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

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

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

Abstract: Kv7 channels, encoded by corresponding kcnq genes, are expressed both centrally and peripherally where they serve to dampen neuronal activity. While Kv7 channel openers have shown efficacy in neurological and neuropsychiatric disease models, the impact of Kv7 channel activation on physiological endpoint markers have not been addressed in detail. In this study we assessed the effect of a range of Kv7 channel openers with different affinity for neuronal Kv7.2-5 channel subunits on body temperature regulation in mice. Female NMRI mice were acutely exposed to vehicle (10% Tween-80, i.p.), retigabine (3–30mg/kg, i.p., pan-Kv7 channel opener), (S)BMS-204352 (60–240mg/kg, i.p., Kv7.4/5 channel-preferring opener), ICA-27243 (1–10mg/kg, i.p., Kv7.2/3 channel-preferring opener), or S-(1) (10–60mg/kg, i.p., Kv7.2/3 channel-preferring opener), and rectal body temperature was measured 15–120min post-injection. Retigabine (>10mg/kg), ICA-27243 (≥10mg/kg), and S-(1) (≥30mg/kg) dose-dependently lowered rectal body temperature with maximal doses of each Kv7 channel opener inducing a marked drop (>4°C) in rectal temperature. The Kv7 channel openers showed differential temporal pharmacodynamics, which likely reflects their different pharmacokinetic profiles. Pretreatment with the pan-Kv7 channel blocker XE-991 (1.0mg/kg, i.p.) completely reversed the hypothermic effect of the pan-Kv7 opener, retigabine (15mg/kg), whereas ICA-27243-induced hypothermia (10mg/kg) could only be partially prevented by XE-991. Because ICA-27743 and S-(1) are Kv7.2/3 channel subunit-preferring compounds, this suggests that the Kv7.2/3 channel isoform is the predominant substrate for Kv7 channel opener-evoked hypothermia. These data indicate the physiological relevance of Kv7 channel function on body temperature regulation which may potentially reside from central inhibitory Kv7 channel activity. [ABSTRACT FROM AUTHOR]

Abstract: The anticonvulsant retigabine has previously been reported to inhibit bladder overactivity in rats in vivo but the mechanism and site of action are not known. In the present study we investigated the effect of retigabine in isolated rat bladder tissue. Bladders from Sprague–Dawley rats were cut transversally into rings and mounted on an isometric myograph. The average tension, the amplitude and frequency of bladder muscle twitches were measured. The bladder tissue was stimulated with carbachol, KCl (5, 10 and 60mM), and by electric field stimulation. Dose–response curves were obtained with increasing concentrations of the KCNQ(2–5) selective positive modulator, retigabine or with the KCNQ(1–5) negative modulator XE991. Retigabine experiments were repeated in the presence of 10µM XE991. Retigabine reduced both the contractility and the overall tonus of bladder tissue independent of the mode of stimulation with EC50 values ranging from 3.3µM (20mM KCl) to 8.3µM (0.2µM carbachol). In support of a KCNQ-specific effect, retigabine had only weak effects after 60mM KCl pre treatment and all retigabine effects could be reversed by XE991. XE991 increased both the amplitude and mean tension of the bladder but was more potent at increasing the number rather than the size of the stimulated twitches. In conclusion, this study demonstrates an efficacious KCNQ dependent effect of retigabine and XE991 on rat bladder contractility. [Copyright &y& Elsevier]

Abstract: The mammalian KCNQ (Kv7) gene family is composed of five members (KCNQ1–5). KCNQ2, Q4 and Q5 (KCNQ2–5) channels co-express with KCNQ3 to form heterotetrameric voltage-gated K+ (KCNQ2–5/3) channels that underlie the endogenous M-current and regulate neuronal excitability in CNS and PNS neurons. Openers of one or a mixture of these channels may be an attractive therapeutic agent for epilepsy and pain. Non-selective KCNQ2–5/3 activators have shown efficacy in pre-clinical and clinical studies. However, more selective pharmacological profiles, including greater KCNQ sub-type-selective activation, could provide efficacy with fewer side effects. One such compound, ICA-27243, sub-type selectively enhances the activation of KCNQ2/3 channels and also exhibits efficacy in pre-clinical anticonvulsant models; Roeloffs et al. (2008) ; Wickenden et al. (2008) . The binding site of non-selective KCNQ2–5/3 openers maps to the S5–S6 pore domain and is altered by mutation of a tryptophan residue (Trp236 in KCNQ2, Trp265 in KCNQ3) conserved among KCNQ2–5 channels; Schenzer et al. (2005) ; Wuttke et al. (2005) . Here we report that the activity of the KCNQ2/3 selective opener ICA-27243 is not affected by these Trp mutations and does not map to the S5–S6 domain. Rather, the selective activity of ICA-27243 is determined by a novel site within the S1–S4 voltage-sensor domain (VSD) of KCNQ channels. The sub-type-selective activity of ICA-27243 may arise from greater sequence diversity of KCNQ family members within the ICA-27243 binding pocket, allowing for more selective small molecule–protein interactions. [Copyright &y& Elsevier]

Voltage-gated K+ channels co-assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 β subunits generates the repolarizing K+ current IKS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of IKS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for IKS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K+ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation. [ABSTRACT FROM AUTHOR]

Abstract: Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K+ channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K+ channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K+ channels. [Copyright &y& Elsevier]

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate their electrical behaviour as a function of external osmolality through changes in the activity of osmosensitive ion channels. We now present evidence that the MNCs express an osmosensitive voltage-gated K+ current (the OKC). Whole-cell patch-clamp experiments on acutely isolated MNCs were used to show that increases in the external osmolality from 295 to 325 mosmol/kg cause an increase in a slow, tetraethylammonium-insensitive outward current. The equilibrium potential for this current is close to the predicted EK in two different concentrations of external K+. The OKC is sensitive to block by Ba2+ (0.3 mm), and by the M-type K+ current blockers linopirdine (150 μm) and XE991 (5 μm), and to enhancement by retigabine (10 μm), which increases opening of M-type K+ channels. The OKC is suppressed by muscarine (30 μm) and is decreased by the L-type Ca2+ channel blocker nifedipine (10 μm), but not by apamin (100 nm), which blocks SK-type Ca2+-dependent K+ currents. Reverse transcriptase-polymerase chain reaction and immunocytochemical data suggest that MNCs express several members of the KV7 (KCNQ) family of K+ channels, including KV7.2, 7.3, 7.4 and 7.5. Extracellular recordings of individual MNCs in a hypothalamic explant preparation demonstrated that an XE991- and retigabine-sensitive current contribute to the regulation of MNC firing. Our data suggest that the MNCs express an osmosensitive K+ current that could contribute to the regulation of MNC firing by external osmolality and that could be mediated by KV7/M-type K+ channels. [ABSTRACT FROM AUTHOR]

We examined whether combinations of K v 7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (−55 to −40 mV; V h = −60 mV; 1500 ms) and K v 7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5–100 μM) and/or Diclofenac (50–140 μM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED 50 : 62.7 ± 3.18 μM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED 50 : 93.2 ± 8.99 μM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 μM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 μM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors. • ED50s for Retigabine and Diclofenac were determined for the first time in Nociceptors • The combination of Retigabine and Diclofenac greatly enhanced nociceptor evoked currents. • Retigabine + Diclofenac substantially hyperpolarized the resting membrane potential. • Retigabine + Diclofenac substantially reduced nociceptor burst discharge. • Combinations of Retigabine and Diclofenac did not safely reduce pain-like behaviors. [ABSTRACT FROM AUTHOR]

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

Abstract: Ion channels encoded by KCNQ genes (1–5) are key regulators of membrane properties in many cell types. The KCNQ5 gene was the last to be identified and has three splice variants that are expressed in human brain and skeletal muscle. The KCNQ5 encoded channel possesses M-current properties and so far no channelopathy has been associated with any of the three variants. We now show that only the shortest KCNQ5 variant, which has exon 9 deleted, was expressed in a variety of murine vascular smooth muscle. In Xenopus oocytes, this variant generated currents with amplitudes, activation kinetics and biophysical properties similar to the full-length variant normally expressed in neuronal tissue. Furthermore sensitivity to block by XE991 and activation by retigabine were also similar between both variants. These data represent an exhaustive characterisation of a truncated KCNQ5 splice variant that may contribute to the native XE991-sensitive channel in murine vasculature. [Copyright &y& Elsevier]

Abstract: Enhancement of membrane K+ conductance may reduce the abnormal excitability of primary afferent nociceptive neurons in neuropathic pain. It has been shown that retigabine, a novel anticonvulsant, activates Kv7 (KCNQ/M) channels in the axonal/nodal membrane of peripheral myelinated axons. In this study, we have tested the effects of retigabine on excitability parameters of C-type nerve fibers in isolated fascicles of human sural nerve. Application of retigabine (3–10μM) produced an increase in membrane threshold. This effect was pronounced in depolarized axons and small in hyperpolarized axons. This finding indicates that retigabine produces a membrane hyperpolarization which is limited by the K+ equilibrium potential. The retigabine-induced reduction in excitability was accompanied by modifications of the post-spike recovery cycle. Most notable is the development of a late subexcitability at 250–400ms following a short burst of action potentials. All effects of retigabine were blocked in the presence of XE991 (10μM). The data show that Kv7 channels are present on axons of unmyelinated, including nociceptive, peripheral human nerve fibers. It is likely that activation of these channels by retigabine may reduce the ectopic generation of action potentials in neuropathic pain. [Copyright &y& Elsevier]

Voltage-dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca2+-binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild-type channels in the ER. Thus, a direct interaction with Ca2+-calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels' exit from the ER. [ABSTRACT FROM AUTHOR]

Abstract: Bipolar spectrum disorders are severe chronic mood disorders that are characterized by episodes of mania or hypomania and depression. Because patients with manic symptoms often experience clinical benefit from treatment with anticonvulsant drugs, it was hypothesized that retigabine, a novel compound with anticonvulsant efficacy, may also possess antimanic activity. The amphetamine (AMPH)+chlordiazepoxide (CDP)-induced hyperactivity model has been proposed as a suitable model for studying antimanic-like activity of novel compounds in mice and rats. The aims of the present study in rats were therefore (1) to confirm previous findings with lithium and lamotrigine, and (2) to evaluate the effect of the novel compound retigabine on AMPH+CDP-induced hyperactivity in rats. In all experiments, co-administration of AMPH and CDP induced a significant increase (191–295%) in locomotor activity. Lithium chloride (0.9mg/kg) and lamotrigine (20mg/kg), which are known to effectively stabilize mood in humans, both significantly decreased AMPH+CDP-induced locomotor activity without affecting basal locomotor activity. The results furthermore indicate that retigabine, like lithium and lamotrigine, significantly and dose-dependently attenuates the induced hyperactivity at a lowest effective dose of 1.0mg/kg, whereas basal locomotor activity is reduced only at doses ⩾4.0mg/kg. In conclusion, retigabine was found to have an antimanic-like effect in the AMPH+CDP-induced hyperactivity model, suggesting a potential role for retigabine in the treatment of mania and possibly in the management of bipolar disorder. [Copyright &y& Elsevier]

Loss-of-function mutations in Kv7.2 or Kv7.3 K+ channel subunits underlies the neonatal epilepsy benign familial neonatal convulsions (BFNC). These two subunits interact to form a functional K+ channel that underlies the M-current (IM), a voltage-dependent noninactivating K+ current. In BFNC, seizures begin shortly after birth, and spontaneously remit in the first few months of life. The nature of this window of vulnerability is unclear. We address this issue using a hippocampal slice model, to study the effects of IM blockade or augmentation on epileptiform activity. We used the Mg++-free seizure model in adult and immature (P8-P15) acute rat hippocampal slices. We recorded from both CA1 and CA3 regions using extracellular and intracellular methods. When M-channels are blocked pharmacologically, the transition from interictal to ictal bursting becomes much more likely, especially in immature brain. We also show augmentation of IM is effective in stopping ictal events in immature brain, at the developmental age that approximates a human newborn in cortical development. IM appears to counter the sustained N-methyl-D-aspartate (NMDA) receptor-mediated depolarizations needed to trigger an ictal event. The increased likelihood of ictal bursting by IM blockade is not shared by other selective K+ channel blockers that increase hippocampal excitability. Voltage-dependent M-channels are activated duringinterictal bursts and contribute to burst termination. When these channels are compromised, interictal burstduration becomes sufficient to trigger the sustained depolarizations that underlie ictal bursts. This transition to ictal bursts upon IM blockade is especially likely to occur in immature hippocampus. This selective function of M-channels likely contributes to the transient window of vulnerability to seizures that occurs with BFNC. [ABSTRACT FROM AUTHOR]

Background and purpose:This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility.Experimental approach:Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta.Key results:All blood vessels expressed KCNQ1, 4 and 5 with hitherto ‘neuronal’ KCNQ4 being, surprisingly, the most abundant. The correlated proteins Kv7.1, Kv7.4 and Kv7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate Kv7 channels, retigabine (2 –20 μM), flupirtine (20 μM) and meclofenamic acid (20 μM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the Kv7 channel blocker XE991 (10 μM). All vessels tested contracted upon application of the Kv7 channel blockers XE991 and linopirdine (0.1-10 μM).Conclusions and implications:Murine blood vessels exhibit a distinctive KCNQ expression profile with ‘neuronal’ KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as Kv7 channel blockers produced marked contractions whereas Kv7 channel activators were effective vasorelaxants.British Journal of Pharmacology (2007) 151, 758–770; doi:10.1038/sj.bjp.0707284; published online 21 May 2007 [ABSTRACT FROM AUTHOR]

Abstract: Mutations in the KCNQ2 gene cause myokymia and neonatal epilepsy, indicating that this K+ channel regulates the excitability of lower motoneurons and CNS neurons. Little is known about the parameters that direct the assembly of this multimeric molecule and other KCNQ subunits. Here, we show that the carboxy-terminal subunit interaction domain of KCNQ2 autonomously folds and assembles into tetramers. This domain contains a bipartite coiled-coil motif. Whereas structural integrity of the second coiled-coil motif is crucial for tetramer formation, that of the first motif is less important. These data suggest a crucial role of coiled-coil motifs in tetrameric KCNQ channel assembly. [Copyright &y& Elsevier]

Abstract: The family of Kv7 (KCNQ) potassium channels consists of five members. Kv7.2 and 3 are the primary molecular correlates of the M-current, but also Kv7.4 and Kv7.5 display M-current characteristics. M-channel modulators include blockers (e.g., linopirdine) for cognition enhancement and openers (e.g., retigabine) for treatment of epilepsy and neuropathic pain. We investigated the effect of a Bristol–Myers Squibb compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1] on cloned human Kv7.1–5 potassium channels expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp recordings we found that (S)-1 blocks Kv7.1 and Kv7.1/KCNE1 currents. In contrast, (S)-1 produced a hyperpolarizing shift of the activation curve for Kv7.2, Kv7.2/Kv7.3, Kv7.4 and Kv7.5. Further, the compound enhanced the maximal current amplitude at all potentials for Kv7.4 and Kv7.5 whereas the combined activation/block of Kv7.2 and Kv7.2/3 was strongly voltage-dependent. The tryptophan residue 242 in S5, known to be crucial for the effect of retigabine, was also shown to be critical for the enhancing effect of (S)-1 and BMS204352. Furthermore, no additive effect on Kv7.4 current amplitude was observed when both retigabine and (S)-1 or BMS204352 were applied simultaneously. In conclusion, (S)-1 differentially affects the Kv7 channel subtypes and is dependent on a single tryptophan for the current enhancing effect in Kv7.4. [Copyright &y& Elsevier]

Voltage-gated K+ channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2–S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress. [ABSTRACT FROM AUTHOR]

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it influences electrical excitability and cell survival. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss in humans and functional k.o.-mice show progressive degeneration of outer hair cells (OHCs). However, characteristic electrophysiological features of the native KCNQ4-carried current IK,n in OHCs are not recapitulated by expression of KCNQ4 channels in heterologous expression systems. This might suggest modulation of KCNQ4 by interacting KCNE β-subunits, which are known to modify the properties of the closely related KCNQ1. The present study explored whether transcripts of the KCNE isoforms could be identified in OHC mRNA and whether the subunits modulate KCNQ4 function. RT-PCR indeed yielded transcripts of all five KCNEs in OHCs. Coexpression of the KCNE-β-subunits with human KCNQ4 in the Xenopus laevis oocyte expression system revealed that all KCNEs modulate KCNQ4 voltage dependence, protein stability and ion selectivity of hKCNQ4 in Xenopus oocytes. The deafness-associated Jervell and Lange-Nielsen syndrome (JLNS) mutation KCNE1(D76N) impairs KCNQ4-function whereas the Romano-Ward syndrome (RWS) mutant KCNE1(S74L), which shows normal hearing in patients, does not impair KCNQ4 channel function. In conclusion, KCNEs are presumably coexpressed with KCNQ4 in hair cells from the organ of Corti and might regulate KCNQ4 functional properties, effects that could be important under physiological and pathophysiological conditions. [ABSTRACT FROM AUTHOR]

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it determines electrical excitability. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss. Several K+ channels including the closely related KCNQ1/KCNE1 channel are regulated by the serum- and glucocorticoid-inducible kinase (SGK) family. The present study utilized the Xenopus oocyte system to explore effects of SGK isoforms on KCNQ4 mediated K+-currents: KCNQ4 channels activated in a voltage dependent manner with half maximal activation at -10 mV. The peak channel activity was significantly increased by prepulsing. Coexpression of wild type SGK1 but not coexpression of the inactive mutant K127NSGK1 significantly increased current amplitudes (by 67 %) and significantly increased the resting potential of KCNQ4 expressing oocytes. Here we describe for the first time a prepulse dependence of KCNQ4 channels with increased currents after hyperpolarizing prepulses. Coexpression of SGK1 significantly attenuated the effect of prepulsing on peak currents. Mutation of Ser to Asp or Ala in the putative phosphorylation consensus sequence in KCNQ4 significantly decreased the sensitivity to SGK1-coexpression. In conclusion, SGK1 regulates current amplitudes and kinetic properties of KCNQ4 channel activity, an effect sensitive to mutations in the SGK1 consensus sequence of the channel. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The non-inactivating potassium M-current exerts a strong influence on neuronal excitability. The channels responsible for this current are made up of KCNQ subunits, and mutations in most of these produce human pathologies. Notably, in terms of excitation, mutations in either KCNQ2 or KCNQ3 lead to benign neonatal familial convulsions. Although a mere reduction of 25% in KCNQ2/3 function can increase excitability to epileptogenic levels, the potentiation of these subunits has anti-epileptogenic effects. After KCNQ2/3 heteromerization, current levels can augment as much as 10-fold, and we have discovered that there are three processes underlying this potentiation. First, there is an increase in the number of channels inserted in the membrane after heteromerization of the C-terminal region. Second, the N-terminal domain from KCNQ2 exerts a negative influence on the current level. Finally, Ala 315 of KCNQ3, a residue located in the inner vestibule after the selectivity filter, plays a critical role in preventing current flow in KCNQ3 homomeric channels, whereas it is permissive in heteromers in combination with Thr at the equivalent 276 position of KCNQ2. [ABSTRACT FROM AUTHOR]

Background and Purpose: Prostacyclin mimetics express potent vasoactive effects via prostanoid receptors that are not unequivocally defined, as to date no study has considered sex as a factor. The aim of this study was to determine the contribution of IP and EP 3 prostanoid receptors to prostacyclin mimetic iloprost-mediated responses, whether K V 7.1-5 channels represent downstream targets of selective prostacyclin-IP-receptor agonist MRE-269 and the impact of the oestrus cycle on vascular reactivity., Experimental Approach: Within second-order mesenteric arteries from male and female Wistar rats, we determined (1) relative mRNA transcripts for EP 1-4 (Ptger 1-4 ), IP (Ptgi) and TXA 2 (Tbxa) prostanoid receptors via RT-qPCR; (2) the effect of iloprost, MRE-269, isoprenaline and ML277 on precontracted arterial tone in the presence of inhibitors of prostanoid receptors, potassium channels and the molecular interference of K V 7.1 via wire-myograph; (3) oestrus cycle stage via histological changes in cervical cell preparations., Key Results: Iloprost evoked a biphasic response in male mesenteric arteries, at concentrations ≤100 nmol·L -1 relaxing, then contracting the vessel at concentration ≥300 nmol·L -1 , a process attributed to IP and EP 3 receptors respectively. Secondary contraction was absent in the females, which was associated with a reduction in Ptger3. Pharmacological inhibition and molecular interference of K V 7.1 significantly attenuated relaxations produced by the selective IP receptor agonist MRE-269 in male and female Wistar in dioestrus/metoestrus, but not pro-oestrus/oestrus., Conclusions and Implications: Stark sexual dimorphisms in iloprost-mediated vasoactive responses are present within mesenteric arteries. K V 7.1 is implicated in IP receptor-mediated vasorelaxation and is impaired by the oestrus cycle., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders. [Display omitted] • ALS patient-derived motor neurons are hyperexcitable • A high-throughput GCaMP screen is used to identify ALS excitability modulators • Kv7 ion channels, AMPA receptors, and the type 2 dopamine receptor are major targets • The D2 dopamine receptor modulates both neuronal excitability and survival Motor neuron hyperexcitability is observed in both ALS patients and their iPSC-derived neurons. Combining a high-content live imaging excitability phenotypic assay, high-throughput screening against a cross-annotated chemogenomic library, and bioinformatic enrichment analysis, Huang et al. identify targets modulating the hyperexcitability of ALS patient-derived motor neurons in an unbiased manner. [ABSTRACT FROM AUTHOR]

Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2)-deficient mice develop spontaneous seizures in early life because of a marked reduction in M currents, which regulate neuronal membrane excitability. We have previously shown that hyper-SUMOylation of the Kv7.2 and Kv7.3 channels is critically involved in the regulation of the M currents conducted by these potassium voltage-gated channels. Here, we show that hyper-SUMOylation of the Kv7.2 and Kv7.3 proteins reduced binding to the lipid secondary messenger PIP 2 . CaM1 has been shown to be tethered to the Kv7 subunits via hydrophobic motifs in its C termini and implicated in the channel assembly. Mutation of the SUMOylation sites on Kv7.2 and Kv7.3 specifically resulted in decreased binding to CaM1 and enhanced CaM1-mediated assembly of Kv7.2 and Kv7.3, whereas hyper-SUMOylation of Kv7.2 and Kv7.3 inhibited channel assembly. SENP2-deficient mice exhibited increased acetylcholine levels in the brain and the heart tissue because of increases in the vagal tone induced by recurrent seizures. The SENP2-deficient mice develop seizures followed by a period of sinus pauses or atrioventricular conduction blocks. Chronic administration of the parasympathetic blocker atropine or unilateral vagotomy significantly prolonged the life of the SENP2-deficient mice. Furthermore, we showed that retigabine, an M-current opener, reduced the transcription of SUMO-activating enzyme SAE1 and inhibited SUMOylation of the Kv7.2 and Kv7.3 channels, and also prolonged the life of SENP2-deficient mice. Taken together, the previously demonstrated roles of PIP2, CaM1, and retigabine on the regulation of Kv7.2 and Kv7.3 channel function can be explained by their roles in regulating SUMOylation of this critical potassium channel., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

• Inhibition of Kv7 channels induce Ca2+ signals through L- and T-type VGCCs in rat and human vascular smooth muscle cells. • Kv7.5 is functionally the most important subunit to mediate these effects. • Phospholipase C mediated Kv7 inhibition is likely to contribute to Ca2+ oscillations induced in VSMCs by vasopressin. • Kv7 activator, retigabine, strongly suppresses [Ca2+ i oscillations induced by vasopressin or direct Kv7 channel inhibition. Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+ i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes. [ABSTRACT FROM AUTHOR]

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca2+ 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 Ca2+. 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. [ABSTRACT FROM AUTHOR]

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.)

The spatial and temporal balance of spinal α-motoneuron (αMN) intrinsic membrane conductances underlies the neural output of the final common pathway for motor commands. Although the complete set and precise localization of αMN K + channels and their respective outward conductances remain unsettled, important K + channel subtypes have now been documented, including Kv1, Kv2, Kv7, TASK, HCN and SK isoforms. Unique kinetics and gating parameters allow these channels to differentially shape and/or modify αMN firing properties, and recent immunohistochemical localization of K + -channel complexes reveals a framework in which their spatial distribution and/or focal clustering within different surface membrane compartments is highly tuned to their physiological function. Moreover, highly evolved regulatory mechanisms enable specific channels to operate over variable levels of αMN activity and contribute to either state-dependent enhancement or diminution of firing. While recent data suggest an additional, non-conducting role for clustered Kv2.1 channels in the formation of endoplasmic reticulum-plasma membrane junctions postsynaptic to C-bouton synapses, electrophysiological evidence demonstrates that conducting Kv2.1 channels effectively regulate αMN firing, especially during periods of high activity in which the cholinergic C-boutons are engaged. Intense αMN activity or cell injury rapidly disrupts the clustered organization of Kv2.1 channels in αMNs and further impacts their physiological role. Thus, αMN K + channels play a critical regulatory role in motor processing and are potential therapeutic targets for diseases affecting αMN excitability and motor output, including amyotrophic lateral sclerosis., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

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.)

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.)

Purpose: Hypoxic pulmonary vasoconstriction (HPV) regulates regional pulmonary blood flow in order to match regional ventilation to preserve arterial oxygenation. HPV is impaired in patients with sepsis or acute respiratory distress syndrome (ARDS). Endotoxemic mice show reduced HPV and recent evidence suggests a central role of voltage gated potassium channel 7 (Kv7) in regulating HPV. Therefore, we tested the hypothesis if Kv7 is induced and inhibition of Kv7 increases HPV in endotoxemia., Materials and Methods: Isolated lungs of LPS-pretreated and untreated animals were perfused with and without specific inhibitors of Kv7 (linopirdine (LI) 0, 0.1, 1 and 10 µM) or Kv7.1 (HMR1556 100 nM). Pulmonary artery pressure (PAP) during normoxic (FiO 2 0.21) as well as hypoxic (FiO 2 0.01) ventilation were obtained. Expressions of Kv7 composing (KCNQ1-5) as well as auxiliary subunits (KCNE1-5) were measured in mouse lungs with and without endotoxemia., Results: HPV was impaired in lungs from LPS mice (16 ± 7% vs 105 ± 13% control, p < 0.05). Perfusion of control lungs with 10 µM LI or 100 nM HMR1556 did not affect HPV (LI 105 ± 12% vs 105 ± 13% vehicle, HMR1556 100 ± 6% vs 98 ± 26%, P = NS). In LPS mice perfusion with 10 µM LI (74.2 ± 7% vs. 16 ± 7% vehicle, P < 0.05) or HMR1556 100 nM augmented HPV (74 ± 28% vs. 15 ± 17% vehicle, P < 0.05). KCNQ1, 4 and 5 gene- and protein expressions as well as KCNE1, 2 and 4 gene expressions were unaltered in endotoxemic lungs. KCNE3 gene and protein expressions were increased in lungs of LPS treated mice (3.1 ± 1.3-fold and 1.8 ± 0.3-fold, respectively, P < 0.05 for both)., Conclusions: Endotoxemia does not alter KCNQ1, 4 and 5 gene and protein expressions but increases pulmonary KCNE3 gene and protein expression. In isolated perfused endotoxemic mouse lungs, perfusion with 10 µM LI or 100 nM HMR1556 augments HPV.

Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca 2+ but until now, the exact role of Kv7 channels in cytosolic Ca 2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca 2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca 2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca 2+ channel inhibitor, nifedipine, or T-type Ca 2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca 2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca 2+ oscillations. AVP and XE991 induced a large increase of [Ca 2+ ] i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca 2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca 2+ signalling in VSMCs with Kv7.5 playing a dominant role., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)