tested the hypothesis that the development of mouse striatal neurons is associated with enhanced expression and activity of M-currents and that determined whether forced expression of Kv7.2/7.3 accelerates the functional maturation of neurons differentiating from human induced pluripotent stem (iPS) cells. Neurons from the developing mouse striatum were isolated at E13, E15 and E17 and cultured in vitro for up to 14 days; functional profiles of the neurons were explored using whole-cell patch-clamp. In some experiments, subventricular and mantle zones were separated by laser microdissection and channel subunit ontogenies determined by gene chip array. Shortly after isolation (1 day in vitro), there was a clear in vivo development-dependent hyperpolarisation of Vm (E13=-24.8±2.3 mV (n=26), E15=-40.0±2.2 mV (n=12), E17=-47.2±2.4 mV (n=9)). Although Vm continued to hyperpolarise in vitro, by 7 days these differences has disappeared (e.g. E13=-52.1±2.8 mV (n=8), E17 =-54.7±1.9 (n=5)). Associated with these observations, were parallel and progressive increases in M-current activity during in vivo and in vitro development, as evidenced by more pronounced responses to retigabine and XE991. Further, both Kv7.2 and 7.3 genes were progressively enriched in the mantle zone during in vivo development. Importantly, human iPS cell-derived neurons nucleofected with a concatenated Kv7.2/7.3 cDNA plasmid demonstrated a significantly hyperpolarized Vm (from −39.9±2.2 mV (n=12) to −51.3±1.1 mV (n=11)) and a dramatic increase in the proportion of cells generating action potentials spontaneously. Taken together, these data indicate that development of striatal neurons is associated with increased M-current expression and that this might be causative as suggested by the effect of forced Kv7.2/7.3 expression in the iPS cell-derived neurons.

All Kv7 potassium channels require membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P2 activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % (P